Advisory Board & Committees
Transcript TRRS 2/27/2008
This transcript was generated automatically and has not been reviewed. Typically, automated transcripts will contain words that were not recognized properly by the software.
Good morning, everybody. I would like to call to order the meeting of the national toxicology program of scientific counselors toxicology subcommittee. We will go around the room now to introduce ourselves.
[ Speaker Faint/Audio Unclear ].
Dave Malarkey, NTP.
Gordon, NTP.
Grace kiss link, NTP.
[ Speaker Faint/Audio Unclear ].
Tracy Bunton.
Mark toreson.
Paul Howard.
Kenny Crump.
Russ Cattley.
Chris Bradfield.
[ Speaker Faint/Audio Unclear ].
Ray Novak.
Barbara Shane.
John Bucher.
[ Speaker Faint/Audio Unclear ].
[ Indiscernible ].
Susan [ Indiscernible ].
Charles allton.
Ron Lawrenceson.
[ Indiscernible ].
Mary Boudreau.
Paul [ Indiscernible ].
Susan [ Indiscernible ].
[ Indiscernible ].
Ron Melnick.
[ Indiscernible ].
John [ Indiscernible ].
Bill Jamieson.
Paul Foster.
Ron Herbert.
Angela king Herbert.
Ruth Lynn.
Matt Stout.
Joe [ Indiscernible ].
[ Indiscernible ].
Charles [ Indiscernible ].
Melton [ Indiscernible ].
Cynthia Smith.
Christine [ Indiscernible ].
[ Indiscernible ].
Greg [ Indiscernible ].
Robert souls.
And I'm Nancy Kerkvliet, Oregon state university. Dr. Shane, do you have any comments before we begin?
You will find the latest agenda, roster, the list of written comments and copies of the comments and a list of oral commenters and the form to fill out for reimbursement. Please make sure you have signed in and use the microphones and identify yourself. If you are making a public comment for the public out there Rio de Janeiroster at the table outside. We will be taking a photograph this morning at the break outside in the lobby. The draft reviews are preliminary comments by the viewers and will not be included in the minutes. The board members must say any comments they want to be in the final reports. I will read a conflict of interest statement, the members of the subcommittee serve as individual scientists. Each member is to exercise jiewment prior to any meeting as to if any [ Speaker Faint/Audio Unclear ] or financial interest. Of yourself, your spouse, minor child, general partner or outside organization for which you are negotiating or arranging for future employment. Should there be conflict of interest [ Speaker Faint/Audio Unclear ] and not to participate in discussion or vote on any action. I just want to identify that Dr. [ Indiscernible ] has a conflict with [ Indiscernible ]. And will not participate in the discussion or vote. Thank you.
Thank you, Barbara. Dr. Bucher, would you like to say a few words?
Thanks very much. On behalf of Sam Wilson I would like to welcome you all. I want to thank Dr. Kerkvliet for chairing this session. I would like to thank ahead of time all of the members of the panel who has put in many hours reviewing the reports. This is a central core activity of the program, where we created our legacy and continue to. This is the most important activity that we carry out. We thank you for your participation in helping us in this challenging activity. I would like to mention that for many years I sat on the other side of Nancy here, where Michelle is now sitting. I would like to introduce Michelle. She's with Dave Malarkey who coordinating the reports for the last three years in that area. Michelle will be taking over my role in dealing with some of the text cooling aspects and working with Dave to put the reports together, I am anxious to have a lot of the credit and of course the responsibility of errors shifted from me to Michelle and Dave. I think they're going to do a terrific job. Thanks for coming. I think this will be a very interesting meeting.
Great, thank you. So just the plan for the morning, um, we hope to get through the first two reports, Dibromoacetonitrile and Bromochloracetic acid before the morning break. Then committee members will have a photograph taken. This will be then followed by the aloe report, we hope to finish it by lunch. If necessary to fit lunch in before the 12:30 we may have to break that report into two sections. The format for the reviews this morning, the first is study scientist and the pathology will make their presentations. If there are questions, um, asked during the discussion -- there will be questions asked during the pathology report. The reviews will present their reviews. Comments will be taken from other subcommittee members and any written comments will be identified. Today I don't believe we have any written comments. Okay. So that's basically the way we'll go. And we'll vote on the final conclusions. Okay. So, I guess we're ready to begin the first report. And it's on Dibromoacetonitrile. Dr. Ron Melnick.
Good morning. A -- for the next two reports I will be discussing the toxicity and [ Indiscernible ] studies we have conducted on water disinfection biproducts. The first is Dibromoacetonitrile. We have had studies in fish or rats or [ Indiscernible ] mice by drinking water route of exposure. On the top you see the structure of Dibromoacetonitrile. It's a disinfection biproduct. There's no real commercial progression of this. Dy infection biproducts are formed by the reaction of chlorine with organic matter present in water sources. A number of factors influence the type of biproducts that you obtain. This is depending on the organic precursors, the nature of the disinfectant itself, PH. Concentrations of Dibromoacetonitrile have been measured in a national survey. They're up to high as 11 micrograms per liter. The U.S. EPA has a rule, but it does not include Dibromoacetonitrile. But the WHO has a guideline of 70 micrograms per liter for Dibromoacetonitrile. I'll be talking about two weeks, three month and [ Indiscernible ] studies. These compounds were nominated to the NTP from the EPA because of the widespread human exposure. [ Indiscernible ] is a representative of the family of [ Indiscernible ] and the compound selected is because of the possibility that [ Indiscernible ] might be more active than chlorinated. Drink water is the route because that is the primary route of human exposure. In our two week studies in fish or rat Dibromoacetonitrile was administered ranging from 12 to 200 ambiguouses per -- migs per liter. This was selected because of preliminary tox studies. 200 was selected as the highest dose. There were exposure related decreases if in water con sujs. There was some body weight gain for the males. There were no chemical related lesions. There was in 205 males atrophy of the [ Indiscernible ] in the high dose group. The same exposure concentrations were used in the three month study. No effects on effect or clinical signs. There was decreased water consumption in the 200mig rat. There were no computer related lesions identified in this study. There were decreases in [ Indiscernible ] sperm mow tilt. The exposure concentrations for the two year study were 200 as the highest, 100 and 50 for male and female rats. Survival was not effected. Terminal body weight was in the high dose groups about 5% less than controls. And from our concentration of Dibromoacetonitrile you can calculate average daily doses, they range from 2 to 8-milligrams per kilogram. The biological effects we observed in this study were predominantly in the upper elementary system. In this study we observed neo plasms in the hard palate and the tongue. In the males there were increases in ska mouse cell carsinomas. The top line are the number of animals examined microscopically. A star in the control group indicates a significant trend. A star in a [ Indiscernible ] group indicates a significant [ Indiscernible ]. We observed an elevation of ska mouse cell papillomas in female rats. Oral cavity neoplasms are uncommon in fish or rats. You can see we've only observed in six studies one male with a ska mouse cell neoplasm. The number is still less than 1% for boat males and females. This response we feel is clearly related to the exposure of Dibromoacetonitrile. Both of these exceed the historical control. The response in males was a clear evidence of carcinogenicity.
Another lesion that we observed was a glandular stomached a know ma in two male ratsive this is rare. In the 1200 rats in the database this lesion has not been observed. So we consider these two to represent an exposure related effect. There were also ska mouse cell papilloma and [ Indiscernible ] basil cell [ Indiscernible ] on the skin of female rats. The trend was positive. The incidence wasn't, this is more of an uncertain finding. Other nonneoplastic regions was hypercare toe sis of the esophagus. And dis, which is a dilation of the glanldzs of the stomach observed in female rats. Our two week studies in mice were at the same drinking water cons rations as for rats. There were no effects on survival or clinical signs. Decreased water consumption again here. No significant effects on body weight. And no chemical related lesions. Same concentrations were used in the three month study in mice. Again, no effects on survival or clinical signs. Decreased water con sujs. We've seen this repeatedly in the 200-milligram mice. No chemical related growths or lesions identified. Again, a small decrease in ep kid mal sperm mow tilt. The survival of mice in the two year study showed a significant increase in female mice in the two higher dose groups. There were decreases in body weight, ranging from about 5 to 12%. In male mice in the highest dose groups. And the effect here is not shown at the end of the study, but there was a decrease in body weight also of 10 to 12% of male mice. These are the exposure levels. And what I was trying to show is if you look at the body weight in males it seems decreased throughout most of the study. But in the females the squares, near the end of the study return to almost control levels. The main observation within this study was the observation of squamous cell papillomas or carns know mas in male mice and female mice. As you can see there were significant trends, significant elevation of the combined squamous cell papillomas or corns know mas and increased hyperplay sha. For this lesion and for the oral cavity lesions we don't seem to see in other studies any type of specificity. That is when a chemical intellectual deuces a lesion in male rats, we see it in female rats as well. With squamous cell papillomas of the stomach, of the number of studies that we have conducted that have shown them, in males and females the correlation is somewhere around 85%. Also, with both of those types of lesions there's good evidence of progression of papilloma to carsinoma. There were carsinomas associated with papillomas for the four stomach, we also used the progression as judging evidence. We can see carsinomas in males, but not in females, about 85% of the time that we see them, they're associated with carsinomas as well. In this case we did not see papillomas in female mice. Another lesion in mice was [ Indiscernible ] or carsinoma, which was elevated in the low dose group [ Indiscernible ] were elevated in the low dose group. There were elevationed relative to control significant in the mid and low dose groups. However, in female Miles the trend for course know mas decreased, this may be related to a decrease in body weight that was observed during the study. In any case, this was somewhat of an uncertain finding and may have been related to Dibromoacetonitrile, we can't really tell. In conclusion, on our studies for rats. In male rats we consider this to be clear evidence. And the two glandular [ Indiscernible ] we considered to be exposure related. In female rats there was some evidence of car know Jenicty. As well as the skin lesions which may have been related. This is our term for ee equivalentcal evidence. In female rats we observed the [ Indiscernible ]. In mice it was clear evidence of cars know Jenty based on the increased carsinomas in the liver [ Indiscernible ] we consider may have been related to exposure. In female mice we considered the evidence to be clear for four stomach squamous cell papillomas in relationship to the lesions observed in male mice. Non[ Indiscernible ] of male mice was the elevation of squamous epithelial [ Indiscernible ] in the forestomach. Food for thought, Dibromoacetonitrile has been examined in [ Indiscernible ] protocols it was found to be an initiator of [ Indiscernible ] in [ Indiscernible ] mice with the disappropriater when administered on the skin. It did not proud lesions when administered orally. Indicating there's something more of that direct effect of the parent compound. As I mentioned, we did not see chemical related lesions in the three month study rats or mice. The effects were in the upper elementary system. Again, indicating probably related to the parent compound or a reactive me tab light formed early. Dibromoacetonitrile is not excreted in the urine. We look for this in the liver of our studies, we did not see a decrease in GST activity. The studies that reported were by [ Indiscernible ]. Ours were drinking water studies. In some disposition studies that were conducted at NTP using regular label Dibromoacetonitrile we found [ Indiscernible ] binding of [ Indiscernible ] to [ Indiscernible ] molecules in the stomach and liver after [ Indiscernible ] administration. The other effects that might also be related is that invitro studies have shown [ Indiscernible ] a DNA repair study and [ Indiscernible ]. So the mechanism we don't really know. This starts to give us some thoughts of terms of what might be involved in that carcinogenic response. Thank you.
Thank you, Dr. Melnick. Any questions for Dr. Melnick, just for clarification purposes before we start our reviews? Questions? If not -- Dr. Novak.
Thank you, Nancy. I have no major comments. I'm pretty much in agreement with everything that was presented. The only comments I would add in some cases it's perplexing there's not more of a dose response. There was an effect of 50, nothing at 100 and nothing at 200. I think those add to the complexity of addressing all of this. I think it's important because of the drinking water aspect of this. And if my notes are correct, from having gone through this, in terms of the terms of the Dibromoacetonitrile concentrations in drinking water a high was something like 11 micrograms per liter. When looking at concentrations where a predominant number of effects were seen, at 100 and 200-milligrams per liter, or an average dose of something like 7 or 8-milligrams per kilogram, I'm wondering if it's possible to relate that to human exposure. What we're talking about here is a cumulative time dependent effect. When the data are finally released and people begin to look at this we suddenly don't have someone running out and demanding that chlorination be terminated. I think that looking at the concentrations and the overall exposure of humans would be important. The second point, perhaps this is more relevant in susceptible populations. Where some conditions might lower the levels of [ Indiscernible ] and increase the prospect for an effect. I think the experiments have been well designed and thoroughly carried out. I have no disagreement with the conclusions.
Thank you. Dr. Cattley.
Okay. Really, I had a suggestion about the conclusions. It has to do with the reporting of the level of evidence in the mice. And, um, because I believe that in the mice there was no evidence of carsinoma formation. So, I think when we review the statement on the conclusion I would propose those two be considered as separate lines rather than being combined for male and female mice. I do think in the case of the female mice in the forestomach papillomas it would be useful to characterize the progression of the [ Indiscernible ]. That is the basis of considering that clear evidence. The other suggestion, addressing the historical control data for [ Indiscernible ] and [ Indiscernible ] in rats that we see the overall numerator and denominator depicted in the text.
Okay, thank you. Dr. Soper.
I thought the study was well done. No comments to add. Thanks.
Thank you. Um, any comments from anyone else? Pardon me.
I have some minor comments, Mike [ Indiscernible ].
Let's have Ron respond to the comments, first of all. Sorry, Mike.
Yes. Certainly we use higher exposure concentrations than those that are found in drinking water for our studies. Largely this is due to the limited power of a [ Indiscernible ] with 50 animals per group. The issue really for drinking water and car know Jenic risk relates to the fact that 200 million people are drinking chlorinated drinking water. And what we are doing is identifying a has -- has yard -- hazard. Dibromoacetonitrile is just one of several hundred disinfection biproducts. This contributes to the overall picture of what is in drinking water, it varies depending. I think EPA is working on these types of issues. Susceptible populations, I think we agreed with that. This needs to be addressed. I think there's emphasis on that in the risk assessment community. For Dr. Cattley, we'll devote a lot for emphasis in our discussion on the evident of production of the four stomach neoplasms. That all fits into the story of why we consider this to be clear evidence. This study shows evidence of production in males. We don't see seem to see a sex difference in response. When we see four neoplasms induced it's accompanied by squamous cell carsinomas. This will be added into the discussion to round it out a bit. And in terms of the data tables on historical control incidence, when we use that there should be a table in the back that provides the information. I noticed the one that you pointed out wasn't in the appendix. I will make sure that is brought forward to the text, as well.
Okay.
Just to clarify in terms of what I was envisioning. In the present study the females had a much more robust response in terms of incidence of papilloma than the males. But the females had no course know mas -- carsinomas. This would say this is not as supportive as other studies that have been conducted. I'm taking that the association. These two lesions in animals from the same treatment groups is considered evidence of production. But to me it doesn't really in a gait -- negate. I'm not familiar enough with all of the studies to say there is or not. The stronger evidence would be coming from not this studdie, but from other studies. In which a better correlation was observed.
I think there's a few reference materials on the continuum of progressive lesions from the four stomach [ Indiscernible ]. And we can also refer to those other references as well as. In this case we also saw somewhat higher, or a trend towards epithelial hyperplay sha in the male mice. Yes, we didn't see carsinoma, but it's all part of that. We're dealing with small numbers of carsinomas as well. I think if there's evidence of progression the evidence specified in the criteria is to assume that progression can occur. I think that's a pretty good assumption in this case. I'll try to address this more in the discussion.
Okay.
Dr. Flake.
I think there's good evidence in the pathology literature that squamous papillomas can progress to carsinomas in the oral cavity and in the esophagus and the forestomach. This is stated in the [ Indiscernible ] and the WHO and the pattology of the mouse book. There's also a book, atlas of tumor pathology of the fish or rat. In there they've got photos showing insight to [ Indiscernible ] changes within the papillomas and in the [ Indiscernible ] book there's a photo showing carsinoma in squamous cell papillomas and lesions that at the surface look like ska mouse papillomas but have [ Indiscernible ] at the base. I think there's -- not that all ska mouse carsinomas arise through that route, but I think there's pretty strong evidence for the latter.
Okay. Dr. [ Indiscernible ] did you still have a comment?
Just a few. For the two week rats. The atrophy that was observed, at the same doses in the three month and two year study there were no changes. I don't think the changes shown were related. There were kidney changes in the two year rat study, increased [ Indiscernible ] and pelvic inflammation that was not mentioned or discussed. Perhaps those should be brought forward. In the summary for the rats, when talking about the skin tumors observed there, it doesn't clearly state that's in the skin. I think that needs to be in there, I think on page 10. It doesn't say that's in the skin. Because you just talked about similar changes in the stomach, I think that should be stated. Then theed a know mas in the mouse. They had to go back and recut them. They didn't show an increased incidence after the cuts. I think you need to show that was not compound related, that's all.
Any other comments? If not, I think we will display the -- okay. That request included public comments. Nothing. Okay. I think we'll display the conclusions on a PowerPoint slide. Then I would entertain a motion to, um, revise or accept these conclusions. Yes, Dr. Cattley.
I'm Russ Cattley. I would move to recommend a revision to the level of the evidence for the male and female mice so the statement separates the two. Because in the statement as it stands it's based on increased incidents of [ Indiscernible ]. There were no carsinomas of the forestomach of the female mice. That could be a point of confusion to the point who might only read this conclusion. If that could be split out, it would read something to the effect there was clear evidence of carcinogenic activity in Dibromoacetonitrile in male mice. There was clear evidence of carcinogenic activity in female mice based on ska mouse cell papillomas and the potential for these lesions to address. Both of those are key to that call of clear evidence.
Any discussion from the subcommittee? Paul?
One point about that. In the case of the female mice there was significant increase in [ Indiscernible ], yet in the males it seem toes be fairly equal. Is this committee comfortable calling it clear evidence?
That was interest Howard. For the record. Any discussion? Yes?
If I can read from the explanations of levels of evidence. Information that is used is progression from [ Indiscernible ] to malignant [ Indiscernible ] as well from [ Indiscernible ] do [ Indiscernible ]. That type of information is used. And where progression is known to be a possibility the most prudent course is to assume [ Indiscernible ] have the potential to become malignant. This is page 14.
That was Dr. Melnick. Russ.
Russ Cattley. Since I brought it up, I'm not really comfortable with the evidence for progression in the present study, even looking at the males. I do think that Dr. Melnick and interest Flake have indicated they will provide additional evidence for the potential for these to progress in mice.
Okay. Um, so, we could take a motion to change the conclusions as stated by Dr. Cattley. Or further discussion? Or another motion.
This is John Mirsalis. I make that motion.
And then we vote on that? We'll change the wording and then you can -- we'll vote on that. Dr. Novak seconded.
Can I ask a question?
Yes.
Compare the changes to the mouse and the rat. Were the papillomas similar? I guess it would be the males that wasn't seen in the females? Or any other kind of evidence to suggest they would act differently?
I don't think there were any differents that were noted in the papillomas of the males and females.
Thank you.
Let's see.
We've lost that sentence --
Do undo.
[ Speaker Faint/Audio Unclear ].
Just undo.
[ Speaker Faint/Audio Unclear ]
Now control C should just copy.
But you cut it before you copied it, Charles.
Can I just see?
[ Speaker Faint/Audio Unclear ]
Where is the text you want to copy.
I might make a suggestion. If the subcommittee has comments like this on other chemicals, write down a suggested change in the conclusions for, um, Charles. Did you have something, Russ? It might just facilitate things.
We'll just repaste that. Then we'll -- okay, have that for the male. [ Speaker Faint/Audio Unclear ]. Is it some or clear evidence for the female?
Clear.
Okay.
We would entertain any [ Indiscernible ] jokes at this point. [ Laughter ]
I have a question, in regard to my comment about the kidney findings in rats, is there a reason why it wasn't brought forward to the summary? And if it should or should not be included in the nonneoplastic effects.
Okay, has everybody read the modified conclusion?
We can add that to the summary. Again, it's a high background rate, the numbers that increased were not as -- you're dealing with a high back ground rate in the -- [ Speaker Faint/Audio Unclear ] but it's worth while bringing forward to the summary.
Okay. I would like to call for a vote on this conclusion. All those in favor of accepting, or more discussion -- sorry. Go ahead and call for the vote? Any discussion? All those in favor of the conclusion as stated? Raise your hand. All those opposed? One. Okay, thank you. Pardon me? Oh, Dr. [ Indiscernible ] would you state why were opposed?
I was okay with the original wording.
Okay, motion carries. So we're ready to move on to the next chemical. Which is Bromochloracetic acid. And Dr. Melnick will again give our report.
Okay, thanks. Good morning, again. This is another disinfection biproduct from water chlorination, the compound is Bromochloracetic acid. I'll be discussing the drinking water studies of this compound in fish or F-344/N rats and B6C3F1 mice. It will exist as the Bromochloracetic acid acetate this is a drinking water disinfection biproduct. Similar to Dibromoacetonitrile. It's formed by the reaction of chlorine with naturally occurring organic matter. Concentrations were up to 19 micrograms per liter. The tox studies, measures showed that levels of not greater than 6.5 micrograms per liter. EPA has a rule and halo [ Indiscernible ] acids are included. The drinking water concentration for the sum of [ Indiscernible ], [ Indiscernible ], [ Indiscernible ], and [ Indiscernible ] should not exceed 60 micrograms per liter. As you can see Bromochloracetic acid is not included in this group. But I suspect the next time they review it will be. EPA nominated to the national toxicology program this. This is because of the widespread human exposure. And there had already been published studies demonstrating it's course know Jenic to the liver of rats and mice. I'll be talking about the two week, three month and two year studies. The water concentration selected for the two year study ranged from 62.5 to 1000-milligrams per liter. This concentration the high is similar to exposure concentrations that have been used in the chronic and toxicity studies in [ Indiscernible ] and [ Indiscernible ]. In the two week study there were increases in the relative liver weights. About 10%. There were increases in absolute and relative kidney weights in male. No gross exposure lesions identified. The same exposure concentrations were used in the 3 month study. There were significant increases in liver weights and kidney weights. The liver weights in rats were increased at the 1000-kilogram per liter group. There was increases incidences of [ Indiscernible ] in the liver males and males exposed to the 1000-milligrams per liter. I believe all animals at that dose showed the [ Indiscernible ]. So the concentration selected for the two year study included the 1000-milligrams per liter and half concentrations and half again as the mid and low dose exposures. No effects on survival. There were decreases in body weight at the end of the exposure period at about 10 to 12% in males and about 10% in females. The average daily dose can be calculated from the concentration in the water and the change in the body weight. You can see they range from 10 to 50-milligrams per kilogram, a finding of particular interest is the observation of increased neoplasms of the large inTess tin. There's been several studies on disinfection biproducts. The most frequent observed affect is with bladder cancer. There have also been reports of associations with rectal cancer. In this study we found increased incidences, a trend for males for theed a know mas in the colon or rectum. And in females a significant trend and significant incidence. The back ground rate in fish or rats is very low. It's a rare finding. Of the 1200 male rats in the database, two showed this particular lesion in males and 2 in 1100 female rats. So the findings of these two lesions exceeded the historical control. A -- similar to what I mentioned with other, with the Dibromoacetonitrile, there does not seem to be a sex specificity for colon cancers when observed in rats. When we see them this males we tend to see in females. The correlation is about 90%. And in studies which have shown colorectal neoplasms most frequently there's the observation of carsinoma indicating the potential for progression of these lesions. So these findings when looking at them together show these are clearly related to the exposure to Bromochloracetic acid in rats, inducing large inTess tinned a know mas. This is a section of a large inTess tin. This is the normal structure. This is theed a know ma growing into the lieu minute. You can see here the glands that are arranged in a lineal parallel pattern in the normal. It's totally disorganized in the adenoma. We don't see the gob let cells. These are inmature cells. And the darker bay sow feela. This is one of the adenomas from this BCA exposed animal.
Two other lesions worth noting was there was an increase in mesoTheo ma. Each of the dose groups showed a rate higher than the historical rate. And the incidence in the 500-milligram group was significant. As I mentioned, mesotheleo ma is a sight associated with Dibromoacetonitrile. Interesting finding in the ma'amry gland. There was no effect on the incidence with exposure. However, in the treatment groups there's a significant increase from the incidence of multiple fiber adenomas in the mid and high dose groups. Looking at the data on those, if we looked at the number of fiber adenomas. Molt policety is part of the evidence used in making an evaluation. This response to us is demonstrates an exposure-related response.
A, in this study we also saw a [ Indiscernible ] adenomas low numbers in male rats as well as in female rats. However, in rats the [ Indiscernible ] adenomas are uncommon. Occurring at a rate of about 1%. In association with the adenomas there were increased in [ Indiscernible ] in male rats and [ Indiscernible ] and [ Indiscernible ] in female rats. We consider this to be of a less certain finding. I point this out as an effect that may have been related to exposure to Bromochloracetic acid. [ Indiscernible ] were increased in the middose group of male rats, it exceeded the historical control rate of 8%, this would be 18%. However, it was not increased in the high dose group. And no effect in female rats. This to us, again, is another uncertain finding. In mice our two week study was at concentrations from 62.5 to a thousand migs per liter. There were no exposure related clinical signs. No chemical differences in organ rates. The same exposure concentrations were used in a 3 month study. There was it decreased body weight gain of females at 250-milligram group and higher. There were significant increases in liver weight of males and females. The increases were as much as 20 to 30% higher in the high dose group compared to controls. There were no [ Indiscernible ] effects in this study, however there were observed increased incidences of [ Indiscernible ] in the spleen and these were not exposure related. But they were observed in several of the dose groups of males and females. The two year study was conducted at exposure concentrations of 1000, 500 and 250migs per liter. There was a decrease in survival in the high dose male mice. This was attributed to [ Indiscernible ]. There were decreases in body weight in ranges of 5 to 12% in males and about 10% in females. Here are the average daily doses throughout the two years. The main finding within this study was effects on the liver. If you recall, in the previous slide the liver was an identified target organ. There were increases in [ Indiscernible ] adenomas or carsinomas combined. There were increases in molt policety of [ Indiscernible ]. And the combination of [ Indiscernible ] and adenomas, carsinomas or [ Indiscernible ] show high significance in this particular study. Included also as reported before, increased sight playsic [ Indiscernible ]. Similar to the males, the females showed the increased incidences of [ Indiscernible ] or carsinomas. I think there were too many to put on this particular slide. As well, as increased sighto playsic [ Indiscernible ] and [ Indiscernible ]. Our conclusions from this part of the study, clear evidence of cars know Jenty in male rats. Based on the increase incidence of the large intestine [ Indiscernible ]. The increase in [ Indiscernible ] adenomas, which was small, we considered it may have been related. And the pancreatic [ Indiscernible ] may have been related. Would we consider to be ee equivalentcal. In female rats the evidence was clear for large intestine adenomas. And as I mention, there is evidence from previous NTP studies of the progression of adenomas to carsinomas, we're all aware of that, as well in humans. Ma'amry gland, we considered to be exposure related. The liver adenomas may have been related to [ Indiscernible ]. What I didn't show, we also observed increases incidents of [ Indiscernible ] in the lung of female rats. In mice, the level of evidence of cars know Jenist was seen to be clear. In female mice the increase was clear based on the inyeased incidences of [ Indiscernible ] and carsinomas. The nonneoplastic effects included the liver, [ Indiscernible ] in male mice as well as in female mice and the [ Indiscernible ] in female mice.
Now the [ Indiscernible ] are an interesting group of compounds. They look simple when you just take a look at their structure. But their metabolism occurred by an enzyme [ Indiscernible ] which causes the initial displace of a [ Indiscernible ]. The first stable product of this pathway is [ Indiscernible ], which can undergo further transformation seen here. In studies where either, any of the three have been administered to rats or mice, what you find is after several days of exposure the clearance of the parent compound is reduced, as much as three to five fold decrease in the clearance of the parent compound. What is shown to be the case is that in this metabolism forming the [ Indiscernible ] that this particular compound can bind covai lantly with [ Indiscernible ] causing this suicidal inhibition of the enzyme. It becomes a consequence of the destruction of the enzyme and its reisn't sis. -- reisn't sis. This is an observation which is also made with people. Because [ Indiscernible ] has been used therapyically for treatment of lactic acid removal. But in any case, the intermediate, when it's formed can bind covai lently to disor release to form [ Indiscernible ]. It depends on how much stays bound, which influences the removal of the [ Indiscernible ]. So, trying to understand what might be happening in the cars know Jenist of [ Indiscernible ], we can note there are several common sites. The mesoTheo mas in rats and the mouse liver tumors with all three compounds. NTP had sponsored some research which demonstrated DNA [ Indiscernible ] in the livers of mice exposed to [ Indiscernible ] and [ Indiscernible ]. This may also have some role in the cars know Jenist of this compound. The compound [ Indiscernible ] is also mute Jenic. Another pathway of interest is that the [ Indiscernible ] has also been shown to be the same enzyme, with a different name, [ Indiscernible ], which is the [ Indiscernible ] enzyme in [ Indiscernible ] metabolism degradation. By suicidal inhibition of this enzyme it's possible that the intermediates of [ Indiscernible ] are also accumulating one of these [ Indiscernible ]. As I mentioned, based on human studies the clearance of [ Indiscernible ] is observed in adults and in children. So, we are in the process of working on developing a physiological model of [ Indiscernible ]'s position. During the course of these studied we collect add fair amount of time course data on [ Indiscernible ]s. Our strategy is to develop a model to simulate these data. The purpose of that is to see if we can estimate values for a variety of dose metrics that might be, a, as occurred during chronic exposure and use them for dose response analysis. Some of the dose metrics we're considering are tissue concentrations of the parent compound, tissue compounds of [ Indiscernible ], liver levels of [ Indiscernible ] activity. There's others that also could be considered. The purpose of that is to see if we can identify dose metrics that might be associated with Bromochloracetic acid or [ Indiscernible ]. We feel this might be pertinent to human studies. So with that, I thank you for your time.
Thank you, Dr. Melnick. Any questions? Yes?
The level in water was 6.5, could you clarify, is that out of the tap? Or is that what the control animals --
Both.
The reverse osmosis water didn't take that out? I'm spiesed to see -- surprised to see that. It's just, it's a con founding variable.
Yeah. We use tap water. It wasn't -- reverse osmosis water for the controls.
Correct. The typical control water is tap water at the laboratories.
Dr. Crump.
Kenny Crump. I had a question about the female rats. There was a consistent dose response for, um, for skin. I think my bro mas went from 112 and 6. I'm just wondering why that wasn't included, or commented on in the report.
I would have to look at the data.
We should ask those questions before we have heard the reviews.
Okay.
Let's move forward. Dr. Soper is the primary reviewer.
I thought the study was nicely done. I agreed with all of the conclusions. I spent some time considering whether it might be more appropriate to classify the [ Indiscernible ] in the male and female rats as being ee equivalentcal evidence. My reason for focusing on that is the [ Indiscernible ] adenoma is a target organ based on observing [ Indiscernible ] and the related compound DCA. For the female rats we had three animals with [ Indiscernible ] adenomas all in the top dose group, that was statically significant. It would be helpful to have a table for the female rats, add that to the report. In the male rats, we did not get statical significance. That was lanchly because we had two animals at a 50. The highest incidence that we have at any prior historical control is about 1 out of 50. There were two dose groups outside of the historical information. I was curious what some of the experts would think about those findings. I thought the study was nicely done.
Okay, thank you. Dr. Melnick, do you want to respond, or wait? We'll go to the reviews and come back.
Dr. Bunton.
Yes, I would agree. I felt the study was nicely done. I did have mainly editorial comments. I won't go through all here. I did want to bring up one. That was in the conclusion, I wasn't sure what you were saying about the incidence of the [ Indiscernible ] adenomas as being some evidence. You had, it was in, I think on page 92, you mentioned how they are the, there was an increase in foecy and the fact that the tumors are rare made you believe there is some evidence. I didn't know if you were associating the findings, or if it was all of the findings together is what led you to that conclusion. So I felt just that we needed more clarification on that.
Okay, thank you. Dr. Novak.
I think the study was well constructed and designed and carried out. I'm in agreement with the conclusions. Again, my concern is just the upper concentrations that were used. That's been noted already. Um, the only other question I would have is were there any other clinical chemistry [ Indiscernible ] to measure the [ Indiscernible ] in the animals in the study. Was there an effect on the lipid profile? An indication that an metabolic disorder might be going on? I didn't remember seeing anything, it's just a question.
Okay, thank you. Dr. Melnick, would you like to respond?
It seems there were two questions related to the [ Indiscernible ] neoplasms in rats. What we were calling the evidence. If it's not clear that we were calling this ee equivalent quail. We will try to work on our language on that. But this, when we use the word may have been related implies an ee equivalentcal response. I'm not sure if from I heard from Dr. Soper you were thinking this was higher level than it -- than may have been related or ee equivalentcal, or whether ee equivalentcal was appropriate.
Some evidence as a response.
A -- largely I think it's based on that finding of for male rats, the incidences were not significant in any of the dose groups. The trend wasn't significant. It's largely based on that comparison to the historic yale database. The only positive affect is the trend in female rats. Although the foecy are potentially precursor lesions, there's not neoplasms. Based on that we feel that the evidence is appropriately uncertain at this point. Perhaps longer time might have shown something different. Based on the two year expose the conclusions seem to fit strongest with ee equivalentcal. I think it -- a -- I don't know -- it's about 1% also in all rats of exposure. By all rats of exposure for male rats we've seen 0 to 4% in male rats. And in female rats there was one study that had several more than 2 animals with a [ Indiscernible ] adenoma, but not by the drinking water route. I don't know which route it was. The evidence to us didn't seem strong enough to call this some evidence at this point.
We didn't see [ Indiscernible ] with [ Indiscernible ]. But they were reported in a study in the literature with [ Indiscernible ]. So it's one which is sort of borderline in our view. Uncertainty is where we're at. [ Laughter ]
Okay, thank you. Any other comments from the committee?
We did not do lipid profiling in the two year study. Our clinical chemistries are limited to our 90 day study. I don't recall any data that indicated there was anything.
I saw the effects that you have on the liver. I was wonder if there were associated metabolic effects to parallel that. That was the reason for the question.
Okay. Yes? Dr. Howard.
Please bear with me. I'm a little confused, not confused. I want you to help me sort thl out. In the male rats, you call this clear. If you look at the [ Indiscernible ] in statistics it shows that only the middle dose, with no trend test on that. If you look at the large intestine adenomas for the males there's no [ Indiscernible ] comparison significance, yet it does have a trend test. I greatly appreciate your evidence at the end of the talk about the [ Indiscernible ] considerations of how this may occur. Looking at boat of those are not real strong. You feel together they go into clear evidence, correct? I'm having trouble with that. There's not both trend and paralyzed comparison evidence with those two.
Yeah. I think the, when dealing with uncommon or rare tumors the [ Indiscernible ] database is important. For mesoTheo mas I believe there's are in the range of 3% in all of the dose groups that were included there was elevations with significance at the middose group. When dealing with ten or 6 -- I don't think these numbers are that different than each other to look for a real close dose response. What we see is elevation with the mesoTheo mas. On the large intestine neoplasms these are rare. These, like I said, occur in 2 out of 1200. The findings within the dose groups here are finding 2 out of 50, even is unusual relative to controls. Seeing 4 out of 50 in another dose group, this is just something which is well beyond expectation for anything anything other than a chemical-related response.
I understand that. I truly appreciate that. I will probably get a beer bought for me by saying that we're driven by statistics. I'm just a little, a little trigger going off, are we, it's just a concern. Are we overemphasizing that? It's not showing statistical significant.
But nor rare tumors we rely on our historical database. In the previous study we saw only 2 in the doats group. -- dose group. We felt that was related to the exposure to [ Indiscernible ].
I'm more comfortable with the females.
Also, when I mention we don't see sex specificity more large intestine neoplasms. When a chemical induces in one sex it tends to also in the second. We see a species difference. But we don't see a sex difference.
what is happening in the other, the other sex. If we saw it in two species that also helps. If we see it in both sexes that's strong.
You may want to check and see, I didn't catch that. One other thing, in your talk where you talked about [ Indiscernible ] you gave the incidence per rat and showed statistical significance. I didn't see that in table 9. Or anywhere else, unless I missed it.
It's in the discussion. It's not something we typically include in our data tables. It's something that I looked into after having the data.
I would like to just ask that you consider putting it in a table.
Sure.
Thank you.
Dr. Crump.
A couple of comments. Just a question for clarification. Is it standard operating procedure to combine [ Indiscernible ] over all sites? In your analysis. Just curious about that. I will bring up again the question about the my bro mas in the skin of female rats, it was -- significant trend, 1, 1, 2, 6. It was never discussed in the report. The reason for that? Or is it a oversight?
We do combine for mesotheleo mas of the pair that knee yum. These are all sites. The incidence is for that particular location that we do combine. On the skin lesion, there was a significant trend. But not a significant elevation in any of the dose group. The trend is driven by the high dose group. I would have to look at the historical group to see how that particular response compares.
The combination of [ Indiscernible ] and [ Indiscernible ] are somewhat common. Probably about 3% back ground rate. The roles here -- controls here are 2%. And our range can go up to mostly around 8 to 10%. Sometimes go up to 12% for combination. It's within what we've seen in historical controls. To have 12% in a group spontaneously.
I think that's worth a comment. A look in the report. There are cases where we would certainly in other situations call that ee equivalentcal evidence. I think it's worth some sort of comment in the report, at least.
We'll do that.
Okay. Any -- yes?
Russ Cattley. I think for the female rats the call of clear evidence is largely based on [ Indiscernible ]. I can appreciate the body of literature in other species, I wonder when the evidence for clear is called if we couldn't say for about the evidence in the report. I think that's why, if I'm understanding what you said, this is the reason this is considered clear evidence. The knowledge. We need to understand what data is being used to characterize that response. Part of that data comes from other studies.
Thank you.
Dr. Pinot.
Just a question. There was no progression. How typical is that for these intestinal tumors? I thought it interesting there was no evidence of hyperplay shas in the intestine. When you talk about the [ Indiscernible ] of the liver. The beginning of paragraph starts with a number of [ Indiscernible ] were considered [ Speaker Faint/Audio Unclear ]. It makes it sound like [ Indiscernible ] is a secondary change. I would clarify that paragraph. Write it so it doesn't sound like it's a secondary effect.
Okay. Thank you. Any comments from the audience? Public? Yes? Would you identify yourself?
[ Speaker Faint/Audio Unclear ].
Hold on.
[ Speaker Faint/Audio Unclear ].
Oop.
Okay. Ron Lawrenceson. I would like to ask Dr. Melnick if the ma'amry gland results were the only results that you had -- how would you have presented that? Where there was an increase in molt policety, but not incidence?
That would have been some evidence.
Let me suggest that we have a sampling problem here. The ma'amry glanldz in the rat is from stem to stern. You know, I think, probably what was reported there was by pal pages, I suppose. That brings implicitly size into the picture as well. Could you see them? Could you feel them? I don't believe typically an experiment does a thorough his toe path sectioning of the ma'amry glands. How do you get those numbers? Have you thoroughly looked at the ma'amry gland issue?
The ma'amry gland response was grossly detected. There were a number of tumors on each animal. We went back to the gross traceable lesions and counted. They were grossly des tebted. -- at the tekted.
I'm just bringing up a hype thetical. When you have a sampling problem, you should worry about how you got those numbers. This is one. Kenny I would just say to those skin FBI row adenomas is a similar thing. You have to be sure that you looked at the whole organ.
I had a comment. A question comes up about the process of our data review. I wanted to tell you how we do it. All of the data, when it comes out it is reviewed by the staff for potential treatment related findings. That's visually and with the flagged findings. We consider each one. Some of them we might decide are not bilollically relevant or important enough to bring forward because they don't fit within historical control ranges or they're not biologically relevant. We bring all of them into the results section. And determine which goes up to the abstract or not. So, um, with that said, the my bro mas we did consider that as a possible treatment related finding. Maybe we counted it and didn't leave it in the results decision. We could write in the review section our interpretation of that finding.
Okay. Any more comments? If not, um, can you display the conclusions?
Dr. Howard?
I'm curious again, why we're combining the male and female mice? There's a slight difference in the data that weighs the arguement.
They both had [ Indiscernible ] and carsinomas, [ Indiscernible ] were seen only in males and that's shown in parentheses.
Is this just conservation of words? [ Laughter ]
Okay. If someone would like to make a motion on the conclusions.
Keith Soper.
Jon Mirsalis, I second.
All in favor, please raise your hand. Looks to be unanimous. Motion carries. Um, Barbara would you like to start the next compound? Or time for a break? Let's take a break. Be back at 20 to 11:00.
There's a photograph, meet in the lobby please.
Session on morning break until 10:40 eastern.
Okay. I guess we are ready for our next report. Dr. Boudreau will be reporting on Aloe vera and total carcinogenesis.
Good morning. The page up does not work. So, enter?
The [ indiscernible ] plan, commonly referred to as Aloe vera has been used as a therapeutic dermatologic agent for more than 2,000 years. Because of its history and popularity, Aloe vera incorporated into a myriad of skin-care and cosmetics products. The Aloe vera plan has more than 75 potentially by active component. Evidence suggests that Aloe vera stimulates skin cell growth, enhances and eugenicists and light energize with ultraviolet radiation to induce skin cancer. The data toxicity of skin-care products and cosmetics is of a concern because of the large surface area of the body that is potentially exposed to sunlight. The National Cancer Institute nominated Aloe vera to the National toxicology Program for study based on its widespread oral and dermal exposure to humans and the general lack of toxicological information. So, the objective of these studies was to assess whether the topical application of Aloe vera planned components, alters the process of simulated cellular light and deuced total carcinogenesis in the [ indiscernible ] mouse. I might add that chronic dosed water studies with the Aloe vera plan components are on going. These will be presented at another date. The length of the Aloe vera plan is covered by a right. Just beneath the leaves, but within the parental tissue of the beet pulp R. Perry cyclic to Bills. These transport owl latex to the peripheral of the plan leaf. The aloe latex--Diaz are of he huge significance and to let the aloe species. The bulk of the leak is the leap pulp and is composed of large, then Wall cells that contain aloe del. Oneof the principal features of aloe gel is the presence of high molecular weight polysaccharides that are composed primarily of glucose [ indiscernible ] linked in a data one --Bond. Three test articles-Sorry, plan extracts, were used for these studies. Age of the plan extracts were obtained from fresh aloe [ indiscernible ] believes. Each extract, however, differed in either the portion of the leaf that it was extracted from or of the method of extraction. The it aloe gel test article is a [ indiscernible ] extract of the inner leaf Pulp. The whole leaf test article was the extract of the whole leaf with the lignified fibers removed. The whole leaf extract contained not only the aloe gel but the speed and 10 aloe latex as well put the decolorized whole leaf test article presented a cost-effective means of producing aloe gel by affording the process of hand filleting and the leafs. However, in doing so, it is treated with activated carbon which removes the aloe latex. It also removes some of the high molecular polysaccharides of the aloe gel. This extract actually differs from both the aloe gel and the leaf. Aloe [ indiscernible ]--of the aloe latex. This compound was selected as a positive control. The Crl:SKH-1 mouse model wasn't selected for study based on its long history of use and other carcinogenesis studies. Each of the test articles was used at two dose levels probably test articles were incorporated in water and incorporated into an oil and water in motion-Based Karim. The topical application of claims was to the dorsal scan of the mice. It extended from the nape of the neck to the base of the tail and midway along the sides of the animal. The source of the radiation in of this study came from filters, six and and a half [ indiscernible ]. Because the emission spectrum of these lands is very similar to that of natural sunlight, they are called simulated solar light. And the following slides, I will refer to that as a SSL. The topical application of the greens to the mice and the exposure of mice to the simulated solar light was conducted five days a week for a period of 40 weeks purpose was followed by a 12 week observation period. Mice that received no cream treatment but received increasing doses of simulated solar light and served as a positive control for the light. Animals that were treated with the control creams with the aloe extract cream or with the aloe in cream either received no light or the same middle toes of like that was also administered to the no cream animals. Survival and mortality in this study do not contain the usual definitions of the terms. Mice were removed from the study due to morbidity or to death. Most often, they were removed because a dorsal skin lesion had reached in the absence of a limit on size. In this study that was five mm. The exposure of mice to SSL resulted in a significant those dependent decrease in survival. This was expected. These and no Green animals serve as our positive controls for the experiment. At the same level of SSL, there was no effect on survival and animals that received the control cream compared with the no cream animals.
Similarly, at the same level of SSL, which again, is the middle dose of light, there was no effect of the Aloe vera text extracts' when compared with the controlled cream animals. The topical application of Aloe-emodin was associated with a significant decrease of survival and female my's only. Neither the exposure of to SSL or the topical applications of grain had any effect on the body weights of mice. Skin lesion data was collected and evaluated in the two phases during this study. During the study, during the in life phase of the study or the clinical phase when the animals were still alive, digital photographs of the mice, individual mice were collected weekly. Visual assessment of these photographs was used for the generation of the in-life skin lesion data. Assessments included skin lesion on set, incidents and numbers. Then, at the end of the study at necropsy, the mice were, again, digitally photographed, growth skin lesions were noted and collected four hits to pathological evaluation. These assessments included incidents in number on the non-neoplastic as well as new plastic skin lesions. We will begin, first, with the results from the in-life.
This side presents the skin lesion on said data. On set refers to the time point in the steady at which 50% of the animals and a particular treatment group have at least one unmeasurable skin lesion that was observed and is consistent with the information of SSL-Induced skin tumors. This is an example the Kurds that were generated from the skin lesion on said. This is for the animals that received no cream treatment and increasing doses of light to. As we go across the slide from the right to the left, doses of light are increased. When we find is that there is a [ indiscernible ] significant decrease in the median time to Ansett. This, again, was expected because the formation of skin lesions is a dose in time effected with SSL. At the same level of SSL, animals that were treated with the control cream did not differ from animals that were treated-Country debt, sorry, that received the note cream treatment ago at the same dose of SSL, animals that received the test articles did not differ from the control cream groups installation on sept. Incidents and multiplicity will be evaluated from this slide. Incident refers to the percentage of animals within a particular treatment group that have at least onedetectable skin lesion that was observed in-life and is similar to the legions induced by exposure to a simulated solar light skin tumors. So, as would be expected, in the animals that receive no cream treatment, as the dose of light was increased, there was a increase in the incidence of in-life-Determined skin lesions. I might add to this, however, that in this snow cream treatment group at both the middle and high dose of SSL, incidence approached 100%. At the same dose of SSL, the animals that were treated with the control cream group did not differ from those with the no cream treated animals, nor did any of the animals that received the test article claims differ from the control cream at the same level of SSL. Multiplicity in this study refers to the total number of skin lesions per animal within and at risk treatment group to go in other words, the total number of skin lesions per treatment divided by the total number of animals in the treatment group. As would be expected, as the dose of SSL increased in the animals that received no cream treatment, there was a concomitant increase in the multiplicity of in-life treatment. At the same level of SSL, the animals that were treated with the control cream did not differ from those that received no cream to in. Nor did the animals that receive the test article claims differ from control cream animals. We will now turn our attention to the history pathological at Data. The exposure of Crl:SKH-1 lies entombed simulated solar light is associated with the diffusive bidding of the epidermis as shown in-This was an normal scan. This is referred to as women sell hyperplasia. In this study it the study was documented. They are not showing up as well on this presentation as they did before. The exposure of Crl:SKH-1 myself to a simulated solar light is also associated with the nodular the thickening in the epidermis, which is usually detected by growth examination. This is Sean and C. This is characterized by this plastic changes and really basil disorganization, as is shown here. This condition was evaluated based on incidents as well as multiplicity in these studies. As would be expected, as the dose of light was increased, there was a concomitant increase in next the incidence of squamous cell hyperplasia. Mice that received no cream a treatment did not differ from animals that received the control cream either in incidence or multiplicity. The topical applications of the aloe test articles in female mice showed a trend toward a decrease in the incidence and multiplicity of squamous hyperplasia, in particular in the multiplicity of focal atypical hyperplasia. The exposure of Crl:SKH-1 mice to simulated solar light forms a dose in time dependent skin lesion proliferation. This proliferation forms a continuum of displastic and pull their Frank changes which forms three types of squamous cell neoplasms. What is shown here is the [ indiscernible ] papilloma which is a solitary focus with barbarized projections, --The photo micrograph of in A shows a [ indiscernible ] papilloma with its stock and mushroom like appearance, where as B shows a example of [ indiscernible ] which is more flattened and does not have a stalk. Papilloma's progress but exposure to light in to squamous cell. These are small, discreet models that rise above the skin surface. They have very sharply demarked borders which often compress the darkness. They are composed of sheets of epithelial cells that's lack orderly arrangement. Again, I am sorry about this broke it showed up clearer before. This is an example of squamous cell carcinoma and is composed of downward projecting sheets and the courts of [ indiscernible ] neoplastic cells. They tend to be a larger type of neoplasm. [ indiscernible ] appears also raided and in greater form. We will look at this as the histopathological data to go in mice that received no cream treatment and increasing doses of simulated cellular light, we saw an increase in the incidence of neoplasms by type, as well this is a combination, some combination of all neoplasms. We saw an increased dose trend. I might add that this reached 100% in animals at both the metal dose and High dose of light. At the same level of SSL, animals that were treated with the control cream did not have an effect on the incidence of neoplasms, either by type or combination. The incidence of squamous cell papilloma is worth significantly increased in female mice that were treated with the aloe gel and exposed to simulated sold their like again at the middle dose of light. There were no significant differences in the squamous cell carcinoma [ indiscernible ]. Nor were there is significant differences compared with controlled animals in the combined, some combination of all squamous neoplasms--Incidence approached 100% and all groups that received the middle dose of like. As observed with the aloe gel cream treatment, mice that received the whole leaf extract and exposure to simulated solar light showed an increase incidence and squamous cell papilloma as. That was observed in males and females alike. There were no differences and carcinoma [ indiscernible ] or carcinomas in some combination of all neoplasms. Similarly, as with the aloe gel and whole leaf, there was a significant increase in the incidence of squamous and cell papillomas in female mice that were treated with the decolorized whole leaves creams and exposed to a simulated solar like. There was no effect of decolorized whole leaf green treatment on the incidence of squamous cell carcinoma [ indiscernible ]. However, there was a decrease in the incidence of squamous cell carcinoma in female mines. This might be partially related to the fact that in this study, animals were removed when the skin lesions reached five mm and squamous teefourteen carcinomas tend to be a larger neoplasm, since the animals were removed early, they might not have had time to develop. In animals that were treated with the Aloe-emodin to creams and exposed to the SSL, there was an increased incidence of squamous cell papillon as in the female mice. There was no effect on Aloe-emodin claim treatment on the--Carcinoma or in India some combination of all neoplasms. Multiplicity, again, it refers to the number of neoplasms pronounced in the at risk treatment group to go in this and the following slides the dapper dagger shown in red preferred to significant dose trends while the times refers to significant [ indiscernible ] comparisons. In animals that received no cream treatment, there weren't significant dose trend increases in the multiplicity of squamous cell neoplasms, regardless of type or combination in male and female mice. In terms of their wise comparisons with mice that receive no cream treatment and no exposure to a simulated solar light, significant differences were observed for all neoplasm types and combination with the exception of carcinomas. Again, this might have been due to the early removal of the animals based on skin lesion sides. --Size. The it multiplicity of squamous cell papillomas and the combination of all neoplasms showed significant dose trend increases in female mice that were treated with the aloe gel cranes and exposed to simulated solar light and and pear-Wise comparison tests, but the three and 6% dose levels of the aloe gel were significant compared with controlled cream animals. Similar effects were not observed with a males. In female mice that were treated with the whole leaf cranes and exposed to simulated solar might as well in the male mice, we saw a significant dose trend increases and the multiplicity of papillomas and in male mice we saw a significant increase in the combination of all neoplasm types. In pair-wise test, their work--Compared to the control cream mice. At these 6% dose level as well as in pair-wise comparison tests in the combination of all neoplasms and all male mice. There was a significant dose trend increase in the multiplicity of squamous cell Pat,s and in the combination of all neoplasms in female mice that were treated with the decolorized whole cream and exposed to solar like an ink pair-wise comparisons, both three and 6% doses of the decolorized whole leaf were significant when compared with control animals. As with the Aloe vera plan extracts, the topical treatment of aloe claims showed a significant dose trend increase in the multiplicity of squamous cell papillomas and in the some combination of all neoplasms in female mice and add the 6% dose level and comparison test of control animals, the multiplicity of squamous teeforteen papillomas carcinoma as well as the some combination of all neoplasms was significant. So, in conclusion, the topical treatment of mice with the aloe gel cranes and the exposure to it simulated solar light had little effect on the incidence, multiplicity and onset of in-life determined skin lesions. Similarly, there was no effect of aloe gel cream treatment on the incidence of squamous cell neoplasms. Of course, this was close to 100% among all treatment groups. There was an increase in the multiplicity of squamous cell papillomas that was detected in female mice. There was no effect on the in-life incidence multiplicity of in-life detected skin lesions or on the incidence of squamous cell and neoplasms and mice that received the whole leaf cream treatments and exposed to simulated solar light. As with the aloe gel, there was an increase multiplicity of squamous cell neoplasms in female and male mice. There were no significant differences on at the in-life determined--Of skin lesions or on the incidence of squamous cell and neoplasms in mice that received the decolorized whole leaf green treatment and exposure to simulated solar like. As with the other test articles, there was an increase and the multiplicity of squamous neoplasms in male and female mice. The it treatment of mice with the Aloe-emodin screens, which was our purported positive control and exposure of mice to simulated solar light had no effect on the in-life determined skin lesion on cent incidence or multiplicity or on the incidence of squamous neoplasm perhaps brother was an increase in the multiplicity of squamous neoplasms in female. That concludes.
Thank you, Dr. Boudreau. I think .
This is a really complicated design and results and is kind of hard to get your hands around. I would like you to comment on the difference between the results for the multiplicity analyses for the specific data and the pathological data, the results are very different. Is a nothing with using the in-life data but do with the pathological data. If I understand the analysis, which I am not assured that I do, I would think that the in-life data would take into account the time in which these regions occur and the pathological analysis would not. We should give you greater power for the in-life analysis than for the pathological data. I was curious about that.
The in-life does give you on said, which, of course, we cannot determine histopathological. Animation on set is different. The incidence and both the in-life and the at histopathological approached 100% in both cases broke and they were not different. In terms of multiplicity, though, we were not able to resolve the differences between the histopathological to murder counts or neoplasm accounts and the skin lesions that were observed on digital photographs. We thought they should be similar, at least show similar trends. We did not find that. We are attempting to resolve these matters.
Dr. Howard?
I just wanted to point out-Let me get my glasses on, on page 121,--For those that wanted to see them and they were not on the screen. To supporting what Mary said is that we are counting bombs on the in-life. They are segregated at histopathological into [ indiscernible ]. We are counting funds that are consistent with the formation of a legion to go so, there are really two different things that are being counted. Oneis looking at a tumor type and one is looking at a bought.
In addition, one pump might be two different neoplasms or more.
Although the multiplicity of tumors increased, the multiplicity of non-neoplastic decrease. If you add the some of them, it would probably be pretty equal to the in-life legions. That is the way that I am interpreting it.
You presumably have the data that you can correlate the bumps with the Legion. You are saying that they do not correlate?
No. We are in the process of attempting to correlate the data. We have digital photographs from both. The trace a gross lesions are studied by the that biologist at the time of necropsy. The in-life bumps that we are counting-A committee examined all of these photographs and counted the lesions on these Ken. We, technically, should be able to trace trace Leeson's with the number of bumps. We have to examine the pathology, digital photographs in order to do that. We have those now. We are in the process of being able to analyze this correlation. That was not part of the steady. The correlation is not part of the study data.
Okay. Thank you. So, people move on to the reviewers. Dr. Crump?
First of all, I think I agree with the conclusions of this report. My comments are pretty much editorial. I do have a lot of editorial comments about this report. This is, obviously, much more complicated than the 2-Year design. Unique features include the dual exposures to light and to the cream. You have the in-life data and the pathological data. To have the multiplicity of verse is the incidence. There are a lot of issues that you do not typically have with the M2-Year-Standard and 2-Year standard to go to the authors have done a great job in synthesizing all of this data and laying it all out for us to look at in terms of tables and figures. The 2-If you're bioassay, we have a standard template and still comfortable with that part of the analyses are standard. That is not the case with these. I think we need to move in that direction. I did have a lot of editorial questions, particularly about the statistical analysis. I will not go into detail on these. I have written comments that I have provided. Just to give you a couple of ideas of what I am talking about, first of all, I am talking about the multiplicity analysis and multiplicity date the. Riding the incidence data are fairly standard. There are no references in the report that I could find to the statistical methods that were used with these data. I do not know-I feel pretty confident that they are all okay. I do not quite understand what was done. There are notation and terminology used in the reports that are not explained. I do not understand what the terminology is. Another example, it says the same method of analysis was applied to the in-life data as to the terminal data without comment to go it seems to me those data are different and you must have time of onset for the in-life data. There is no statement that those data were analyzed differently. So, I would really like to see a much better job done of explaining exactly what was done in the statistical analysis and lay it out very clearly. I think what we should aim for is a given the explanation and given the data, someone should be able to reproduce the analyses. I think that is true with the two-year bioassay that we have been using. We need to move towards that with this data as well. Regarding the conclusions, as I said, I think I am generally in agreement with them. As stated, they are very difficult to follow. I need the mistake, I think of reading the conclusions first before I read the report. I was sitting there scratching my head trying to figure out what the conclusions are not contradictory. Then, I realized that the key phrase there is in-life versus one sentence says in-life and the other does not have in-life at the that is the key difference. I think there are some ways that we can simplify these analyses and make them more clear. We can just say right off-Right at the front is that nothing was significant using the in-life data and not have to repeat that every time. I guess I am also a little bit not an easy with the distinguishing between incidence and multiplicity put up there is no evidence on incidence but there is evidence on multiplicity. Basically, my thinking is multiplicity is just a more general way of looking at incidence. The role critical question is, did you get an increase in these lesions? I do not really think we need to get into that Lovell of detail in the summary conclusions. I think that would also simplify the conclusions. Actually, I tried my own hand at wordsmithing. I will hold off on that and see what is presented here. That is all. Thank you.
Thank you. Dr. Cattley?
Dr. Cattley: I have some comments about information that I thought would be useful to clarify the findings and gain perspective on them. I do not have any question about the actual data itself. Let me just mention that you did talk about the positive control Aloe-emodin. I might have missed it, but I was curious as to how much Aloe-emodin was present in the other three treatments? It seems like you are left with a result where your positive control did not exactly to what some of the other treatments. I do not know if that is due to the amount-Whether the amount of Aloe-emodin might be different between these things or not. I would expect that there might have been less in the other one's. I have no way of relating those doses with what I think you presented as the active ingredient. The other thing is, I was interested in some of the literature that you reviewed in many of the studies used, rather than using simulated solar light, and you used something called UVB rate of exposure. I am interested in the SSL in the current study and UVB in the scientific literature. Did have a reference to a study of Strickland and colleagues that used UV and Aloe-emodin. It produced melanomas. I am baffled by that difference in tumor response. Maybe you have some ideas about why that is. Since you raised the study, I think it would be good to close the loop as to what the difference is, or maybe it is and unexplainable differences. I feel like I might have missed reading, but I was not sure about the study design and why there was a 12 week recovery period after the end of treatment. I think it would help the report if the readers understood what the rationale was for that. I could guess, and it might be there, and I might have missed it. Finally, when it comes to the conclusions, this is a very different study design. I guess I am not sure how we applied some of the criteria that have been used in and the two-year studies. From my standpoint, it would be helpful if the conclusions-I do agree with the comment about the in-life not contributing their much to how we would interpret the it steady at this point. From my standpoint, it would be of interest to delineate the benign from the moment and turn malignant neoplastic in these groups. For the concluding statement when you talk about the significant effect on multiplicity, you are really looking at a combination of all neoplasms. I would be interested in knowing for each active ingredients, which the differentiation was between the response of the papillomas versus the other two malignant tides of lesions, the [ indiscernible ] and squamous cell carcinoma. I think that is it for me. Thank you.
Thank you. Dr. Bunton?
Some of my comments have already been mentioned by Dr. Crump and Dr. Cattley. I will just reiterate them as I have them listed here. I did have the same question about what to appeared to be a recovery Group and using a title of a one-year study when the animals were actually exposed four 40 weeks and there was this 12 week period that I was assuming was a recovery, but there is no mention of it that I could see or any explanation of why that occurred or what the results of that were. I think that would be worthwhile to bring forward. I also had some of the same comments about the conclusions. To me, as they are written, they are not really conclusions. They are stating, again, what happened, what the results are. You have not taken the next act like we have done with these other reports and saying what it means. I do not know if that is just the way this is going to be done to. I think we should discuss how we should handle it in this case. There were other editorial things that I did not understand, such as the tables that included whole areas that said, systems examined with no neoplasms observed. Every system was listed. Then, you have not done histopathological. I was not clear about what exactly had happened. There was an area that said histopathology had been stopped, but no reason. Of like to hear more about why. The part of it like to make with that is that this is a large study. If, in the future, there would be any kind of a plan to assess, which is a systemic effect, a toxic effect due to these compounds, you have that data set, you have those tissue samples. I would like to see those used, rather than repeating and doing another steady progress that is an overall impression of what was used in this study as big and complicated as it is. I would like to avoid or at least have considered, if it needs to be taken up again in the future that this information might exist in the tissues that you have from these animals. I did have a question about testing all of the different components, but I think I figured it out afterwards as far as the differences between them all. You also clarified it here. I appreciate that. I think that is about it that is necessary to bring up.
I am going to try to cover the points one at a time. In terms of Dr. Crump's comments, even though they are mostly editorial, we have discussed this, and we will expand the description and the report to provide a better description of the statistical methods that were used, in particular, for the [ indiscernible ] regression model, I believe what you are referring to. In terms of the Aloe-emodin, we do have that data. We will incorporate it into the report to. This was in the characterization. I just fail to include that in the report. I will be happy to do that. That would also include the amounts in the various test articles. I believe you mentioned in terms of the conclusions, that you would like to see the different skin lesion types separated as well as the combination. I believe we can look into that and see if we can include all of that in the conclusions. Then, in terms of the 12th week period following the 40-Week exposure, it was actually an observation period. It was not to recover. This was the induction of skin tumors, a time and those response time to the light to. You do not expose an animal to light and see the tumor develop immediately. This 12 week period was to observe the development of skin lesions on the animals before and sacrificing [ indiscernible ]. In terms of the systems that were mentioned that no histopathology was conducted on, that is a formatting, a template formatting issue that we will try to address. It is automatically produced. We will try to see if there is a way to eliminate that. In terms of the tissues and slides and cassettes, all of that is archived by ID, by treatment. Those tissues, slides, the cassettes that were used on the study, we have those available for any further studies on these compounds.
Before you move on-during that Wellfleet observation. , then, did you see any incidence of regression?
No. We did not seek regression. I am not sure-I would have to go back and look. I am not sure that we saw any further progression either. Perhaps [ indiscernible ] would be able to.
[ AUDIO/SPEAKER NOT CLEAR].
This is Paul Howard. Dr. Crump, you mention that this was not a standard bioassay. You are correct or the this is not a traditional two-year bioassay. This [ indiscernible ] has been used since, I believe, the mid-1980s for drug nominations to the U.S. Food and Drug--This was developed in the mid 1980's and has been perfected to this design received today. Given a similar to those of solar light and then you had this observation period for 12 weeks purpose has been worked out with how many--It is a considerable number. What the NTP has added, those are typically only a in-life observations. We have added is a pathology consideration of these bumps. That is what the NTP has done is bring in the pathology to ask the question of traditional in-life, can we enhance that information with pathology? That is where this differs from what is considered to be a--You are adding pathology.
I believe that I addressed-the UVB rate. Yes, we are going to calculate the UVB rate of these lights and include that in the report. In terms of the Strickland study, there were numerous differences between the study. Besides the type of light, there was a broad band UVB. Ours is simulated solar light. There be Haeckel, they use ethanol and reused water in a cream base. The method of exposure, they exposed vertically and we exposed horizontal the. Our animals are in cages and our light comes from the simulated solar light directly to the front of the cage. The duration of exposures were also a different. It is a difficult to be able to compare the two studies in terms of why they developed melanomas, which, I believe they subsequently said were not really melanomas, but rather, I believe [ indiscernible ] came back and said they were actually deposits of the compounds in the skin at lesions. I feel certain that is what she said. I believe that is about it.
The difference between the Strickland study and Mary's and study is the light intensity. They are delivering three times a week about twice as much effect of light to. You have to remember the fact that UVB is very effective in producing European and tumors. We are mimicking some like. UVB is much less effective. We are giving human exposure--They are delivering a dose of light which it will be more wavelength by wavelength--Overall, they are delivering twice as much light that only three days a week as we discover five days a week. There is much more impact on the skin, if I can use that term.
Okay. Are there any other comments?
These are pretty minor comments. On the summary on page six, the discussion of the aloe gel agreement being discussed, the significant increase in multiplicities squamous lesion neoplasms but on page 82, there is a decrease in multiplicity of the non-neoplastic--It seems for consistency that that should also be mentioned here. Again, I do not think it is a big deal. It reflects the overall decrease in the neoplastic lesions. On the map on page 37, it says that growth skin tumor lesions core by the skin pathologist. --At this point with the grows exam,--Finally, the least minor point is just for figures on tumor multiplicity, the female comes first and then the male comes first. It--
Keep you on your toes broken any other comments from the audience? So if not, can be put the conclusions up on the board? On the screen? Let me ask if there are to be any motions to change the text, specifically?
I would like to hear what other people have to say. To me, maybe it is just me, but they are stating what happened in the study. What does that mean here? As it does with the other studies that we are reviewing, is there carcinogenic activity? It is just stating, again, what the results are, in my mind.
I would second that. With the categories of evidence that we used for the other bioassays, the summaries [ indiscernible ] apply here. Are there some other thing we should go by in order to give a bottom line is to what this all means? The same question.
So, I can comment. This is the second [ indiscernible ] study that we have reviewed. The first was quite Kollek [ indiscernible ] that were used as a defoliant, I guess. In that instance, Dr. Howard was the steady scientist for that one. We came up with some descriptive language that did not really fit the template that we have for the two-year studies. I do not think that we, in trying to fit this information into the template for a two-year study, have been successful in figuring out how they relate, either in the use of the words themselves or from the standpoint of the fact that we do not have a lot of experience in interpreting the outcomes of these. We do not have a sense, clear evidence, in this kind of study, what it really needs in terms of the NTP making a call on it. Paul might want to comment on how the committee dealt with the [ indiscernible ] studies. In that instance, there were actually no changes or protective effects. That is the other thing that you can obviously see in these studies is protected in facts. There were not any hints there that would lead us to see how to best utilize the criteria that we have for the two-year studies. We would like to move in that way. We would like to make these kind of study interpretations more consistent with the interpretations of the two-year studies. In fact, I know that Kenny said he took a shot every writing something. We have taken a shot at writing something. The panel might want to consider these other alternative suggestions at this point.
This is Paul Howard. On behalf of the Food and Drug Administration, and suitable does not defoliate women to go [ LAUGHING ].
I am sorry to go exfoliates.
Thank you. [ LAUGHING ] the go this is on the record ago actually, what Dr. Bucher is saying is in the--Report 524, the conclusion we made states taking into consideration the survival data and pathology results, glycolic acid did not--SSL is the benchmark. We are including it in that light, no pun intended, that it did alter. 84 in [indiscernible], it decreased the effectiveness of the light. --Of the simulated subtler and Chancellor of like. There is no mention of a son, it is stating the fact, the most obvious fact. Dr. Bucher, I do not know if you want to put up the one that you and the NTP staff constructed after you received your report. At more reflects that type of conclusion.
I would ask Dr. Crump, if there is alternate language that he would like to offer first?
Well, I do have alternate language that I put together. Do I want to offer it [ LAUGHING ]. Sure. I will offer. It does not really deal with the question that Dr. Bunton has raised. I think it does-I think it simplifies-I think it makes it more clear on the best way to do it. I am looking at it here on my screen.
While we are rethinking this, why don't we put the language that we have up and you can see if what you have written agrees or disagrees with what we have posted. Can you move these screen over a little, Charles, so that it includes.
Yeah, this is just part of it.
Yeah.
I kind of like the wording. If I wonder if the second sentence is really necessary. Does that add anything? We're talking about incidence here, right? Wait, no, we still have the [ Indiscernible ] life part. Okay.
[ Speaker Faint/Audio Unclear ].
Yeah. Okay. I think they're all saying the same thing. I think people can read that and they can figure out what is going on, so.
Can you go back, Charles, to the first one. Let people really have a chance to read through this. Dr. Bunton, does this address what you are getting at?
Yes. I think it does, certainly. I can appreciate the difficulty with this. This gets better at an actual conclusion based on the results that you found from this study.
Impliflt in this, we struggled with coming up with words to describe the response. The issue is related to whether you put your money on the traditional way in which these studied have been done and interpreted. But does not include the his toe pathology. In that case the words weak effect may not be sufficient. Or if you come down in the middle, which is basically where these come, I think, in that there is no increase in the traditional measures in the study, but an increase in the multiplicity -- therefore at this point this is weak evidence. Or evidence of a weak enhancing activity. But we're open to a complete discussion of this particular issue. This is key to us. We don't have a lot of experience with these studies.
Jon Mirsalis. I like this is a lot better. I read the conclusions and didn't know what to make of it. We're so used to reading this [ Indiscernible ]. These are such different studies, it would be misleading to put it into the conventional. When we talk about an NTP study, this clear, sum equivocal. If you try to apply that to a 40 week study with recovery, it gives maybe more strength to it than it deserves. I don't mean that in a negative way. These are different studies. We need different language to avoid using the conventional NTP language.
Kenny Crump. I think this is better than the first versions. I still have a problem with the second sentence. And the last sentence. Particularly, there was no, did not increase the incidence. I think that's misleading. The reason it didn't is because the incidence was 100%. There was no power to increase the incidence. Pointing that out is giving a misleading direction, maybe, to some people. So, a, as I said earlier, I will prefer to present these results without distinguishing between multiplicity and incidence, they're two sides to the same coin. If you didn't see it with incidence, I think it's a little misleading.
Can I add to that? I think at the lower levels of light intensity the incidence was not topped out. You could have picked up something there, if there was a tendency to increase incidence.
You could say you got it without reference to whether you saw it with incidence or multiplicity. You would also see it with multiplicity there, you should. Is there some biological reason to look differently at incidence and multiplicity? I can't, I don't know of one -- but --
Dr. Howard?
Trying to think how to phrase this. One thing we do know is at a point in time incidence is related to dose of light. When incidence is Maxed out, I think also during the course of onset of tumors multiplicity is also dose related. I don't think they're necessarily connected. The first tumor you see happens to be the one that a biologically uncoupled from growth control. Whereas, I think they're not giving the same information. I think it's a different type of information. One is multiplicity is much more dose-related. And incidence is much more time [ Indiscernible ] dose related. I can't describe it. I wouldn't want to lump them together. In this case where you had near a hundred% incidence your power of deteblghting a difference is so diminished. I would agree with you. If we had used a lower dose of light, definitely incidence would have played into this. [ Speaker Faint/Audio Unclear ].
Any more discussion?
Is it possible to see the second page again?
Yes.
So would anyone like to suggest a change in verbage for the incidence versus multiplicity issue? [ Laughter ] Dr. Crump?
An idea. What my version is. See if you like it.
That's a great idea.
I can read it. I'll read the first part. Once you get the flow you can see. Males and females SKH-1 mice were treated topically with aloe jell creams. Data on skin lesions were [ Speaker Faint/Audio Unclear ] no effects of creams upon skin lesions were identified from data collected during [ Indiscernible ]. Next paragraph, all these paragraphs are the same. Squamous cell [ Indiscernible ] identified in [ Indiscernible ] were increased in female but not male mice treated with aloe jell cream and exposed to SSL. [ Speaker Faint/Audio Unclear ]. The statement is simplified. It doesn't say multiplicity or incidence. Just says that they were increased. And the other conclusions with the other creams are similar.
Yeah, I guess the only difference it gets away from making a conclusion. It's more a statement of the data. Is there more discussion?
I think with the statements up there, what I like it's saying there's a weak enhancing effect. It's okay to use as a different language with these types of study. On the photo carcinogenic activity, to me those are the keys. You are saying what the effect is, and specifically this is -- what you believe is a result of these data. At least in my mind. Versus restating that you have more tumors in a specific group, or increased multiplicity. I'll stop there -- you know what I'm saying. [ Laughter ]
Uh-huh.
You can't read this once. The way it's written. You have to read it twice. You get to the second sentence you feel that you are reading the opposite conclusion. You have to go back and see there's a distinction between multiplicity and incidence. That's always a cumbersome way to present things. I don't know what the solution is. It might to be drop the last sentence. I don't see how to get through this reading it once. If you read it cold.
Russ Cattley. I guess I would like to know that it was based on multiplicity in a conclusion statement. You know, there's ways to word Smith and make it readable. Just dropping the last sentence would also fix it.
This is Mike Pinot. I agree. I do agree with Kenny's comment. I already almost at a hundred%. It's meaningless here.
I agree.
This is Paul Howard. In a tradition assay onset is the most important term. Not incidence.
Because incidence does go to high levels, usually.
It doesn't Max out at the lower doses.
I would also say from modest experience in the field that multiplicity is an enpoint of -- end point of interest to researchers in that area. As any other carcinogenic end point.
Is there any support for the simple solution of dropping the second sentence and saying what we didn't find. But saying what we did find?
I think from a public health stand point [ Speaker Faint/Audio Unclear ] it also serves some utility to state a negative result. As we did in the previous study. That also needs to be weighed as carefully as a positive affect that was detected. That's just my -- I think sometimes negatives are just as important.
This is a different kind of negative. We saw it one analysis, but not in another. We're looking at the same end point. In that sense I wouldn't totally agree with you on that.
It seemed to me, if we use Dr. Crump's first paragraph. And into the second paragraph state the two positive findings, leave off the negative secondary findings, would that work? You summarize the in life data, which indicates latency and incidence as having no effect. If I got it right. The second paragraph was -- or we could add a second paragraph that would speak to the multiplicity and the conclusions there from. Would that work?
[ Speaker Faint/Audio Unclear ].
Yeah. You have one sentence to summarize the in life findings. That was really good to start off with.
No effect of [ Indiscernible ] upon skin lesions were identified from data collected during life.
You could maybe expand on that as to what data you get out of an in life study. In a second paragraph you could do the positive findings.
We could break for lunch and work on a combination statement over that hour.
Jon Mirsalis. I like Kenny's statement. You could probably say that no in life affects were observed. However, [ Indiscernible ] revealed increase in multiplicity -- period. We can argue about whether we're all callout incidence [ Indiscernible ]. But the point is, it's a good point, Paul makes a good point. You want to get in life we didn't see anything -- but, or however -- in a [ Indiscernible ] evaluation some multiplicity was seen. A single sentence that nothing in life but -- [ Indiscernible ] would give the best feel for what we are trying to convey.
Use the term weak? Weak enhancing then?
You could say however, weak enhancing effect was seen based on increased multiplicity in the his toe pathlogical evaluation, something like that.
John? Did you want to say something?
I think it's important to make sure we can agree on language so we can work on this.
[ Speaker Faint/Audio Unclear ].
Will you identify yourself?
Ron Melnick. You have an unidentified modifier. Which could be problematic in future studies. If future studies come you need to define that. What are the criteria more weak or heavier different level of evidence. I would uncomfortable with using the word weak without a definition of what you mean.
Ron always keeps us honest. [ Laughter ]
Okay, whoever will word Smith is -- we'll break for lunch.
What time back?
1:00.
Session on lunch break until 1:00 eastern time. Please standby for realtime captioned text.
As we are waiting, please read the version that is on the slide.
Good afternoon. I'd think we can resume the discussion on the revised conclusions of the Aloe vera study. If everybody on the committee has read the revised version, is there any discussion lacks Dr. Howard? The map on the last word of the paragraph, should it be a life or in-life?
I suggest during the in-life phase.
I am still thinking about Ron's comment before lunch about where do we mean by weak? I confess that I do not have a better word for it to. It gets to the old minimal, my, moderate Mark. What is weak? What is strong? Without a clear definition of what that means, this kind of ambiguous. On the other hand, the sentence is kind of ambiguous. I am a little uncomfortable with the word weak, unless I know what weak means.
When should never disagree with one's Supervisor. In all of the conclusions on the NTP studies, it has modifiers of strong, equivocal, clear. It is consistent with that.
It is consistent in the sense that we have a modifier. I am not sure that is consistent in any other way. If we leave it there, we will set a precedent of what we mean by weak. I am not sure that we have thought about this enough to really set that precedent.
Dr. Mirsalis: I do not recall the statistics on this. Was is it statistically significant effect? Do we say a statistically significant enhancement and not get into weak, strong, super strong, moderate, clear and as a statistically significant enhancement?
Can I make a suggestion? We had intended to quantify the defects in this study and even back to the alpha and beta hydroxy studies. Basing them on the SSL equivalent, that is how much the [ indiscernible ] compound represents as a change in the SSL activity. Would that be sufficient to quantify weak? That had been suggested originally in a [ indiscernible ] meeting. It was always a good idea. We have not put it into the report yet.
Dr. Howard: We can do that for Ansett because we do have multiple doses of like. Can we do that with multiplicity on the pact ology?
Sure. Can do it with any endpoint in the study. We measure it on all of them to go in a sense that is quantifying something against nothing. People will have a understanding of what a particular light level represents as a carcinogenic activity. Is it sufficient to resolve the issue of weak? -week-old I am not sure how that would translate here.
You have a certain level of activity on the, let's say, multiplicity. We have the scale. Oneof the reasons we did the no cream SSL group is to affect the compound to have a like equivalent to it. We would quantify the effect of Aloe vera, whole leaf, let's say, on a unit basis of how much like it would take to give the equivalent effect. If that is a seemingly minuscule amounts of light, then, probably, you would generally agree that that is a weak defect.
Dr. Crump: I think that is a good idea but is estimating potency, which goes beyond what NTP normally does. I could go with not having a quantifier and could go with statistically significant.
How about statistically significant? Does everyone buy into that?
I guess I would not use statistically significant therefore the enhancing effect. That is not what you measure the statistically significant. That was for the increase multiplicity of tumors. If you stay distich Lee on--Increase and the multiplicity of tumors, what was actually significantly increased, not the enhancing effect-That was the conclusion based off of that observation.
I will just add one more comment. This is Dr. Kerkvliet. There was a we enhancing effect. We are defining a weak enhancing effect. If you put one more modifier in there, you could qualify it. There appeared to be. There was a potential for, one more qualifier that takes it away from the defining weak. I'd like to just throw that out.
This is an expert panel to go this is your subject of expert opinion on weak. Do you agree that it is a weak defect. You are trying to get to define specifically what weak is. You are getting into a subjective interpreted call here and not the point of we of what we are asked to do based on the data.
I guess I would agree with this. And a lot of time you are going from an average of 6 to 8 or nine. To me, that is a weak defect.
So, with someone like to put a motion for to accept the conclusions as written?
Dr. Mirsalis. I will accepted as written.
Any more discussion? A second? Thank you, Dr. Novak. Shall we vote? All those in favor of the conclusions as written. It is a unanimous approval. So, moving on. We will now be discussing the Chromium picolinate monohydrate Report. Dr. Stout?
Good afternoon, everybody. I am a Dr. Stout. I will talk about the toxicology and carcinogenesis studies of the Chromium picolinate monohydrate. Before I get into the study, I would like to talk a little bit about the form of chromium in this compound. Chromium picolinate monohydrate is a dietary supplement that contains chromium and the unsettled state. To my recall that at the last technical report meeting we talked about the [ indiscernible ] which have the chromium and a different [ indiscernible ] state. Is an essential element. It is found in many foods and is involved in lipid metabolism. It is taken for weight loss and the entire diabetic effects. The oral absorption of this metal is poor. [ indiscernible ] is thought to increase the tissue absorption. It is at present in a [ indiscernible ] form. The typical daily doses contain approximately 2 00 to 600 micrograms of [ indiscernible ] chromium which comes out about 3 to 9 micrograms of chromium three per kilogram per day for a normal individual. So, chromium picolinate was nominated by the National Cancer Institute and a private individual because of the potential for widespread consumer exposure through its use as a supplement. The monohydrate form was selected as the test article because that was what was commercially available and feed was selected as the route because humans and just chromium picolinate and supplement's. I have the structure at the bottom right corner. As you will see the [ indiscernible ], and insulated by three molecules of a and associated with one molecule of water. The NTP conducted a pre year and its--And beat in both female and female F-344/N fish and B6C3F1 Weiss. The concentrations 43 month studies went from 0 to 50,000 parts per million. That top dose of the 50,000 or 5% is considered to be a limit doughs and feed studies. So, in male and female rats and mice, there were no biologically significant changes in any of the end points that we measured. These included survival, body weight, speed conception, hematology, clinical chemistry, where and which, histopathology or the sperm apology and vaginal cytology parameters. The lack of a body weight effect is of no--People in just this supplement in part for weight loss defects. So, the NTP conducted genetic toxicity studies as well. Part of these studies were part of the three month studies. In vitro--Were conducted in salmonella, on both Chromium picolinate monohydrate and a compound characterized as not the model hydrate form. All of the strengths tested in the presence and absence of the metabolizing fraction. There was no evidence of Munich tenacity. [ indiscernible ] were also conducted in mice as part of the 13 weeks a study produced these results were negative and male mice and equivocal and female mice. An additional study was conducted--Three times one they are part. These results were negative as well. Following the three month studies, two-year studies were conducted. The exposure concentrations were the three highest concentrations from the three month study is. No defect was observed. Again, there were no biologically significant changes in the survival, body weight, feed consumption common incidence as of non-neoplastic reasons--Or in male or female mice. Again, I would note the lack of a body weight in effect in terms of what the compound is consumed for. So, in male rats, we did observe an increased incidence of t he--At the 10,000 doses, which we considered that might have been related to exposure to the compound. I have these data listed on a table. Deeply to show gland is the male accessory sex glands and program--Is the corresponding gland. Adjutancy in both male and females, there was no evidence of hyperplasia. In female, there were no increases in neoplasms. And males at the meadows and we have an increase that was statistically significant and exceeded the historical control range when which went up to about 10% for the studies and studies of all [ indiscernible ]. We considered that to be an equivocal finding. In order to support the toxicity and carcinogenesis studies, w e--and excretion studies as well as tissue distribution studies to look at the distribution of the compound. For the [ indiscernible ] studies, rats and mice were given a single dose of a--Of Chromium picolinate monohydrate and urine was collected for 48 hours. Chromium and--Each part of the complex could be determined separately. In looking at, and excretion, a common expression in the species was must greater than The urinary excretion, while in the case of the radiolabeled the excretion was similar to the urine PCs and most of the urinary excretion was in the form of [ indiscernible ] which was proposed to be the reaction of a [indiscernible]. These data suggest there was--That led to the conclusion that most of the acid was not bound to the during absorption--Is thought to be very well and, also suggests that this was a study of not only chromium picolinate and picolinic acid acid, separately. As part of the two-year studies, coming to shoot distribution was analyzed. Additional groups of male rats and female mice were treated the same as in study animals and were removed from a study on days of four, 11 and 180. Adding in a 48 hour watch out three in to remove unobserved chromium were common during was collected on basics, 13 and 182. In addition to urine and feces, erythrocytes, plasma, liver, kidney,--Were collected as well for a total chromium Analysis. So, in terms of looking at the results of these studies in both rats and mice, increases in total chromium were most apparent and kidney, liver and plasma and these concentrations were highest and right kidney and mice Oliver. In these tissues, primarily, but there was also evidence and other tissues, you saw when looking at both tissue concentration against exposure concentration or exposure to ration, you saw a plateau of total chromium concentration. On both of these Graves, either the exposure to ration goes from day six, 13 or 182. Of the doses go from control of two 50,000. You can see the leveling off of tissue chromium, which suggested to us that the IRA come in concentrations could not be achieved. These results also did indicate that there was systemic exposure to [ indiscernible ] chromium, despite its low absorption. So, the conclusion of the two-year studies on Chromium picolinate monohydrate, there was evidence of carcinogenic activity and Mail F-344/N rats in precocial planned adenoma. There was no evidence in female rats or male or female mice.
Thank you, Vt.. That was very nice. Are there any specific questions before we go into the reviews? If not, Dr. Cattley?
Dr. Cattley: This is a fairly straightforward study, survival and body weight gain was a reasonable. I think it was a very adequate test of the carcinogenicity of this model. At the level of treatment, certainly, was achieved at a very high rate and definitely covered the range of concern that probably lead to its nomination for evaluation. I think that the findings are adequately dealt with in terms of your explanation and also the interpretation put go I have one slight commented in the discussion where there is a mention of explaining a lack of exposure concentration response relationship for the proposal of land adenomas. If it is an equivocal finding, I would suggest we do not talk about an exposure response relationship on it. Other than that, I would agree with your conclusions.
Dr. Mirsalis?
Dr. Mirsalis: I would echo most of those comments. It was a well written report and straightforward. I agree with most of the conclusions with one minor point. I would say that the correlation in the chromium tissue levels and the results was very nicely done. Kudos to the people who designed and conducted the studies. I think that adds a lot to the report. Just a couple of someone in a picky things. We offer our outside experiences to those proposed last year our institution had its triannual [ indiscernible ] at inspection and a FDA inspection applicable points that I will bring up relative to that purpose might just be a point of clarification. I notice that we do carcinogenicity studies, we will spell out two or three animals per cage and the 13 weeks study is five animals per cage. It is a note to the people during the studies, is that keeping a five per cage four 13 weeks within the current NRC guidelines. We are getting hammered-We have to start pulling them down to three or two per cage when they reach a certain weight. We are doing it for the carcinogenicity studies, I would look at it for that and all studies whether or not-We are in fact, monitoring weight and when you reach that capacity, which is spelled out pretty clearly in the [ indiscernible ] regulations, that you are pulling down the number of animals per club that is something that struck me, I am sure I have read these reports 50 times before but after getting dinged on it by [ indiscernible ], it is now on my mind. Another thing, related to feed, this is a common form of reports but generally on this, this is a feed study, but you do not sit with the feed as. If you go back into appendix K, you can say that is [ indiscernible ]. Is a dispelling it out in the body of the report when the safety, especially when it is a dosed study. And other trivial point. I know that we officially claim these studies are all under FDA [ indiscernible ]. FDA is now mandating that you do independent verification of the impurities of feed. Simply getting a certified feet with a Certificate of Analysis is no longer adequate under FDA guidelines. You have to do an independent assessment provided another one we learned the hard way last year produced of this has been brought on by the Chinese but the debacle of a couple of years ago put that is a requirement that I guarantee no one is complying with including any of your contractors. We got a 483 on it to go anyway, that is just something to bring a three-year program, go back to your contractors on a feed. You have to do periodically analysis, even if you are buying and analyzed Chou. They say they no longer trust the vendors and their analysis. Another, again, this is what my staff calls a Johnism would like to see a number-I will bring this up on body weights. It would be easy to say increased 8% over control instead of saying increased predictably 3% with a type variants. Again, that is something else. My one subject of a point is about the micra nucleus of results. We are calling the male negative and female equivocal. I did a little quick analysis of all the reports we are covering in these two days. The range of Micron nucleus for the seas and mice is part five to 3.9 per thousand. One to 4 comment with the industry gets in the Reports. That is fine. In the male, the council was to play eight and today was 2.9 to [ indiscernible ]. We call it not significant role in the female the control was 2.1, a little while and the range was one printery to 3.4, all the [ indiscernible ] were negative to go now, all the sudden, it is a equivocal did not toxic agent. Certainly, if you sent is that as a plan compound to a testing laboratory, this would not be as-Well within the historical ranges. If that control instead been to .1 had been 2.4, which is about three micra nuclei out of 10 animals scored, that equivocal would have gone away. I question equivocal put my opinion on it would be that it would be a clear negative because it is so well within the historic ranges. I understand you are erring on the very conservative side for risk identification. I do not think the data hold up to the difference in the males between the controls and the highest response was greater than the females yet you called the male negative. Other than that, I thought it was a very good report.
Thank you Dr. Novak?
Dr. Novak: I think this was an excellent study with the total analysis and the data and everything. I support the conclusions. I have no other further comments.
Great. [indiscernible], which like to respond?
I would like to divide the reviewers for their comments. In response to Dr. Cattley a comment about responsible, delete that from the text, in response to the comments on housing, we went back and looked. Our five per cage does meet the [ indiscernible ] requirements.
Just to clarify, it is based on total body weight in the cages broke you tend to use of Fishers, which stand to stay small logger. You very, very quickly within two or 36 into the study by the time they are about nine weeks of age, will not need it. It is not stray animal number, it has to do with what the weight of the animal is. If you are checking and are fine, I am good. I just wanted to call it to your attention to check it.
Thank you. In response to the comment regarding the diet, we do analyze each lot of seat independently. There is an independent analysis per vote during the course of each study, an animal will receive several lots of the. We go beyond the periodic analysis that is recommended and the GOPs. Speaking of the comment about micro nucleus, we do agree that this response is not impressive. We considered a response equivocal if there is a significant trend or a single group is elevated. It does meet our criteria for an equivocal response. Again, we agree that is not tremendously an impressive finding. We will at the detail on the increases in Oregon leads to the report as well. --Organ weights to the report as well.
Thank you. Either any other comments from the committee? Yes, Dr. Howard, please.
Dr. Howard: I have a minor one. On page 17 you refer to the typical daily dose of Chromium picolinate monohydrate to--Of a fight to have a reference at and see if that is a dietary supplement or normal diet. You do say recent reviews--Just please for clarification point out where you got that information and if it does to refer to regular diet or dietary supplement. Thank you.
Any other comments? They're being none, which is like to display the conclusions? --Would you like to display the conclusions? If I could have a motion on the conclusions.
Dr. Cattley so moved.
Dr. Mirsalis has second.
Any discussion lacks all those in favor? --Any discussion? Opposed? It is unanimous. Let's move on to the next chemical, one, 2-Dibromo-2, 4-Dicyanobutane. Dr. Dunnick?
Good afternoon. We did toxicology and carcinogenesis studies of Dibromo to [ indiscernible ] dicyanobutane. I served as a study it scientist and Dr. Herbert did the pathology. This chemical was nominated for study by the NIEHS because of its widespread use in over-the-counter healthcare products and because of the lack of toxicology and carcinogenesis studies reported in the literature. The demo group of administration was to mimic the human exposure. The chemical was not mutagenic in salmonella or in other tests for Gina toxicity. The study design for this chemical was that the chemical was administered by the turmoil administration to male to a female in which the doses were from zero, up 37.52600 milligrams and rats and--In these studies we administered the chemical at a constant concentration varying the dose based on body weight per go toward rats, 0.5 milliliters per kilogram of weight and an mice 2 milligrams per body weight. Intestine the studies showed quite a bit of skin toxicity. In subsequent studies in the 13th week studies, the high dose was 18 milligrams per kilogram and rats and mice and the to your studies, the high dose is 18 milligrams per kilogram and rats and six mg per kg and mice. In the 14 day and studies, the primary finding was skin toxicity, as I just mentioned in all dose groups of rats and mice. The body weight presented here as a percent of control body weight was generally similar to that of controls and not statistically different by pair-wise comparison to the control group to go up the high dose with 18 milligrams per kilogram per go the survival was good and all dose grips. Oneanimal did die early in a female rap group and were not able to determine the cause of death provoked the body weight by a pair-wise comparison were similar in the treated groups to the control group. In male rats as presented in this finding there were a series of skin lesions including hyperplasia in the treated groups, [ indiscernible ] and some information. And female rats, the similar type of lesions were seen. At the high dose groups, while there were skin lesions, they were not considered to be severe enough to compromise a two-year study. The high dose was kept at 18 milligrams per kilogram for the two-year study in rats. Annette mice, skin lesions were, again, seen here presented for male mice including hyperplasia. However at the height dose, there were some Nicklauses and ulcers in the male mice. Thus, the high dose selected for the two-year study was 6 milligrams per kilogram. In female mice, we also have one or two animals with the necrosis as well as these other lesions of hyperplasia and some inflammation in the dermis as with male mice, a 6 milligrams per kilogram. Now, at turning to the two-year survival data in rats-Add to your body weight data and rats, the body weights were, in general, similar threat most of the study, a little decrease in some of the high dose and survival was a good. But, again, we saw the scan lesions that we have seen in the pre-Chronic studies including hyperplasia some hyperkeratosis and inflammation of the dermis and male rats as was found and female rats, the hyperplasia, particularly at the high dose. 82 of the animals presented with other findings such as the necrosis that we had seen and the inflammation in the dark as. Even though there were the series of skin lesions, there was no evidence for a carcinogenic response at the site of application or in other target organs and throughout the body. We did a full pathological examination. Survival was good interested groups of mice. And body weight were, in general, similar between treated groups and controls brought most of the studies. The primary target organ was the skin in mice common share presented for male mice, where there was hyperplasia and a few other animals presented with inflammation of the dermis and one or two ulcers. Again, no carcinogenic response in the skin or other organs in male mice. While there again boar's skin lesions and female mice, there was no evidence of a carcinogenic response. In conclusion, there was no evidence of a carcinogenic activity in male or female rats or mice. Dermal administration of Dibromo dicyanobutane--In both rats and mice.
Thank you, a very much. Are there any general questions before we move to the review? If there are none, Dr. Bradfield.
Dr. Bradfield: Overall about this was an inappropriate toxicology and carcinogenic study's purpose of the protocols and this is an exposure route all seemed rational. I really did not have any substantive issues with the report. I did notice one scientific criticism, which I think maybe had some fire my way. It seems to me that this time that NTP start to include transcription all profiling in their toxicology reports. It seems like a great example where I start to say to myself, did this compound get anywhere? I realize there are all of these sort of arguments about half life and absorption rates that are in the literature. Is started to make me think that I could get a lot of information out of this and all of the other studies I have reviewed if I had some information on profiling. I think it could be as sensitive, as powerful as any of the slides we are looking at that happen to be stained with [ indiscernible ]. I think we could stain them with [ indiscernible ] and get it some, I think, very powerful data that might drive some people's decisions. That is my one scientific criticism of the reports that I am reading is that I'd like to see profiling data on all of them.
Thank you. Dr. Mirsalis?
Dr. Mirsalis: This is another very nice report. If you keep reading very good reports without any errors in them, you do not need as. You ought to sneak in something every now and then to try to trip us up. I do not have any major criticisms, it is a couple of minor points per I put a note in the written comments about it being confused and between the varying the volume and concentration and not that I read the text it makes % to me. When I reviewed it, it did not. I do not know if it is the wording or not. Ignore my written comment on that. Again, another point I made on the previous report, when you refer to something been significant in the text, but to seek a number like the main body weight was significantly less, I would say, 8% or something like that. That would be helpful to tell the reader while you are reading it what the actual value as. Some of these are approaching the 10% mark, which is a fairly significant decline in body weight. The one Scientific comment is that I know in the past NTP has done something like PCNA--I would love to have seen self proliferation on this. I do not know if you have that data and did not put it in the report. I know you still have the blocks and could do PCNA. For something like this it could have been a useful and point to go in the written comments I gave I gave to comment on the [ indiscernible ] Miller paper where they did--and then did proliferation side-by-side with it. It makes for a nice story. I do not know if you have that data and did not put it in. That is an observation that I think would have read the report better. Otherwise, I put it was a very nice report. It was a very nice report and I agree with the conclusions.
Thank you. Dr. Bunton?
Dr. Bunton: I agree. It was a nicely written report. I was curious. There were comments that I have that I did add to this sheet. In your presentation you answered them all. So, I am not sure, maybe we have a psychic connection or something I did have a general question, though, about the negative trends that we are seeing in this study, and if you have any thoughts on that? And even as you went through-I believe and some of the non-neoplastic lesions they had a lower incidence at higher doses. So did some of the tumors.
Okay. Is that the end of your comments? Are you finished? Okay.
Dr. Dunnick?
Dr. Dunnick: Thank you I very much for your comments. I will add the comments of that were suggested. On the negative trends, the one that we did bring forth to the abstract was a decrease in mammary gland tumors and female rats where we saw 17 in the control, 19, eight and nine in the high dose. [ indiscernible ] we have a special statistic we do to make sure if this was related to any body weight per call this a decrease was not related to any body weight in fact. The eight and nine in the mid and high dose Group, a [ indiscernible ] was lower than that, seeing the historical control data that we have for this. Therefore, we do feel it might be chemically related. We do not have a mechanism that could explain this to decrease. We do know it this in the discussion. There were some decreases also observed in the pituitary gland tumors and female rats. These decreases that work in the historical range--We did not feel that this could be related to the chemical as also was the case for a liver tumors Annette mice. Their work, perhaps, some of decreasing in male mice by numerical analysis, 41 in the control and a 31 in the high dose. This 31 was within the historical range for liver, as I think you might have seen, there is some variability from a study to a study. We could not determine that this was clearly related. We do note of this in the discussion that these incidence, for some of these tumors are within the historical control range and these decrees incidence could not be clearly related to chemical Administration. We do want to do studies where ever warranted that further explain results, such as further analysis by Texaco --We appreciate your recommendations on this. We do have a NTP [ indiscernible ] faculty which [ indiscernible ] heads up. He has gathered a number of experts to assist him. There are many people here property expert in the field. We do bring projects forward for consideration for microarray and I will take your suggestions on this, more interest in this whole topic and Dr. Walker and his colleagues will discuss it further as we will your recommendation for PCNA staining.
Great. Are there any other comments? From the audience, any comments?
I have no one question for clarification. In the two week rats said there was a decrease in the thyroid gland weight I went to the end civil study to see if there was anything there. The thyroid was not wait in the three month study. It is typical that you do not weigh the the rights but they are weighed in the two week study is. I was curious as to why that was done like that?
Over the years we have had several target organs that we do away. We do additionally add organs to this. Midweek could have done that in the 13 weeks studies additionally. I guess we were not able to capture that data.
That is interesting. I did not realize that. I will have to go back and look at that. Concerning the issue of the routine microarray analysis, I do not know whether Nigel by to make a comment about this. I suspect we will have a chance to talk about this tomorrow. I would encourage you all, actually, to give this thought overnight to. We have considered throughout the years the rather routine collection of tissues during to your studies--Two-year studies for genetic analyses and for a number of technical reasons have not done not. The [ indiscernible ] faculties that Nigel had of has argued the point whether they should be a screening activity or hypophysis driven activity perked up as we have always come down on what we thought was a more prudent approach with a high profit this driven [ indiscernible ] that we would have studies specifically that were for that purpose. As technology moves along and you are able to do more with tissues that are fixed and things of that nature, I would encourage you all to give thought to it and give us advice as to what you think might be appropriate at this stage as we take this program forward.
Any other comments? If not, may we see the conclusions? I would entertain a motion on this conclusion.
Dr. Mirsalis, I move to accept.
Second? Dr. Bradfield? Of those in favor of the motion? It looks unanimous to me. Thank you, very much. Dr. Bucher is going to speak for a moment.
When we put together the schedule for this meeting, we had anticipated having one more report today and perhaps a three tomorrow probably put out a Federal Register announcement suggesting we would have three reports for tomorrow and we have some public comments on those reports. At this stage, I think all of those folks that have been emitting you and today with the knowledge you could not respond are going to be disappointed. I think you will have time to respond to your e-mail. We will have to take up the rest of the business in the morning, I believe. You can be more efficient wintertimes.
Thank you, John. Do you have closing comments for today's?
I do not have any comments. I need to know what time I can take you back to the Hotel. I will have to scurry around and see if we can get the bus. If you can hang around here, and as a novel so that I can let you know.
Barbara, did you want to say anything about dinner?
As far as dinner is concerned, the bus will pick you up at the hotel at 6:30. We will meet at 7:00 at [ indiscernible ] for those that are coming on their own, separate the part of the bus will take you back to the hotel around 9:00 or earlier depending on what time we are finished. Tomorrow morning, the bus will pick you up at 9:00 to take you here for the meeting to start at 8:30. No breakfast briefing tomorrow, just read into the meeting. Thank you.
Thank you. Thanks, everybody.
Good morning. I would like to call the meeting to order. We'll begin the day with introductions.
[ Indiscernible ].
Dave Malarkey.
John Peckham.
Grace kiss link.
[ Indiscernible ].
Tracy Bunton.
Mike Pino.
Mark [ Indiscernible ].
Paul Howard.
Kenny Crump.
Russ Cattley.
Chris Bradfield.
Keith Soper.
Mike Novak.
[ Indiscernible ]. John Bucher.
Mary wolf.
Susan Elmore.
Charles all Don.
[ Indiscernible ].
[ Indiscernible ].
Bill Iverson.
Ron Lawrenceson.
Robert [ Indiscernible ].
[ Indiscernible ].
Rick Irwin.
Angela king Herbert.
Bill Foster.
Matt Stout.
Cynthia Smith.
[ Indiscernible ].
[ Indiscernible ].
[ Indiscernible ].
Christine [ Indiscernible ].
Ron Herbert.
Greg [ Indiscernible ].
Okay. Thank you. I'm Nancy Kerkvliet. Dr. Shane, did you have announcements?
Good morning. Please make sure that you have signed in again today. Please use the microphone and identify yourself when you make a comment. It's being taped and video cast. If you are making comments from the public and you have not registered, please register outside if you need to make a comment today. The draft reviews are preliminary reviews and will not be included in the minutes. The board members must be verbalize any comments they wish to be stated. I will now read a conflict of interest statement. The members of the NTP board of scientific counselors technical review serve as individual scientists. Each member is to exercise judgment prior to any meeting as to whether a potential conflict of interest might exist relative to topics for discussion by the board. Should there be a potential conflict the member is to decline services is a reviewer for the relevant report and not to participate in the discussion or vote. There are no conflicts of interest today. Thank you.
Thank you, Barbara. The format for today is the same as yesterday. We'll have the study scientist and if necessary the pathologist make presentations. There will be questioned asked of them. Then the principal reviews will present their reviews and make recommendations. Then others may make comments and then continued discussion. The first chemical this morning is Estagole with Dr. Bristol.
Thank you. I'm here today to present the results of our 3 month toxicity studies of Estagole, which is a naturally occurring exon. It -- compound.
It occurs in plants. It's 95 to 97% of the oil from the buds of this plant. It occurs naturally in a variety of foods. It has commercial sources. The distilled from the oil of term tine -- terpintine. Its uses are derived from its odor and sweet taste. We call it a diettary chemical. It's in a variety of foods and beverages. It's in baked goods, for example. It's in a whole variety of personal care products. It's used in central and south America as a medicine. In the southern U.S. it's used as a spice. It has antimicrobial activity. I wanted to make a moment to look at structures here. In general these compounds are called [ Indiscernible ], in the botany world and whatnot they're referred to as [ Indiscernible ] compounds. They generally, the size chain double bond, its location defines two sets of analogs here. So with the analogs we have the saturated [ Indiscernible ] group in between the double bond of the propeen and the air mattic system. The double bond here is congregated with the fennel system. This could be called a beta methyl sty reen. [ Indiscernible ] then is one of the [ Indiscernible ] compounds like methyl [ Indiscernible ]. Later today we'll hear about Isoeugenol. Here I have shown the structure of [ Indiscernible ]. Safrol has this group. In the late 50s and through the 60s is when toxicity tests were run and it was realized that it was a rodent carcinogen. With regard to the toxicity, the metabolism of these [ Indiscernible ] compounds in particular has been studied. They form a [ Indiscernible ] at that [ Indiscernible ] carbon. That gets sulfated, which produces the active compound that leads to the [ Indiscernible ] in rodents and punitively in humans. NTP performed studies with methle [ Indiscernible ]. There was [ Indiscernible ] in the liver of male and female rats and in mice. Both Estagole, Estagole is known to form the [ Indiscernible ] compound. And it's also known to be [ Indiscernible ] in the [ Indiscernible ] mice assay that the millers used back in the 80s. The [ Indiscernible ] is inhibited by pretreatment with [ Indiscernible ] or by using [ Indiscernible ] that do not have the ability, they don't have the [ Indiscernible ] to convert the alcohol to the sulfate Esther. That's the theory.
In general the aloe Ben design compounds form DNA [ Indiscernible ]. So Estagole was selected by NIEHS. We've done [ Indiscernible ], then [ Indiscernible ] and Isoeugenol was underway. So Estagole was added to that because of its widespread human exposure and the literature knowledge about it being [ Indiscernible ] in mice, um -- some of the aloe benzine compounds have been listed in the report on carcinogens. They're anticipated to be carcinogenic in humans. We want to compare it with the results from methyl ugole.
We used the gavage route of administration. We wanted to design a studies and conduct them so it would be easy to compare the results. The dose levels ranged up to 600-milligrams per kilogram. Corn oil was the vehicle. Also, because special toxicity studies were done with the other compounds, we did them in this set of experiments. They were designed around one month exposure to look at stomach PH. Serum [ Indiscernible ] production and the histology that might occur at the stomach and [ Indiscernible ]. Which were the salmonella tests in vitro, negative. In vivo the test on bud smears taken from male and female mice and Estagole was negative for micronuclei in both sexes.
We saw after 3 months of exposure all of the animals exposed the high dose. There were decreases relative to controls in body weights. Down to 73% in the males and 87% in the females. With regard to the hematology affects, there was a pattern that reflected anemia [ Indiscernible ]. There were increeted plate let counts. There was an iron deficiency as sublghted by the total of iron binding capacity. There was decreased serum iron. In general the effects were more pronounced in males than females. The mice hematology was unremarkable. There were other liver affects in the rats. The organ weights increased in the two high dose groups of males. And down to the 75, or middose group of the females. For clinical chemistry there was increased [ Indiscernible ] and [ Indiscernible ] activity in the serum. And the [ Indiscernible ] concentrations were increased, indicating liver injury had occurred. Um, the most surprising finding in the study was neoplasia after three months of exposure. Two of the high dose male rats had multiple [ Indiscernible ] carsinoma. A third male rat had [ Indiscernible ] adenoma. The occurrence of these lesions was supported by the pattern of [ Indiscernible ] fibrosis here in the high dose. [ Indiscernible ] in all of the exposed groups. There was also [ Indiscernible ] down to the 75-milligram per kilogram group. The [ Indiscernible ] inflammation and infill trace of [ Indiscernible ] was also noticed in all of the exposed groups of males. There was a pattern of foecy in the higher dose groups, generally.
Um, and so we checked the historical controls for rats and saw there were no liver tumors whatsoever in the control rats from three month studies that involved 662 males and 677 females. That's by all routes and vehicles. And so these are, this is truly a rare occurrence here that we're looking at. With regard to the female rat liver, the pattern of nonneoplastic lesions was similar to the males. But it lacked 9 neoplastic response. And we had the [ Indiscernible ] and [ Indiscernible ] at all levels. And [ Indiscernible ] at the higher levels. [ Indiscernible ], [ Indiscernible ] occurred and we have this smattering of foecy. The denominator is ten animals in each group. Um, we did obe gland lar stomach effects in male and female rats. After one the [ Indiscernible ] were both increased in the high dose males and females. Gas trick glanl atrophy occurred at the two highest doses in males and females. In the three month exposed animals we also saw the gas trick gland atrophy down to one 50-milligram per kilogram level. These are slides that show what the atrophy looked like. So there was a group of lesions that involved reproductive and endocrine affects in the male rats. The test ease had decreased weights and had [ Indiscernible ] of the gem nail epithelium. These are in the two highest groups. And this collection of lesions indicates some potential there's reproductive toxicity of Estagole in male rats. Although that study has not been performed yet. Other nonneoplastic lesions that were occurred include [ Indiscernible ] of bone marrow, at the kidney there was weight increases, and renal tubal lesions at the higher dose levels, there was also Sal vair gland and [ Indiscernible ] of the two higher dose groups of males and females.
Turning to the results. All of the female mice died during the first week of the exposure. Whereas the survival of the males, there was no problem. There were dose related decreases in body weights, down to 79% in the males. And down to 82% in the survival females. Organ weights, the only significant change was the relative liver weight, which was increased from the 75-milligram per kilogram level up in males and females. So from this it appears that the females might have been more sensitive to the toxicity than the male mice. With regard to the nonneoplastic lesions, at the libber we have the [ Indiscernible ] again. Along with degeneration at the two higher dose groups in the males. The high dose females that died early all exhibited necrosis.
Other lesions in mice included at the glad lar stomach [ Indiscernible ] and at the forestomach [ Indiscernible ]. In the highest dose groups there was olfactory epithelial degeneration in the nose. And the two highest groups of males and females. So to summarize this, from the literature we know there's ample evidence that Estagole is [ Indiscernible ] in mice. But this is the first evidence that it is also carns know Jenic in male rats. The toxic efforts of Estagole and methyl [ Indiscernible ] turned out they were similar. I have a slide at the end of the Isoeugenol talk that compares across the chemicals and columnlike fashion. Estagole and methyl [ Indiscernible ] are very much the same in their toxicity affects. The [ Indiscernible ] for male mice was [ Indiscernible ]. But it could not be determined for the other sex species in the experiment. Our conclusions from the three month study are that Estagole is [ Indiscernible ] to male F-344/N rats based on the occurs of [ Indiscernible ] carsinomas and he pattic cellular adenoma. Because they were exposed for three months these do not expose the full potential of Estagole. The combined evidence from this studdie and other previous studies make it highly likely that Estagole would be carcinogenic to additional sites in rats and mice after longer exposures. So that's, concludes my presentation. I would like to ask Dr. Malarkey to come up and show you slides about the his toe pathology that was seen for this study.
Thanks, Doug. Do you want to sit?
I'll wait.
Tell me if he does anything behind my head. Thank you. I would like to share with you the features of [ Indiscernible ] and [ Indiscernible ] in this study, the Estagole toxicity study. Of course this was a lesion in the rats. What is [ Indiscernible ]? This is an example here. There's a proliferation of bile duct. There's inflammatory cell reaction. You can see the blue dots, they represent inflammatory cells. Now this epithelial is atypical sometimes because it's piles up and you will see crescent shaped areas. These are usually small in portal areas. That's how they begin. They will progress and expand in the liver. They're [ Indiscernible ] to [ Indiscernible ]. Sometimes there's dilation with mucous accumulation in the center of these ducts. They can have gob let cells. This is from another study to demonstrate the gob let cell. It was not prominent in the Estagole stud Y. this is probably the source of the [ Indiscernible ] in the middle. You can see the duct here is partially lined. Over here there's a partial lining. It looks like a rupture might be going on here. Here's inflammation percolating through that collagen matrix. This is an example of a mild case from the Estagole study. You can see over here in this portal area, you can see the fibrosis of the imflamtory cells. Notice here the nod ewe lar appearance to the liver. This nod ewe lar appearance is due to hypertrophy. This is another case of a similar looking lesion. This nod ewe lar here. I wanted to show you what these bridge be areas look like, that's shown here, depicted here. These are he patio cites. Those are the oval cells of the liver. You've probably heard of them. Now you can see them. If you look closely you can see some, there's a formation of billary hyperplasia. It may be the stem cell in the liver. Um, that's, that's another liver lesion to show you that may be related to the [ Indiscernible ]. [ Indiscernible ], a common lesion usually found in the rat with treatment. It's rare in the mouse. But it has been reported in the mouse. It's not reported in 90 day studies, whether it's control animals or treated. We don't really see it. These were two year studies, some were reported at early as six months to begin to show [ Indiscernible ]. It's believed to be a [ Indiscernible ] lesion. It progresses on to [ Indiscernible ] carsinoma. In all of these studies there was an increased incidence of [ Indiscernible ]. What do the [ Indiscernible ] carsinomas look like? They were large masses am they had floored growth. The third bullet here, there's infrequent me tas 'tis has been demonstrated. [ Indiscernible ] had a low metastatic rate reported. But it appears to be infrequent. Here's [ Indiscernible ], this is one of the two cases in the Estagole study. You can see the flower arrangement. That's where the word florid comes from. If you look at higher magnification it's this atypical structure separated by a collagen matrix and [ Indiscernible ]. It's invading and replacing the liver. These are larger masses. This is the other case. The entire liver lobe has been pretty much replaced by the infiltrating and expanding [ Indiscernible ] carsinoma. At a higher magnification in that second case, again you see the atypical [ Indiscernible ] epithelium. There's invasion and replacement of the [ Indiscernible ], which is up here. That brings us to think about the origins of the [ Indiscernible ] in the Estagole study. Perhaps this is true in other studies as well. There's some evidence because of the histologic progression to the [ Indiscernible ] course know ma -- carsinoma. It may be arising from a stem cell, oval cell or billary cell. There may be progression right from this billary proliferation right to the [ Indiscernible ] carsinoma. So that's it for [ Indiscernible ] and [ Indiscernible ] carsinoma. Doug, you left.
Okay. Thank you. Any questions from the committee for -- yes, Dr. Howard.
Did you see any evidence of increased necrosis or apoptosis?
In the glad ewe lar lesions?
In the nonknee owe plastic?
In there was degeneration in the mouse that probably was involving ep toe sis. Or necrosis. Is that what you mean? There was in the mouse. Not as much in the rat.
Okay. We'll go to our first reviewer. Dr. Pino.
All right, Mike Pino. First of all as scientific criticisms, I don't really have any. Moving on to the results and information to be added or further discussed. Start with the clin path. [ Speaker Faint/Audio Unclear ]. I was just wondering, the finding was discounted even though it seems there could be a good explanation for why the white cells were elevated [ Speaker Faint/Audio Unclear ]. I guess before dismissing the white cell counts I would like to see the evidence for discounting that. And why it was discounted. The blood smear information. In discussion of the organ weights, it needs to be separated which effects were correctly related to the compound and what were secondary effects. The changes in the kidney, [ Indiscernible ], heart and lung was secondary. I think they should be split apart and discussed. Um, in discussing the histopath results only the statically significant findings were discussed. Whereas at the lower doses there were clearly evidence of an effect going on. I think that should be mentioned. For example, the [ Indiscernible ] foecy in the rat, in the female rats at 75-milligram had an incidence of 3 out of 10. None in of the controls. To me in my mind that's compound related and should be mentioned. The same goes for some of the other findings. In the kidney and the bone marrow in the rat and the -- deJen raitive [ Indiscernible ] in the mouse. Um, the renal pigment, or the pigment in the kidney [ Indiscernible ] cells. What pigment was that? Could that have been related what was going on in the hematology data? In the mouse study, I have the same comment about the organ weight affects. The liver should be clearly identified. In the discussion, I think when discussing the liver affects at the [ Indiscernible ] inflammation and [ Indiscernible ] infiltrates in the liver it should be brought up they weren't seen with [ Indiscernible ]. I don't know if could be involved in the patho genesis or not. In the mettle [ Indiscernible ] study they did report [ Indiscernible ], which in the report says it was similar to [ Indiscernible ]. Maybe that should also be brought up in the discussion. There's similarity to the methyl [ Indiscernible ] study. With a conclusion, I do agree. Since this wasn't really a carcinogen sis study. You did see tumors, it was designed as a toxicity study. You need to say what the target organs were as part of the conclusion. So that's all.
Okay, thanks. Dr. Crump?
Kenny Crump. I don't have any comments on the conduct of the study. Seemed like it was well conducted. This is another one of those special cases that we're not quite used to dealing with. I did have some concerns, I guess, about the conclusions. I think the flat statement that Estagole is carcinogenic to male rats, we don't use that kind of terminology with a two year bioassay, I think it would be understood to be clear evidence. I think in a two year bioassay the evidence we have [ Speaker Faint/Audio Unclear ] it wouldn't be rated as clear evidence. It may be even equivocal. I would feel for comfortable if we had qualifiers in that statement. Saying it occurred in three animals in the high dose. It would give better understanding of what actually, what the evidence was in the study. And, um, I guess I don't agree with the last statement in the conclusions, either. It was, it was sort of speculation of what would happen if you did a longer study. I feel like fill solveically, it's okay to speculate, I'm not sure it's appropriate to include that kind of information in just a very short summary of what happened in this study. That's not what happened in this study, what you can conclude from this study. And another reason is I think when people read that they will well, this is what happens in a two year bioassay. We would see a lot more lesions, which may be the case. I think you also have to think about the fact, I don't think yeve even seen these in the two year bioassay, I think we've exceeded the NTD. You had 25% lower rate in the high dose animals, I think would not have used that dose. You wouldn't have even seen these lesions. So, I would feel more comfortable if we didn't have that kind of discussion in the short summary of the report. That's all, thank you.
Okay, thank you, Dr. Bradfield.
Profiling, here we've done [ Indiscernible ] on [ Indiscernible ]. I would have found it to have a global transcriptional profile, rather than a more old-fashioned approach. I come away confused how it fits into the greater picture of the flavoring agents. I wasn't sure what exactly was gained from this study. I wasn't sure why this experiment was done, other than a vague comment how this would fit into the -- exact wording, better characterize the toxicity. It all seems vague to me. It would be helpful how this information is going to be used in this larger picture, in these reports.
Is that it?
Yeah, sorry.
Dr. Peckham.
All right.
First I want to say thank you for the review information. We appreciate the thoroughness and insights that go into providing those kinds of comments. So first, with Dr. Pino's comments -- starting with the three month rat study the comment about page 25, um, adding the target tissues that were examined microscopically in all groups, we'll do that. With regard to the increased Luke sight counts, on page 35, in the original evaluation that Dr. [ Indiscernible ] did for the study, he noticed some unusual effects like that and went ahead and performed counsels analysis on a subset of the blood smears. That turned out to be informative. He's here to share the results of his counts with you. And also give a rationale for why the elevated Luke sight counts are considered to be [ Indiscernible ] in this study, Greg?
Hello. So there's always a short answer and a long one. I tend to always give the long. I'll try to be as briefs a possible.
So we did notice the apparent increase in Luke sight counts. And in conjunction with the fact there was a liver effect with an inflammation in the liver it would have suggested that the Luke sight counts were related to the information. We did discount them, or I discounted them, more pointedly, based on -- based on how the data looked. We see, this was noticed in the rats and in the mice. But more prominently in the rats. I'm showing you the male rats here. This is a substantial increase here. We don't normally see that. We look and see the increases here. We don't know how these are derived. This is instrument derived data. The blood goes through the instrument. The red cell count is counted first. Then a licing agent leaves the white cells to be counted by an [ Indiscernible ] method. The instrument used in this case was a dual. It counted by impedance for most of the variables but it also had an optical reading for the white cell counts. We don't use the [ Indiscernible ] for the instruments. We read a 100 cell dif. You end up with the counts for the different cell lines. Interestingly enough, the percentage, or the percentage of the Newt fills and lymphocytes were the same regardless of dose. If this is related to inflammation you would expect this to be higher. That raised the flag. Was this a real affect regarding the increased white counts? That's a pretty substantial increase. There are chemicals that are compounds that cause this. For example, an ep enough rin response. That's usually a short duration response, usually acute. This is happening at study termination, didn't necessarily make sense. Not to say there couldn't be a compound that could cause it by changing distribution within the circulation. So this stimulated the estimated counts. Now this method is semiquantitative at best. But should have been tell qualitative the different between a 6000 and 15,000 white count. I did the male rats and then the male mice, just to see if I could demonstrate this really high white count. It doesn't hold up. You could argue maybe Tallahassee increase in the rats, these are estimated counts. There's a lot of variability. The same thing was found in the mice. That leads to the question, why do we have this over doubling in the white count in the high dose rats, male and female?
Okay we have the documented [ Indiscernible ] anemia in this study. Caused a very markedly change of the profile of the blood. It's known, I have to lice the white cells. If the red cell membrane is changed so it's more resistent to the licing agent some of the cells will be left in the count to be counted. This instrument and other instruments have been shown if there's resistance to the licing agents you can have them counted as particles in the white cell channel. We have moverlogical changes. Maybe it's the microsights that are more resistent. It wouldn't take many. Red cells are in millions and the white cells are in thousands. So it wouldn't take many red to be resistent to cause the increase this white cells. Well, that was my speculation based on those, on those three points. Any questions?
I have a question on the slide before that. What is a target cell?
It looks like a target. Target cell, target cell, target cell. [ Laughter ]
Is that your name for it?
No, that's an accepted name. It's related to distribution of hemeo glow bin in the cell.
When you did your white cell counts by hand did you see red cells?
To review the method, I took the blood smears and I just did the control and high dose, that was the most dramatic. Looked at eweny layer fields of the [ Indiscernible ] on high power. This is high dry power, which is a 40X field. Did ten random fields and I counted the number of white per field. Took an average. There was an algorithim. Basically did it that way.
Okay. Any other questions? Okay, thank you.
Thanks.
So then we'll go back to Dr. Bristol.
Thank you. Proceeding on. Dr. Pino made the comment about the, when we discussed the organ weights to distinguish clearly which are treatment-related and which are not. And we will do this in our report. For each sex species. And a similar general comment, there were several lesions where we had not commented on the extent to which the lesion occurred in lower doses, where they were not statically significant. We will review all of the lesions and for biological significance to support a statement like that, and where appropriate it will be included. With regard to special stains done, the renal pigment in the males Dr. Malarkey and Dr. Peckham have arranged to do that, that will be done. In the mouse study, some of the general comments are already taken care of. And then, um -- let's see, in the discussion, yes, we will mention the [ Indiscernible ] infiltrates in rats. I noticed you just commented about the billary [ Indiscernible ] and the methyl [ Indiscernible ]. I recall reading that session, I agree. We'll review that and try to incorporate that, that's a good point. Also, your comment about the conclusions. We can incorporate that into the conclusion statement. And I think that Dr. [ Indiscernible ] addressed your comment about the [ Indiscernible ] counts as well. Now for Dr. Crump's comments, with regard to our flat statement that Estagole is carcinogenic in the conclusion statement, we feel that's consistent with statements made for studies that had similar indications of carcinogenic effect in other toxicity reports. Um, specially where we do not plan to carry out followon with a full blown carcinogenicity study. The liver tumors in the male rat, we feel are, they are the result of Estagole exposure. Based on the striking similarity with the methle [ Indiscernible ] study, that's why we projected in the two year study we would see those results. With regard to the dosage used in the [ Indiscernible ], they were appropriate for the subchronic study. The 600-milligram per kilogram dose would not be used in the chronic study. One of the lower dose levels would be used. In the, at the 600 level, after three months there were 3 out of 10, the denominator was 10 for that group. That's very unusual and striking evidence. In the methyl [ Indiscernible ] study the highest dose was a 1000-milligrams. We have a similar situation. The high dose in methyl diswould not be appropriate for the chronic. And the lower doses in the chronic produced the full spectrum of [ Indiscernible ] in male and female mice. That is expected for Estagole as well. Um, one other point about our conclusion, about Estagole being carcinogenic in male rats is that regulatory groups, for example, California EPA in 1999, have said that Estagole is a carcinogen. Um, now for Dr. Bradfield's comments, the comment about global transcriptional profiles being generated with each study like this is interesting. And, um, the NTP does have a [ Indiscernible ] project in place. And it looks, in fact there's a project that looks at these [ Indiscernible ] compounds in particular with the goal of identifying patterns that can be used to correlate the liver carcinogen sis affects. This afternoon there's a seminar at 2:30 down in F193 that deals with that general topic. If you are interested, I brought along the announcement with me. I can show that to you. We have that as an ongoing study. It's just one indication of where this goes. With regard to the information that should be added, I alluded to the slide at the end of my Isoeugenol presentation that will summarize the similarities and differences. In a follow-up journal article we would expand on that to bring in more information from the literature so that's an indication of where this information will go besides the technical report here. The intoxicate cogenomics study on this group of compounds, I think, goes in the direction that you are projecting. That's it. I think.
Any more discussion from the committee?
Jon Mirsalis. Two comments. One is echoing what Kenny Crump would say. I would strongly object to the statement we will probably get a different [ Indiscernible ]. There are drugs that produce tumors fairly early. You take it out two years and you see the same ones. I don't think we should be trying to second guess what we might get. The other comment, you reported the gene tox results. About two decades NTP cut the list down to [ Indiscernible ]. For those two tests are poor at predicting liver cars know Jens. A long list are missed in those two assays. I would like to see a inveeo liver. Something, invivo liver is the way to pick up these compounds. In the absence of that data you have to make the statement that you haven't really assessed the [ Indiscernible ]. I think these are probably good, very nice Gino toxic carcinogens, based on their [ Indiscernible ].
Thank you. Anytime discussion? Dr. Howard?
I agree with the comment from Kenny and from Jon. If we look at the studies done in the past there's essentially one study by gavage. It was by the millers in the 80s. Would probably be considered inadequate for risk assessment. The rest of the studies in your report are subcutaneous or [ Indiscernible ] injection. The thing I'm concerned about is, is where do we go from here? I gather there are no plans to go forward on a two year bioassay study. Two out of 10 animals having tumors, or 3 out of 10. The hallmark of the NTP is a rigorous number of animals. And if ten animals per dose group were adequate for risk assessmentals we would have been doing that for the past couple of decades. I think this is an extremely adequate study for doing dose response and for setting up a two year study. I'm not sure it's adequate for risk assessment. If you look at the tumors, the carsinomas and adenomas as a measure of where the dose response lies, then you are somewhere between [ Indiscernible ] and [ Indiscernible ]. If you look, however, at what you are proposing then your left with no dose response. In the lowest dose you had a response. So you leave, you leave yourself juxtaposed between two conclusions. I question why a two year study was not proposed to move forward. The doses will be somewhere at 37-milligram per kilogram or lower. Which will drop your no observed level level orders of magnitude, perhaps.
Thank you. I wonder if you want to respond.
Sure. We've studied a number of these compounds. It was based on the similarity and the metabolism of this compound. And similar purported mechanism of [ Indiscernible ] and also seeing three tumors out of ten animals in the three month study at an early point. Other groups have also determined based on the literature and miller studies that it is considered to be a liver carcinogen in mice. This study now demonstrates it appears to be in male rats.
I want to respond to that one point. I agree with you as far as hazard identification. My issue is not with that. It's where the dose response lies.
Paul, I believe that Doug stated this was an NIEHS nomination's part of this class study. I would certainly would want to entertain from FDA if there's a serious request for dose response. You have to remember that Doug mentioned the genomic studies with this particular class of compounds. What we're trying to do as a program is move beyond doing a two year study on all compounds. When we have responses like this in three months. With we see this three months, we generally, would like to avoid what we think is sort of a redundant study and taking it forward with a 50 animal, two year bioassay.
Yes?
Paul Howard. I totally appreciate that, John. I understand why spend another several million dollars. I did not get a response from our risk assessment group. I will bring that back up to them. See if we will move forward. I want to encourage. I'm not criticizing the study. I'm looking at the adequacy of where we go from here. Since the pathway is most likely what you are talking about, then biomarkers might, I think biomarkers of exposure might be a more appropriate, or equally appropriate way of going, as well.
Dr. Crump?
Kenny Crump. Want to add to our comment, or what Paul has said. Often we may be, we overestimate the information that risk assessors use from these studies to come up with numbers and risk assessments. Basically it's some rough indication of the doses at which you saw some tumors. This chemical is known to be carcinogenic in mice. I'm not familiar with those studies. I'm guessing there's enough there for regulators to do what they need to do. I certainly wouldn't take the evidence that we have here as a necessity to go on and do a two year bioassay in rats. I feel just given that it's carcinogenic in mice and the information that you have that can make that call, that is what a regulator would use. I would basically doubt if anything you got out of a two year bioassay would change the quantitative results. Very crude approach that is being used.
Any other discussion?
I guess, Jon Mirsalis. Maybe a compromise position would to be propose rat carcinogenicity studies. FDA largely ignores now the presence of mouse live ever tumors as evidence by the very, very large number of mouse liver carcinogens. Rat tumors it makes you take note more. A compromise position, there's cost and animal use here. I agree with John's position. Where do you draw the line? A compromise might be one rat two year study to find an appropriate lower dose. Paul is right you might find a know eel that [ Indiscernible ]. If you did that.
Um, just to comment a little bit on the mouse liver tumor issue. I think that you are correct with regard to the two year mouse liver responds we see in many, many studies. I'm not sure that the protocol that is used by the millers is one that could be thought of generating the typical mouse liver tumors.
And just to at to that. In the methle [ Indiscernible ] we saw other target organs. There's a similar mechanism that we're seeing here in this 90 day study of Estagole. I wouldn't want to eliminate the mice if it would provide other information, about additional target tissues.
I would like to ask a question about structure activity relationships and how much data do you have on that for shorter term end points and you could make good predictions for long-term?
I've been in the literature in detail. I come away from that with some skepticism. It might be best illustrated by talking about methle [ Indiscernible ], [ Indiscernible ] and Isoeugenol. Those three, are similar in structure, are different with respect to the carcinogenic response that we see in a full bioassay. The [ Indiscernible ] study and studies might help us with that regard. But, um, it gets complicated, like satisfy roll has a [ Indiscernible ] group on the [ Indiscernible ] and the related compound says mol which has the [ Indiscernible ] of [ Indiscernible ], gives [ Indiscernible ] tumors which are rare. Is somehow a related effect or not? There are similar patterns for metabolism, but with Isoeugenol there was a study done using the uniformly ring labeled compound and using HPLC and radio chemical detection they identified at least 40 different peaks, or me tab lights that appeared in the urine. That's complicated. These studies that are done all produce information, I find it hard to put it together in a comprehensive way that I would be able to use it with confidence to make predictions. So, I like to rely more on the actual whole animal studies.
Okay, thank you. Any more comments? Any comments from the audience? Yes?
Is this on? Yes? No?
Yes.
Ron Lawrenceson from the center for food safety at FDA. I hardly know where to begin. Are we being taped? [ Laughter ] I'm here as a scientist to judge the veer asty of the performance and the interpretation of these studies and so are. If any of you want to, after you leave this room, join some vigil anti-group to remove all of the spices and herbs that we use every day, you are welcome to do that. I think that's still a civil right. But personally I wouldn't recommend it. So what do these studies give us? What they give us is some reflection of the carcinogenic load for humans, okay? That is attained by every day activity, namely, the way we eat. I would personally love to separate out all of these flavorrants, I'm not sure how you would do it and do a risk assessment to get some idea of what this is. This is not a trivial task. If I didn't have anything else to do I might take it up on the weekends, but I do have other things to do. But I certainly can't use a study like this Estagole study, which seems to be, even though the doses are fairly high, I believe that everybody is right, we would expect that a few ors of magnitude lower we would see results. And you know, these results are also [ Indiscernible ] by profound toxicities that make it very difficult to interpret what this means from a risk point of view. So while we didn't request these studies, this is a local nomination, um, the value in the studies of all of these safrol compounds [ Speaker Faint/Audio Unclear ]. I'll try to answer any other questions. We're here to assess the voir'sty of the performance of the studies. And the interpretations that are made here.
Thank you. I'm a little confused as to your point.
The scientific truth of the study. You are here to assess the science.
Of this study though?
All of them. Not the risk management. The risk management is what we do to deal with this issue. That is not issue. It's not my issue. Even though I give advice on it. You may feel strongly about it. Okay? In this room that's not issue. Outside this room it is your issue.
Okay. Thank you. Any other comments? Discussion? If not, display the conclusions.
Okay, um, any discussion about the, um, I guess maybe I should say is there a motion on the conclusions?
I guess I, Mike Pino, I would agree with Dr. Crump. Strike the last sentence. About speculation. I would also add wording maybe similar to page 55, about the target organs, what organs those were seen in.
Dr. Crump?
Kenny Crump. I would support removing the third sentence. I would also like to see the first sentence be a little bit more specific. I would suggest wording, Estagole showed carcinogenic activity to male rats based on the occurrence of two [ Indiscernible ] carsinomas and one [ Indiscernible ] in the liver of three male rats. And I would also suggest that the second sentence be modified, not only to show it was three months, but it was at doses higher than a normal two year bioassay. Because rats and mice were exposed only three months and at higher doses than a full life bioassay.
Yeah, I would add that you might want to say 3 out of 10. And Dr. Howard?
I agree with the suggestions. I don't think we want to say these are doses are higher than what would be used in [ Indiscernible ], because some have used 50,000 parts per million. These are higher doses since there was an occurrence of [ Indiscernible ] in all dose groups. This is really, I think, the point is these are high doses because of the [ Indiscernible ] that was noted. Not just because they are high doses, knew mayorically.
I didn't mean high doses, I meant high relative to the toxicity that you see here.
Dr. [ Indiscernible ]?
I wanted to clarify the intention of the program with regard to the third sentence. That basically is we didn't want to, by the virtue of the response in the liver only we didn't want to give the impression this is the only organ that could respond. Based on the overall similarity with the responses to methyl [ Indiscernible ]. That was our intention. If that didn't come across, then we need to -- fix that.
Mike Pino. The second sentence seems to cover that though.
Yeah, I think so.
Dr. Howard?
I agree with that, Michael. John, the problem I have with that is for this particular compound the biomarkers of exposure were not measured. We don't know dose response there. The carcinogenicity was only in mice in other studies. I understand structure-related exons -- compounds being brought into this. It's not showing a common intermediate. Unless that's really brought into the discussion, a really good multiple compound comparison where a reader can see where you are drawing a conclusion, I'm not comfortable with that last sentence.
I was referring to the issues related to the gas trick PH and other measurements we did in this study that were related directly to the methyl [ Indiscernible ].
Jon Mirsalis. This makes it sound like you, it's similar to the other analogs, when in fact your studies show it as negative. I don't believe that's the right answer. There's so much confusion about what we know and don't know. Comparing studies that are done by totally different mechanisms. I just, I think the whole, I think Mike is correct. The second sentence addresses it. Period. I don't think we, this group should start to predict what we might see based on other studies done a long ago by different methods. And try to determine what this is going to do. I think the last sentence really needs to go.
So is there anybody that would like to wordsmith it?
[ Speaker Faint/Audio Unclear ].
This might be a good point for me to mention, with regard to the [ Indiscernible ] toxicity of Estagole, we did the design in the study was our standard design for this three month study. And the DNA, in vivo studies have not been done for Estagole, as far as I'm aware. If they were done I think most people would feel that would bolster the analogy. It's easy to accept that kind of correlation for the [ Indiscernible ]. We need to beef that up in our discussion, like one of the comments suggested earlier.
I would add this statement is perfectly okay to put in the discussion section of the report. I see no problem with putting it there.
Okay. Did you want to -- Kenny, did you want to change the first sentence?
My recommendation, Estagole showed carcinogenic activity in male rats based on the occurrence of two [ Indiscernible ] sar comas and one [ Indiscernible ] carsinoma. And three rats, three male rats, in the liver of three male rats, um, in the high dose group.
Okay, Paul.
Paul Howard. Do you want to put a denominator on that?
I meant to say 3 out of 10. 3 out of 10 male rats in the high dose group.
Okay. Um, so do we really need to change the first few words? Just insert the number of animals. Or do you not like the phrase?
I don't like that very much. I think it carries a lot of bag and with it. I would prefer something like showed carcinogenic activity.
Okay. The rest of the committee good with that? Okay.
Okay.
Yes.
[ Speaker Faint/Audio Unclear ].
Okay. If the committee is happy we could take a 15 minute break while they wordsmith it. We'll come back and approve the conclusions. That should save a little time. How about we take a 15 minute break? Your chair needs it. [ Laughter ]
Session on break until 10:15 eastern.
If we could come back to order.
If you would like to read the revised conclusions and offer any comments.
Jon, Mirsalis, I move we we accept these.
A second?
Keith Soper.
Any discussion?
I would like to ask for clarification about the definition here of the NTD. The NTD in this three month study, or as is would relate to a two year. In my mine they don't [ Indiscernible ] based on duration of the study. So I'm just wondering what NTD, what you were trying to represent here.
Um, I was trying, thinking it was trying to represent the fact that the doses are higher than a typical use in a normal two year bioassay. NTD is what people think of when they think of the NTD, so a -- I feel there needs to be something that indicates the doses were higher. If there's better language, we could go to it.
Tracy Bunton. I think what now, as it's written it's a confusing. A couple of ideas. What is highlighted now belongs in the first sentence. Or at least as I understand what the two concepts are. And the original statement that was the second sentence, or whatever will remain in tact. Now it confuses, a little bit, what people are trying to say.
This is Mike Pino. If you put in the liver of F-344/N male rats at a dose that exceeded the [ Indiscernible ] instead of high dose group. Leave the rest of that second sentence the same. Would that take care of it?
I would take issue with the concept that we've exceeded the NTD. [ Speaker Faint/Audio Unclear ] where the accepted practice for selection of doses has taken doses that would be double that, one would pick for a two year study because of the trunk Kateed nature of the study. And the fact that the NTD was originally put forth so you could maintain a sufficient number of numbers to the end of the study to attain statical power. In this case we're talking about a three month exposure. None of those things make sense in a traditional way. It's a confusing, the issue to introduce that concept into this report. This is a statement of what happened in this three month study.
Paul Howard. I agree with that. The reader will go to the text and will see what doses were used.
Point of order. Do we need to withdraw the motion?
No, this is a discussion.
All right.
Nancy?
Excuse me. The motion -- was to accept it as written.
Jon Mirsalis, I'll make it easy, I'll withdraw the motion.
I would like to comment.
Dr. Bristol.
The data that we're reporting and the concept of NTD in the female mice there were deaths. You might say that, you know, that exceeded some NTD. But it is hard to apply the concept to this. In the male rats, were they sick? Was it effecting the power of the test? Those are the considerations we apply to assessing whether or not NTD was reached or exceeded in a chronic study. We're not talking about those here. In we did, the male rats, I don't think exceeded an NTD at this three month exposure duration.
Thank you. If we remove that phrase. Then I would entertain a motion to accept the conclusions.
I , I, maybe I'm arguing against the forces here that are going the other direction. The second sentence says exposed only three months. When people read that people will wonder what we get in a two year bioassay. It's legitimate to say this is study. And it used higher doses. I think those things are, are --
Jon Mirsalis. Because rats and mice were exposed for only three months and using doses higher than would be expected to be used in a two year study. Is that what you are trying to get at?
You're talking two different sides of the question. That doesn't make sense. If you want to qualify intoxicatist or whatever the problem is, do it in the first sentence about where you saw, or at what dose you saw the tumors. Then you say they were exposed for three months, you don't know what would happen at low doses -- or at any dose. You don't Novak what would happen.
I'm okay with it as is. The first sentence is under the conditions of these three month studies. That has a lot wrapped into it. I'm trying to address Kenny's concerns. At some point you have to read the report to see what was done, so --
This is Mike Pino. I agree, based on that first phrase. I make a motion that we accept the conclusion as as.
Jon Mirsalis. I second.
They don't have bone marrow. Would you considered aking those in there? -- adding those in there?
Short answer is yes.
[ Speaker Faint/Audio Unclear ].
Would you mine giving us the latitude to put the correct nonneoplastic effects in the second paragraph and just give that to us? [ Laughter ]
Yes.
That's part of the motion, to accept this revised conclusion.
Okay, I make the motion that we accept it, with the caveat that the nonneoplastic lesions will be added.
Jon Mirsalis, I second.
Okay. All those in favor? Of accepting the conclusion. Looks unanimous to me. Thank you.
All right, let's move on to Isoeugenol. Dr. Bristol.
While I'm getting ready, one thing I thought of that I didn't mention earlier is that nyingle walker is, he heads up our project. He's here at the meetings too. If someone wants to ask questions about that project, he'd be happy to answer. Turning now to Isoeugenol. This has widespread natural occurrence. Um, and the essential oils from plants and seeds and particularly [ Indiscernible ] provides Isoeugenol extracts that are used in the world's most expensive perfume, joy. Another source is nutmeg. And back in the 16th century European nations went to war trying to control the source and supply of nutmeg. So, it's had an important effect on history. It's used as a food additive. It enhances the sweet spice and clove flavor in foods and in gums, beverages and so on. It's a fragrance in many personal care products, like detergents used in the house. More recently it's been used in aqua culture to sedate fish in the rearing and harvesting of them. That's a growing food source. The FDA has given temporary approval for that source. With regard to isn't sis -- it's catalyzed by heating with either disor [ Indiscernible ] compounds. It produces the double bond shifts and the congregation shifts from being no congregation there to being congregated. This is more [ Indiscernible ] stable. You have to heat it up with some catalysts and bingo you are there. I've shown what we consider to be the parent compound for the aloe. The [ Indiscernible ] on the air mattic ring seem to have less of an effect on the activity of these lower molecular weight [ Indiscernible ] compounds than on the ones that as you add, um, you know [ Indiscernible ] or [ Indiscernible ] groups to the ring the effects, some of the analogies drop off. But now we're talking about Isoeugenol, which has that rop
Based on -- the general lack of information about 2. It's surprising how, seems like it's been overlooked, by and large by people who have done studies back in the 80s or 60s.
So the studies that NTP did include the three month toxicity studies, along with the kind used for -- andestrigol. The double -- how it's used, and how it occurs in nature. We use dose levels up to 600-milligrams per kilogram, based on information frommest eugenol and -- the special toxicity studies were done in rats with one-mont excoz month exposure. We did the two year toxicity and car sin cars nighsity.
The E. Coli assays and -- were negative without the usual vaikz, didn't use special activation as is sometimes done, can produce positive results, but we don't know, done didn't do do it here, not in the literature.
The negative result in males, positive result in females.
With regard to -- dropped to about 90% of controls. The weight of the liver increased about 14% in the two highest groups of females. The clinical pathology was in general unremarkable. The only histo olg in male and female -- the top three doses and female's liver, cyto plaza mick -- how the special toxicity studies turned out. There was a small dose-related decrease, not increase, decrease in the stomach PH at 150-milligrams or higher, females only. The magnitude of decrease. The serum gas trine levels were unchanged in male and females, no stomach lesions. The P 450 levels were slight decrease and activity of the CYP 182 -- bodies of males, females, relative to controls decreased in the high-dose group down to about 90%, liver weight increased in the end males, two top doses by 14 and 22%. Clinical pathology again was unremarkable. Histopath owe logic lesions only in the nose this case. males, females, minimal to mild severity. Minimal to mild in [indiscernible].
So, here are the doses selected for the 2-year studies, same doses for rats and mice, up to 300-milligrams per kilogram. These were based on the changes in body weight. At the high dose group in the mice, and the male rats, and there was concern that because ole faction is important in feeding behavior the higher dose might effect the body weight gain and the general health of the animals during the 2-year study, so they got below that effect or at least to where we thought it would not be a significant effect in effect in the study. No effect on survival, and in body weight, in fact the exposed animals had body weights that were approximately equal to E even greater than the controls, especially in the male rats here. At the end of the study they were about 10% greater than the controls, and the females, you can see not much effect at all at the 300 per -- kilogram level.
The male rats, after two years there were thigh mommas, [indiscernible], mammary gland carcinoma -- both of these lesions are rare in occurrence compared to historical database. It's small for the studies, not observed, all routes of exposure, for the thyme thy momma -- and the mammary, seen three times out of 1100, those are very e very low rates. And the assay -- rates for the trend.
Turning now to the mice, the survival rates in the males, high-dose males versus control, the decreases there were significant, and the trend with those was also significant. We have a survival effect that appears to be treatment related in the male mice. There was no effect on the survival of the females. With regard to the weight decreases in the males, they decreased to about 90% of the controls. In the females to about 85% of the controls. By the usual estimates of NTD, those did not exceed NTD. The neo plastic and nonneo plastic lesions in together liver for the male mice -- all that was there was hep at cellular add noma, carcinoma or combined Aden only a or cars carcinoma, all significant, and also the trend was significant. The only other nonneo plastic results for these neoplasias for Aden only a, ma, using the -- the rate is 50% using routes, and 48%, up to as high as 84% 84. For carcinoma all routes, the rates up to 38%.
With regard to the combined incidence of adden only a or carcinoma significant events for both, trend and also individual exposed groups versus controls.
For the female mice, histio sittic car commas, here in the controls, the asterisk indicates the occurrence in the other groups, 1, 1, and 4, statistically tistically significant trend.
It's not been ob served in the other Cornell study, good sample there, the rate historically is 2.5. So this is another rare tumor.
The conclusions then, we reached, for the results of the 2-year studies in the roots, evidence of carcinogenic activity in male rats based on increased incidences of the rarely occurring did I momma and mammary gland carcinoma. No evidence of carcinogenic activity in the female rats at any dose. And the lesions in the nose, male and female rats were increased.
In the mice, excuse me -- carcinogenic. Clear evidence of increased carcinogenic activity based on the -- or combined and -- carcinoma. Any equivocal evidence regs based on incidences of -- and again there were, well, there were more nonneo plastic lesions in the mice, they occurred in the nose, fore stomach, glan lar stomach and -- the mice.
Summary slide was promised. Here it is. We are looking at meth eugenol, Estragole, and Isoeugenol, here we have various endpoints of toxicity, the columns show, I think, that convincingly the toxicity of meth eugenol and Estragole are very similar, and different from the toxicity observed for Isoeugenol. So, this will be incorporated into our report, paper we publish, expand expanded on, drawing more from the liter lit ra. Approximately eight different chemicals in this class. Thank you.
Does the committee have questions at this time?
Dr. Crump: Thank you for the presentation. I have a general question. In the course of your presentation you indicated the NTD was not exceeded --
How does the NTD define the maximum tolerated dose, what basis for that comment?
What comes to mind for me in response to your question, back in the mid-80s there was a committee set up to address the design of these studies. It was called in the report in general the do-all committee report published in 84, 85. In there they talk about NTD survival, body weight effects. In general if there's no effect -- survival in the body decreases to do not exceed 15%. There's a published citation, authoritative on that point.
I just wonder where the 15% figure, good to know.
That part, and general experience, where I am coming from with that, statement. Seemed to be a concern with the est -- I added it on the fly.
Kenny Crump: As far as I could study, was well-conducted. I agree with the consolutions, think that's all the comments I have at this time.
Okay, Dr. Pino?
Dr. Pino: As far as any scientific criticisms, it was acknowledged in the discussion the lack of effect at high dose in rat study suggested high dose could be tolerated. That is acknowledged. I don't recall in the dose -- data, used as part of rationale, if that's not there maybe you could add that to part of the rationale.
Then again, I had some similar comments about -- clearly identifying those considered directly compound related, also secondary data, body weight changes. Then also, as far as identifying some of the, for the histo logic findings, lower doses not significant, clearly related to the compound. On page 78, discussing the finding in the spleen about cellular depletion, due to decreased -- cells. I want to ask the pathology is if that was really hem at owe pettic cells or limp owe cites, if it was lymphocytes, I would change it to reflect that maybe.
As far as the kidney findings, there was some neo plastic -- in the kidney, I think PapO and tubular nee cro sis, I would bring those, something you don't usually see as a background finding that often. I would bring that forward to the summary and mention non-neo plastic effects for the mice, kidney effects.
Then just a comment on the statistical methods section, calculation of incidents. This was also in other reports as well, but where it says when macroscopic examation required to detect certain -- denominator consisted z of certain number of an mals in which neo cropsy per formed -- the -- routine test necessary test necessary mammary gland or -- unless I am interpreting wrong, an example that should be included there, that's all.
Doctor
Dr. Bradfield field: Nothing to add.
Dr.
Yes. Again, thank you very much for the thorough review and comments. I would like to respond for Dr. Pino, some of the general comments you made about effects of toxicity, we will address all those as indicated earlier. Thank you for those. I think with regards to your question about the distinction between cellular depletion in spleen and liesm liesm owe lymphocytes --
The word was incorrect. Should have been lymphoid. We will change that. It was a mis-- the wrong word. We will indicate it was lymphoid depletion. While I have the mic, can I address -- the protocol required tissue, that statement is not entirely accurate. We will change it. Those organ systems are [audio choppy] thank you for finding that.
Jon Mirsalis: An issue brought up yesterday, identifying positives in the micronucleus. This is a little more A egregious than yesterday. You are calling the female -- grace will smack me, but the POO 1, anybody looked at the numbers? The studies we have done in the last few days, the controls, space.five to 2.7, typical. This happened to be low, .5. The highest response was 1.6, we're classifying this size a clear positive based on a single group with 1.6 per thousand micronuclei, means you did eight mice, half had one, half had -- I accept there's an increase -- this is so -- even on the low end of normal control range, I cannot imagine how one would call this positive. I think we have to look at these things in the context of what wlab historical ranges are. You have an unusually low control, very low. That 1.6 on which we are calling this positive, the low-dose mail eugenol had 1.6 and the male is clearly negative.
I just -- I think you really have to -- significant, however well within the control range -- the same as the low dose on the male which we are calling negative, lower than the typical control. I think there's too much of a knee-jerk the P level says -- you have created a [indiscernible], in vivo vive tox Ico -- you have to put caveats on the report.
So we will look at this issue closely after hearing -- thank you.
Okay, anymore discussion? Dr. Howard? I think this is a very nice study. Enjoyed reading it. The one question, you make a good issue about the incidents of the thigh momma and mammary malig Nancy malignancy in the male rats --
I don't have an answer for that. Grace, do you --
Verification, are you asking separately or animals where you have seen both in the same --
Within the same dose.
The nature of rare occurrences makes answering that difficult, but we will search.
You do go into good length explaining how it's different than historical controls, but looking at induced might add weight to your argument.
Okay. We also had written comments on this report submitted by Dr. [indiscernible], New Zealand Limited, and Dr. Mark tp ton bee, MIT, and in Sweden. Are there any oral comments from the public? From these two individuals? Dr. Goodrich? Did you want to present --
You have -- minutes to present.
Goodrich: Thank you. I thank you for the opportunity to speak. I represent -- their U.S. agent responsible for providing regulatory consultation. A question ease New Zealand has -- they have been e been in the -- in their efforts to register the product internationally, and my charge for the United States, they have had to go through, obviously, quite a number of regulatory hurdles and in reading the report, we found something we felt was inconsistent with the types of things we are required to do in our studies. Basically, Isoeugenol, the methodology which Dr. Bristol indicated was one of the methods, the method we use, catalyzed, typically ice Jen Isoeugenol through oxidation goes through -- one of the things is -- can be effected by light, water, and by oxygen or exposure to air. It's this latter which led us to -- and the statement within the test article, used 99% pure, led us to the conclusion that's you may want to consider that particular statement.
This has to do with the methodology used, the gas cro mat graph methodology used to analyze the article. As I indicated, AQUI-S has a -- to meet regulatory requirements internationally. Although we acknowledge that the methodology the NTD used or -- used in the study is consistent with what's found in the literature, it's really inadequate to detect the polymerized compounds. These are the type of compounds we would see in a polymerized sample. In the bottom two, neither of those are detected in the methodology used in this study. Essentially what we are looking at is the NTD starts at roughly 80-degrees, ramps up over the first eight minutes, held for a total of 17. The methodology developed, modified to look for these polymerized compounds by AQUI-S New Zealand, starts at -- held for 13 minutes, over the next three ramped up, held for 14 additional minutes, total of 30-minute time continuum.
In looking at the -- fresh material by both methods, and polymerized. This is the typical for Isoeugenol in NTP method and the AQUI-S method. The scales are different because of the temperature and time differences. Even in fresh you are missing at least 9/tenths of a%. Doesn't show up because of the scale. 1% of the material that [lost audio] when we look at polymerize material with the NTP methodology you see the primary peaks of -- some mine oar weak peaks occur, but -- looking at the same material, polymerized color with the AQUI-S method, as we get out past 13 minutes, there's 6.5 of the material that's polymerized.
This is just an expanded scale with the NTP, and with the AQUI-S method showing this roughly 6 and a half percent polymerized. The conditions we normally look at is that for our product, we have about a two-year shelf life, pretty standard for a compound, the volatile nature of this particular compound. As we read the report, the understanding is a single or actually two lots were purchased back in 1997, these were stored, used throughout the course of the study, which, depending upon how much shelf life was left when first purchased, could be 7 to 9 years, exceeding normal shelf life. Light, water, the other one, keep forgetting -- air. Can cause breakdown or polymerization, when AQUI-S -- they purge with nitrogen to get rid of the air, I understand the conditions used in this one [indiscernible], trying to use a single-source for the entire study, but I would submit this is not in keeping with a typical gerks LT for a study we do for the GLP studies we conduct for regulatory studies. I appreciate your time ask my opportunity to present this.
Thank you very much.
Dr. Tone bee?
I appreciate the opportunity to make comments. I am Mark Tom by. in the regulatory work we have been carrying out I have also laid out a number of studies for this product, and we have a recent study sponsored by [indiscernible], which could be of interest for the -- we have
I can add that -- full copy of that report, but for proprietary reasons it is kept at least confidential until now. the report concentrates on the -- study, and when it comes to the conclusion also presenting quite a lot -- some information on this -- which you have already seen.
The new study, which I will present some data on is also in vivo Jen owe toxicity study. The test made according to the -- 474 study, carried out by the laboratory in Denmark, now days called, LAD, carried out in MN MRI mice.
After preliminary study it was decided -- use corn oil as vehicle, and oral dosing by -- the design of this study, it's according to the -- guidelines, also design from the -- studies, the -- studies, 2-year studies. As you see, the vehicle and the doses, eugenol, and -- body weight -- they are not significantly different from the control, and pretty similar to each other. [indiscernible], a positive control at the lower dose, which is significantly different from the control. This is, once again, polly chromatic studies, carried out according to G EP. We have a second -- exposure of 48 hours, the original was 24 hours. Again, no significant difference. The conclusion of this report is that, Isoeugenol did not show any geno toxic activity in the test in the mouse. It is a citation from the actual report. Then I have also added the studies we have already seen today in the main presentation by Dr. Bristol, that are here are two entity studies, in vitro studies, both negative, and the in vivo study which is partly negative, in male mice, the report written as positive at highest dose, 600 million PKO. But when I looked on the actual data on page E nienl 9 of the report, I found the results were not easy, so to say, for the female to interpret as the clear positive result. The difference between control male and female mice is greater, actually greater than between the -- male and female mice treated. In order to understand the results here, I think that it's necessary to have some historical control data to compare with, and positive control data to compare with. I have not found such data and tried to search because it is quite new information.
on the other hand, we have from Dr. John -- your -- you have hit on this point in the report as well.
We have got at least verbally some historical control data on the control. We think that the weight of evidence clearly suggests that Isoeugenol can be regarded as known geno toxic. Based both on the NTP studies and we have this problem with evaluation of the female data. We have to mention. But still, and of course we base this -- on study. So, we have come to the conclusion that we would like to suggest that the report which has been so far -- toxicity, not only two-year, but all the other toxicity studies, but in the final conclusions, there is not written anything about the unitoxic aspects in the study, not even on the rodents. Therefore we would like to suggest that it is added, sentence here, where it is -- there is strong evidence to support geno toxic -- new strong evidence to support geno toxic -- statistically speaking -- in the evidence of -- mice. Thank you.
Thank you very much.
Is there discussion from the board?
Are there anymore comments from the public?
There being none, we will display the conclusions and entertain a motion on acceptance.
While he's getting that up. A point of clarification for Doug. I am a little troubled by the discussion about the polymerization in the exper pirration of the material. What may not be evident to the gentleman who made the comment is how long it takes to get from a study to this report stage. My guess is it hasn't been seven to 10 years, but could you comment, Doug, on when the in-life phase ended relative to the purchase of -- is it a couple years or was it in fact many years that the material was sitting on the shelf?
Dr. Smith went over reports, so on, that are pertinent to this, and put together the timeline that answers the question. I think the -- what we really need to refer to is the information included in the appendix of this report. Some of D r. Goodwin's information came from a different study of Isoeugenol, and the information from that report. But from this report here, this technical report and records in the NTP archive for the study, the [choppy audio] took three weeks to perform the identity, and -- study, up to the present it's been stored at low temperature, minus 20-degrees in sealed containers. Also, initial analysis for puter purity of the test article -- would have detected polymer if present, and saw nothing that would have exceeded 1%. Those same analysis were reported on stored samples of this lot, recently, in connection with another study, reproduced those analyses done more than two years ago, but approximately two plus years. So we feel confident about the test article and that it was handled properly. Looking at details of records, study, I think, clears up that question.
I have a question in had terms of what is the implication of having polymers there in terms of toxicity?
There's some reports in the literature of biological activity of the DI-Isoeugenol, one more step, dehydro Isoeugenol. As model compounds for studying lignin chemistry, kind of interesting. And thal in of -- nothing is very noticeable, but they have some biological, some commercial interests are exploring.
Thank you. Anymore discussion?
Paul?
If a statement should be added to a materials, section -- page 31, 32 to reflect an analysis of polymer content was conducted and what was found, give that information to the public at least.
Is there any discussion -- for the conclusions?
There's actually nothing in the conclusions, typically you don't put -- I have no other comments.
I entertain a motion to motion to accept the conclusions?
One comment. I still suggest adding the kidney as non-neo plastic finding for the mouse.
Dr. Crump made the motion to accept the conclusion, is there a second?
I will second, Keith Soper.
All in favor, raise your hand.
opposed?
Dr. Pin Dr. Pino?
I would like to add --
Carries, with the assumption that will be added.
All right. Then I guess we are ready to move on to 5 high to hide hydroxymethyl. Furfural.
Conditions present during the cooking, baking of most food stuffs. Examples of commonly occurring food stuffs containing five hide drox meth -- breakfast cereal, coffee, pastries, dried fruits, beer. Many people don't consider that a food item, but I thought worth mentioning. It's ubiquitous in the human diet.
The major metabolites in it humans are normal constit wepts of human your in. Some industrial uses in the synthesis of several types of pollins and resins, undocumented, and major exposure is through the diet.
NTP first became interested in the compound, based on a series of papers published in the early 1990s suggesting that compounds form during cooking could possibly be involved in the promotion of colon tumors. In the first paper here, by, what was done is to initiate a group of rats with oxy meth -- feed different diets, one with -- one with [indiscernible], in ole, and one control, that study showed that the rats receiving either -- increased numbers in -- the gastrointestinal tract, the colon. They also showed 5 hide drox meth alone had the -- the activity much weaker than oxy methane. In the second paper the authors hypothesized possible intermediate, they ingt sized the sulfate ester, showed it was mute owe Jenic, and also singt sighed the clor owe metic, but since the pathway of that particular metabolite was not clear, whereas the cul fa transfer as and involvement of -- is well-known, they hypothesize the the cul feat ester might be ght be might be a -- so this suggested -- for mages of sulfate ester, of course, the active compound, DNA mutations, and lead to cancer.
So it was against this backdrop that this compound was nominated by NIEHS, and based on the papers I just summarized. The fact there was essentially no data on the cars carcinogenic -- widespread, probably everyone on the planet. And additionally would provide information about the role of naturally occurring dietary chemistries that would be potentially cars car carcinogen. So it was administered by gavage and water. We did absorption distribution, metabolism, discretion studies. I summarized the studies in the report, the details were actually published in a separate publication. We did three-week and three month toxicology studies. During the 3-month study we collected urine three different --
The range findings indicated that in rats there was really no treatment related pathology, a slight decrease in the mean body weights of -- males, but very little else happened in that study. So we used the same doses from 94 to 1500 -- in mice no treatment related pathology, however we saw reduced survival at 1500-milligram in male and females, therefore we backed off for the three-month study to use 750-milligram per kilogram as the high dose.
During the 3-month study there was in rats, reduced body weights at the top two dose levels, reduced survival at 1500-milligrams per kilograms, but no biologically significant reduce in weightings or -- females, reduced survival, again no clinical signs, biologicalally significant body weight changes, or histopathic lesions. We found at 375-milligrams per kilogram and above the ease trough cycle was prolonged in females. Because of the reduced survive will in both sexes we selected 750 as the top dose in the two-year study.
In mice, males, we saw reduced body weights at 750 in the 3-month studies. Didn't see treatment related changes in organ weights or -- there was histopath logical region, at 180 and above. There was no significant treatment related pathology in females, a slight body weight reduction. We selected 750-milligrams per kilogram as the high dose for mice.
The results of the A DME studies confirmed -- is met metabolized. The urine analysis conducted at the thee different time points indicated no evidence of glia keen depletion.
During the two year stud nee rats there was no significant treatment related to survival or mean body weight, no treatment reported neo blast lesions -- of the nose, degeneration and -- both male and females. Scwameous and hype -- in male and [indiscernible]. Disbliefer during the two-year study in mice, the survival and body weights of the 750 oar 750 -- therefore these groups could not be used -- seizure activity -- occasion, other neurological signs, initial pathology review indicated no neo plasms in the prain but currently involved in a much more in-depth review of the brain, central nervous system, and we will include the results of that review in this report which it's complete
In male and females we saw significant increase in nonneo plastic lesions of the nose in -- and in females there was a significant increase in the incidences of and add noma and carcinoma combined ombined in the -- I note this trend, the control and the 188 and 375 group, nothing happened as far as carcinomas were concerned.
So based on those results, we conclude there's no evidence of carcinogenic activity in rats, no evidence of cars carcinogenic activity in male mice. Some evidence of carcinogenic activity in mice based on incidences of -- in the -- groups, and administration of 5 hydroxy -- the ole factory olfactory and -- the end.
Thank you very much. Are there comments from the boards, to begin with? We will go straight to review. Dr. Mirsalis? I have no major criticisms. This is a nice study, nice report. A couple things. You saw a lot of what I would call clinical neuro behavioral effects, unconsciousness, salvation, cat tone yea, some death, you are studying that, that's excellent and you should include that in the report. In light of the human exposure, I would like to see a little discussion on the average dietary intake in humans, what it might be in milligram per kilogram. Can would be useful, pull a calculator, determine average dietary intake the average person consumes from this to this, that would help in the discussion.
A trivial point, something I brought up yesterday, more to add. I would like to see you mention in the body of the report what the feed was used. You have to dig in the appendix to find that. Under the, now the way FDA is enforcing analysis, we subsequently determined after talking -- the -- are not doing what FDA recommended, secondary independent analysis of feed constituents. Something for your entire program to talk to -- go back, talk to contractors, something FDA is issuing a tions on now.
Didn't see lesions in the colon, some discussion, calling that out, one of the first things I looked for and you don't really address it other than to say there was carcinogenic -- and another thing -- put nip% 9%, very nice report. Otherwise I agree with the conclusions.
Dr. Soper: I thought this was a nice report. I thought it was well-done. I had one comment on the statistical analysis, typically the trend test. Since these 750-milligram per kilogram dose was deemed above the maximum dose in mice, not included in the evaluation of carcinogenic potential. It would also be appropriate for them not to be included in the polly 3 trend test. You will come up with the correct conclusions with the pair-wise comparisons, but to show the difference it can make, for the hep at owe cellular, carcinoma and -- you got a value of -- and I think I saw in the report you gave without the top dose the P value is .001, big change. Thanks.
Thank you. Dr. Pino?
Dr. Pino: My comments mainly concern the rat study. In the report, the results, there was talk about some -- adenomas not addressed anyplace else. I would like a better idea of bhoots thought to be going on there. Also, there were some clear cell -- in the liver, I think mentioned in the discussion buts not mentioned in the summary, in another report they were. So for consistency maybe they should be -- not in conclusions, at least the summary.
Then there's a statement that says two hep atee sight -- in 375, but not, no indication of if those are considered compound related or not. I think they are not, but that should clearly be stated and why.
Thank you. Would you like to respond? D r. Irwin?
Let me start with Dr. Pino. The add adenomas in male rats, a single occurrence of two animals, no dose response. We will definitely explain that, why we considered it not treatment related in the report. As far as -- cell adenomas, again, there's really -- they were increased only not in highest dose group but in the 375 group, and there's no dose response there. The control rate in the study, 1 out of 50 is very low, the historical control is 14%, about seven out of 50. There were no effect s in females, the reason we considered the adenomas not to be treatment related. I will delineate in the report. We agree they increase with dose, not preneo plastic, but we will -- [indiscernible].
Dr. Soper, I put that in the slide for your comment. Actually in table 13 we show the P value, but will make sure we get that in the body of the report, mention it so we use the correct P value just using the control and the 188 and 375 group and delete the high dose.
I have tried, Dr. Mirsalis, specifically looked for quantification of the two metabolites, in -- attempt to calculate what might be considered an average exposure. I will continue to look at that. Depending on what dietary substances you want to add together, but you can put together what you might consider an average human diet, figure something from that. I will certainly pursue that.
I think it's really -- I agree, worth mentioning the fact we found no lesions in the colon. Actually, was, when I first started going through this, discovered that HMF wasn't all that stable in feed it made me wonder about the first study when they administered in feed, I thought we might find something different here. I would be glad to answer any other questions.
Anymore discussion? Dr. Howard? Dr.Howard: I agree with Dr. Mirsalis' suggestion, putting the daily average intake. The discussion, as far as drawing comparisons with the doses using the study, but based on comment you just made I would also suggest if it's not already published, you put a statement in the -- no reports of average daily intake could be found. I do not recommend you try to calculate it.
I really looked, almost all publications are concerned with determining the levels, various types of food, but almost none deal -- except one particular one in which it was actually shown that these were components of the human urine, but not quantitated. It's a good suggestion.
Not try to move beyond what's published as far as average daily intake. That could be fraught with a lot of problems.
Comment from the public?
Ron Lawrence from FDA. Is since this material is a product of thermal degradation of food, it's going to occur in all cooked food, processed food, they turn out to be quite good once you get there, but you have do a lot of Monte Carlo A flail sis, analysis to get a good figure. You start taking the figure, level, 1000 foods, add them up, you don't get what you want. You get -- there's -- gluttony has it's limits, you know. I suggest that unless it's been done, I doubt it has, that you don't do this. I will just point out that once you start going down that road, folks, you are doing risk assessment. But because what you want to do is compare this to -- what humans are getting to what rats are getting, that's risk assessment to me, and whatever --
Thank you for those comments.
Anymore discussion? Dr. Crump?
I wanted to make another comment on the C cell. Adenoma, carcinoma, and the tie thyroid. That's very striking response, in the controls, one to 14 in the mid-dose. I am not sure that's dose related, but I don't think we can attribute that to chance either. It either says it's dose related or some of the assumptions about our study are not being met. If you start distributing animals at random, dose has no effect, not one time in a 1000 will you get that large a difference. To me, just -- if it's not dose related calls into question some of the assumptions or some of the ways we're conducting our responses or something. I think it's a very striking response. I will also comment on the liver tumors, female mice. Going back to the thyroid, no effect in the male rat -- female rats. But in this case, if the liver adenoma, mice, significant decrease in males, significant increase in all struck me as quite strange.
Well, the control here is really far below what the historical control is. That would probably not be a significant increase if that control were nearer what the historical control is. Now, that combined with the fact that in my mind if this were really treatment related certainly we should see some type of dose response. It's hard to imagine why it would be so high just in that one mid-dose, not increase also at a higher dose. I realize it seems to be sort of a freak response, but occasionally we see these types of things. So also the fact that actually the carcinomas did not increase at all, they were 0122. No indz station Indication this is -- led us to conclude it was not treatment related.
Could you repeat the historical background --
14.16% -- 750 --
We're basically almost twice that in the --
Correct.
Dr. Howard: I can agree with your argument in relation to historical controls, but I I don't using the one middose, you use the same logic to drop the high dose in the mice from your trend test, because of a significant decrease, looking at historical controls is probably the best way to look at --
Did that make sense?
Well, when you say high dose in the mice --
Table 13 you are dropping the 750-milligram per kilogram, using two doses. You use that same logic to -- to say I can drop the high dose and do polly K test --
In the case of rats there were no mitigating circumstances in the high dose, like in the case of the high-dose mice. Many of them died. There was obvious, significant compound related toxicity. Nothing to indicate there was a problem in the high-dose rats at all.
Good response, thank you.
Thank you.
Sometimes a little disconnect comes across, different end points get --
I will try to explain that better.
In the context of in the historical control data or not --
Thank you. Anymore comments before we display the conclusions?
Public comments?
Okay.
Any discussion?
Just asking the question. Adenoma incidences sufficient for some level of carcinogenic activity ? yes, okay.
There being no discussion, I will entertain a motion to accept the conclusions?
Jon Mirsalis salis, I move we accept.
Second?
Somebody brave?
Keith Soper, I second.
All in favor?
carries unanimously. Brings us to end of reports. Dr. did you have comments?
I can't express enough our appreciation of the staff, the, you put into the review, I thank the staff. The presentations this time were really excellent and I hope enjoyed your stay with us this time and you have a nice trip home. Thank you very much for your efforts.
Thank you. I would echo the presentations were outstanding, very clear, very thorough. Dr. Shane, did you have parting comments?
Thank you very much for coming, and the bus will be ready in about five minutes of noon. To take you to the airport.
Thank you.
Thank you, and the meeting is adjourned.
Contents
Day 1
Event Started: 2/27/2008 8:15:51 AM ETGood morning, everybody. I would like to call to order the meeting of the national toxicology program of scientific counselors toxicology subcommittee. We will go around the room now to introduce ourselves.
[ Speaker Faint/Audio Unclear ].
Dave Malarkey, NTP.
Gordon, NTP.
Grace kiss link, NTP.
[ Speaker Faint/Audio Unclear ].
Tracy Bunton.
Mark toreson.
Paul Howard.
Kenny Crump.
Russ Cattley.
Chris Bradfield.
[ Speaker Faint/Audio Unclear ].
Ray Novak.
Barbara Shane.
John Bucher.
[ Speaker Faint/Audio Unclear ].
[ Indiscernible ].
Susan [ Indiscernible ].
Charles allton.
Ron Lawrenceson.
[ Indiscernible ].
Mary Boudreau.
Paul [ Indiscernible ].
Susan [ Indiscernible ].
[ Indiscernible ].
Ron Melnick.
[ Indiscernible ].
John [ Indiscernible ].
Bill Jamieson.
Paul Foster.
Ron Herbert.
Angela king Herbert.
Ruth Lynn.
Matt Stout.
Joe [ Indiscernible ].
[ Indiscernible ].
Charles [ Indiscernible ].
Melton [ Indiscernible ].
Cynthia Smith.
Christine [ Indiscernible ].
[ Indiscernible ].
Greg [ Indiscernible ].
Robert souls.
And I'm Nancy Kerkvliet, Oregon state university. Dr. Shane, do you have any comments before we begin?
You will find the latest agenda, roster, the list of written comments and copies of the comments and a list of oral commenters and the form to fill out for reimbursement. Please make sure you have signed in and use the microphones and identify yourself. If you are making a public comment for the public out there Rio de Janeiroster at the table outside. We will be taking a photograph this morning at the break outside in the lobby. The draft reviews are preliminary comments by the viewers and will not be included in the minutes. The board members must say any comments they want to be in the final reports. I will read a conflict of interest statement, the members of the subcommittee serve as individual scientists. Each member is to exercise jiewment prior to any meeting as to if any [ Speaker Faint/Audio Unclear ] or financial interest. Of yourself, your spouse, minor child, general partner or outside organization for which you are negotiating or arranging for future employment. Should there be conflict of interest [ Speaker Faint/Audio Unclear ] and not to participate in discussion or vote on any action. I just want to identify that Dr. [ Indiscernible ] has a conflict with [ Indiscernible ]. And will not participate in the discussion or vote. Thank you.
Thank you, Barbara. Dr. Bucher, would you like to say a few words?
Thanks very much. On behalf of Sam Wilson I would like to welcome you all. I want to thank Dr. Kerkvliet for chairing this session. I would like to thank ahead of time all of the members of the panel who has put in many hours reviewing the reports. This is a central core activity of the program, where we created our legacy and continue to. This is the most important activity that we carry out. We thank you for your participation in helping us in this challenging activity. I would like to mention that for many years I sat on the other side of Nancy here, where Michelle is now sitting. I would like to introduce Michelle. She's with Dave Malarkey who coordinating the reports for the last three years in that area. Michelle will be taking over my role in dealing with some of the text cooling aspects and working with Dave to put the reports together, I am anxious to have a lot of the credit and of course the responsibility of errors shifted from me to Michelle and Dave. I think they're going to do a terrific job. Thanks for coming. I think this will be a very interesting meeting.
Great, thank you. So just the plan for the morning, um, we hope to get through the first two reports, Dibromoacetonitrile and Bromochloracetic acid before the morning break. Then committee members will have a photograph taken. This will be then followed by the aloe report, we hope to finish it by lunch. If necessary to fit lunch in before the 12:30 we may have to break that report into two sections. The format for the reviews this morning, the first is study scientist and the pathology will make their presentations. If there are questions, um, asked during the discussion -- there will be questions asked during the pathology report. The reviews will present their reviews. Comments will be taken from other subcommittee members and any written comments will be identified. Today I don't believe we have any written comments. Okay. So that's basically the way we'll go. And we'll vote on the final conclusions. Okay. So, I guess we're ready to begin the first report. And it's on Dibromoacetonitrile. Dr. Ron Melnick.
Good morning. A -- for the next two reports I will be discussing the toxicity and [ Indiscernible ] studies we have conducted on water disinfection biproducts. The first is Dibromoacetonitrile. We have had studies in fish or rats or [ Indiscernible ] mice by drinking water route of exposure. On the top you see the structure of Dibromoacetonitrile. It's a disinfection biproduct. There's no real commercial progression of this. Dy infection biproducts are formed by the reaction of chlorine with organic matter present in water sources. A number of factors influence the type of biproducts that you obtain. This is depending on the organic precursors, the nature of the disinfectant itself, PH. Concentrations of Dibromoacetonitrile have been measured in a national survey. They're up to high as 11 micrograms per liter. The U.S. EPA has a rule, but it does not include Dibromoacetonitrile. But the WHO has a guideline of 70 micrograms per liter for Dibromoacetonitrile. I'll be talking about two weeks, three month and [ Indiscernible ] studies. These compounds were nominated to the NTP from the EPA because of the widespread human exposure. [ Indiscernible ] is a representative of the family of [ Indiscernible ] and the compound selected is because of the possibility that [ Indiscernible ] might be more active than chlorinated. Drink water is the route because that is the primary route of human exposure. In our two week studies in fish or rat Dibromoacetonitrile was administered ranging from 12 to 200 ambiguouses per -- migs per liter. This was selected because of preliminary tox studies. 200 was selected as the highest dose. There were exposure related decreases if in water con sujs. There was some body weight gain for the males. There were no chemical related lesions. There was in 205 males atrophy of the [ Indiscernible ] in the high dose group. The same exposure concentrations were used in the three month study. No effects on effect or clinical signs. There was decreased water consumption in the 200mig rat. There were no computer related lesions identified in this study. There were decreases in [ Indiscernible ] sperm mow tilt. The exposure concentrations for the two year study were 200 as the highest, 100 and 50 for male and female rats. Survival was not effected. Terminal body weight was in the high dose groups about 5% less than controls. And from our concentration of Dibromoacetonitrile you can calculate average daily doses, they range from 2 to 8-milligrams per kilogram. The biological effects we observed in this study were predominantly in the upper elementary system. In this study we observed neo plasms in the hard palate and the tongue. In the males there were increases in ska mouse cell carsinomas. The top line are the number of animals examined microscopically. A star in the control group indicates a significant trend. A star in a [ Indiscernible ] group indicates a significant [ Indiscernible ]. We observed an elevation of ska mouse cell papillomas in female rats. Oral cavity neoplasms are uncommon in fish or rats. You can see we've only observed in six studies one male with a ska mouse cell neoplasm. The number is still less than 1% for boat males and females. This response we feel is clearly related to the exposure of Dibromoacetonitrile. Both of these exceed the historical control. The response in males was a clear evidence of carcinogenicity.
Another lesion that we observed was a glandular stomached a know ma in two male ratsive this is rare. In the 1200 rats in the database this lesion has not been observed. So we consider these two to represent an exposure related effect. There were also ska mouse cell papilloma and [ Indiscernible ] basil cell [ Indiscernible ] on the skin of female rats. The trend was positive. The incidence wasn't, this is more of an uncertain finding. Other nonneoplastic regions was hypercare toe sis of the esophagus. And dis, which is a dilation of the glanldzs of the stomach observed in female rats. Our two week studies in mice were at the same drinking water cons rations as for rats. There were no effects on survival or clinical signs. Decreased water consumption again here. No significant effects on body weight. And no chemical related lesions. Same concentrations were used in the three month study in mice. Again, no effects on survival or clinical signs. Decreased water con sujs. We've seen this repeatedly in the 200-milligram mice. No chemical related growths or lesions identified. Again, a small decrease in ep kid mal sperm mow tilt. The survival of mice in the two year study showed a significant increase in female mice in the two higher dose groups. There were decreases in body weight, ranging from about 5 to 12%. In male mice in the highest dose groups. And the effect here is not shown at the end of the study, but there was a decrease in body weight also of 10 to 12% of male mice. These are the exposure levels. And what I was trying to show is if you look at the body weight in males it seems decreased throughout most of the study. But in the females the squares, near the end of the study return to almost control levels. The main observation within this study was the observation of squamous cell papillomas or carns know mas in male mice and female mice. As you can see there were significant trends, significant elevation of the combined squamous cell papillomas or corns know mas and increased hyperplay sha. For this lesion and for the oral cavity lesions we don't seem to see in other studies any type of specificity. That is when a chemical intellectual deuces a lesion in male rats, we see it in female rats as well. With squamous cell papillomas of the stomach, of the number of studies that we have conducted that have shown them, in males and females the correlation is somewhere around 85%. Also, with both of those types of lesions there's good evidence of progression of papilloma to carsinoma. There were carsinomas associated with papillomas for the four stomach, we also used the progression as judging evidence. We can see carsinomas in males, but not in females, about 85% of the time that we see them, they're associated with carsinomas as well. In this case we did not see papillomas in female mice. Another lesion in mice was [ Indiscernible ] or carsinoma, which was elevated in the low dose group [ Indiscernible ] were elevated in the low dose group. There were elevationed relative to control significant in the mid and low dose groups. However, in female Miles the trend for course know mas decreased, this may be related to a decrease in body weight that was observed during the study. In any case, this was somewhat of an uncertain finding and may have been related to Dibromoacetonitrile, we can't really tell. In conclusion, on our studies for rats. In male rats we consider this to be clear evidence. And the two glandular [ Indiscernible ] we considered to be exposure related. In female rats there was some evidence of car know Jenicty. As well as the skin lesions which may have been related. This is our term for ee equivalentcal evidence. In female rats we observed the [ Indiscernible ]. In mice it was clear evidence of cars know Jenty based on the increased carsinomas in the liver [ Indiscernible ] we consider may have been related to exposure. In female mice we considered the evidence to be clear for four stomach squamous cell papillomas in relationship to the lesions observed in male mice. Non[ Indiscernible ] of male mice was the elevation of squamous epithelial [ Indiscernible ] in the forestomach. Food for thought, Dibromoacetonitrile has been examined in [ Indiscernible ] protocols it was found to be an initiator of [ Indiscernible ] in [ Indiscernible ] mice with the disappropriater when administered on the skin. It did not proud lesions when administered orally. Indicating there's something more of that direct effect of the parent compound. As I mentioned, we did not see chemical related lesions in the three month study rats or mice. The effects were in the upper elementary system. Again, indicating probably related to the parent compound or a reactive me tab light formed early. Dibromoacetonitrile is not excreted in the urine. We look for this in the liver of our studies, we did not see a decrease in GST activity. The studies that reported were by [ Indiscernible ]. Ours were drinking water studies. In some disposition studies that were conducted at NTP using regular label Dibromoacetonitrile we found [ Indiscernible ] binding of [ Indiscernible ] to [ Indiscernible ] molecules in the stomach and liver after [ Indiscernible ] administration. The other effects that might also be related is that invitro studies have shown [ Indiscernible ] a DNA repair study and [ Indiscernible ]. So the mechanism we don't really know. This starts to give us some thoughts of terms of what might be involved in that carcinogenic response. Thank you.
Thank you, Dr. Melnick. Any questions for Dr. Melnick, just for clarification purposes before we start our reviews? Questions? If not -- Dr. Novak.
Thank you, Nancy. I have no major comments. I'm pretty much in agreement with everything that was presented. The only comments I would add in some cases it's perplexing there's not more of a dose response. There was an effect of 50, nothing at 100 and nothing at 200. I think those add to the complexity of addressing all of this. I think it's important because of the drinking water aspect of this. And if my notes are correct, from having gone through this, in terms of the terms of the Dibromoacetonitrile concentrations in drinking water a high was something like 11 micrograms per liter. When looking at concentrations where a predominant number of effects were seen, at 100 and 200-milligrams per liter, or an average dose of something like 7 or 8-milligrams per kilogram, I'm wondering if it's possible to relate that to human exposure. What we're talking about here is a cumulative time dependent effect. When the data are finally released and people begin to look at this we suddenly don't have someone running out and demanding that chlorination be terminated. I think that looking at the concentrations and the overall exposure of humans would be important. The second point, perhaps this is more relevant in susceptible populations. Where some conditions might lower the levels of [ Indiscernible ] and increase the prospect for an effect. I think the experiments have been well designed and thoroughly carried out. I have no disagreement with the conclusions.
Thank you. Dr. Cattley.
Okay. Really, I had a suggestion about the conclusions. It has to do with the reporting of the level of evidence in the mice. And, um, because I believe that in the mice there was no evidence of carsinoma formation. So, I think when we review the statement on the conclusion I would propose those two be considered as separate lines rather than being combined for male and female mice. I do think in the case of the female mice in the forestomach papillomas it would be useful to characterize the progression of the [ Indiscernible ]. That is the basis of considering that clear evidence. The other suggestion, addressing the historical control data for [ Indiscernible ] and [ Indiscernible ] in rats that we see the overall numerator and denominator depicted in the text.
Okay, thank you. Dr. Soper.
I thought the study was well done. No comments to add. Thanks.
Thank you. Um, any comments from anyone else? Pardon me.
I have some minor comments, Mike [ Indiscernible ].
Let's have Ron respond to the comments, first of all. Sorry, Mike.
Yes. Certainly we use higher exposure concentrations than those that are found in drinking water for our studies. Largely this is due to the limited power of a [ Indiscernible ] with 50 animals per group. The issue really for drinking water and car know Jenic risk relates to the fact that 200 million people are drinking chlorinated drinking water. And what we are doing is identifying a has -- has yard -- hazard. Dibromoacetonitrile is just one of several hundred disinfection biproducts. This contributes to the overall picture of what is in drinking water, it varies depending. I think EPA is working on these types of issues. Susceptible populations, I think we agreed with that. This needs to be addressed. I think there's emphasis on that in the risk assessment community. For Dr. Cattley, we'll devote a lot for emphasis in our discussion on the evident of production of the four stomach neoplasms. That all fits into the story of why we consider this to be clear evidence. This study shows evidence of production in males. We don't see seem to see a sex difference in response. When we see four neoplasms induced it's accompanied by squamous cell carsinomas. This will be added into the discussion to round it out a bit. And in terms of the data tables on historical control incidence, when we use that there should be a table in the back that provides the information. I noticed the one that you pointed out wasn't in the appendix. I will make sure that is brought forward to the text, as well.
Okay.
Just to clarify in terms of what I was envisioning. In the present study the females had a much more robust response in terms of incidence of papilloma than the males. But the females had no course know mas -- carsinomas. This would say this is not as supportive as other studies that have been conducted. I'm taking that the association. These two lesions in animals from the same treatment groups is considered evidence of production. But to me it doesn't really in a gait -- negate. I'm not familiar enough with all of the studies to say there is or not. The stronger evidence would be coming from not this studdie, but from other studies. In which a better correlation was observed.
I think there's a few reference materials on the continuum of progressive lesions from the four stomach [ Indiscernible ]. And we can also refer to those other references as well as. In this case we also saw somewhat higher, or a trend towards epithelial hyperplay sha in the male mice. Yes, we didn't see carsinoma, but it's all part of that. We're dealing with small numbers of carsinomas as well. I think if there's evidence of progression the evidence specified in the criteria is to assume that progression can occur. I think that's a pretty good assumption in this case. I'll try to address this more in the discussion.
Okay.
Dr. Flake.
I think there's good evidence in the pathology literature that squamous papillomas can progress to carsinomas in the oral cavity and in the esophagus and the forestomach. This is stated in the [ Indiscernible ] and the WHO and the pattology of the mouse book. There's also a book, atlas of tumor pathology of the fish or rat. In there they've got photos showing insight to [ Indiscernible ] changes within the papillomas and in the [ Indiscernible ] book there's a photo showing carsinoma in squamous cell papillomas and lesions that at the surface look like ska mouse papillomas but have [ Indiscernible ] at the base. I think there's -- not that all ska mouse carsinomas arise through that route, but I think there's pretty strong evidence for the latter.
Okay. Dr. [ Indiscernible ] did you still have a comment?
Just a few. For the two week rats. The atrophy that was observed, at the same doses in the three month and two year study there were no changes. I don't think the changes shown were related. There were kidney changes in the two year rat study, increased [ Indiscernible ] and pelvic inflammation that was not mentioned or discussed. Perhaps those should be brought forward. In the summary for the rats, when talking about the skin tumors observed there, it doesn't clearly state that's in the skin. I think that needs to be in there, I think on page 10. It doesn't say that's in the skin. Because you just talked about similar changes in the stomach, I think that should be stated. Then theed a know mas in the mouse. They had to go back and recut them. They didn't show an increased incidence after the cuts. I think you need to show that was not compound related, that's all.
Any other comments? If not, I think we will display the -- okay. That request included public comments. Nothing. Okay. I think we'll display the conclusions on a PowerPoint slide. Then I would entertain a motion to, um, revise or accept these conclusions. Yes, Dr. Cattley.
I'm Russ Cattley. I would move to recommend a revision to the level of the evidence for the male and female mice so the statement separates the two. Because in the statement as it stands it's based on increased incidents of [ Indiscernible ]. There were no carsinomas of the forestomach of the female mice. That could be a point of confusion to the point who might only read this conclusion. If that could be split out, it would read something to the effect there was clear evidence of carcinogenic activity in Dibromoacetonitrile in male mice. There was clear evidence of carcinogenic activity in female mice based on ska mouse cell papillomas and the potential for these lesions to address. Both of those are key to that call of clear evidence.
Any discussion from the subcommittee? Paul?
One point about that. In the case of the female mice there was significant increase in [ Indiscernible ], yet in the males it seem toes be fairly equal. Is this committee comfortable calling it clear evidence?
That was interest Howard. For the record. Any discussion? Yes?
If I can read from the explanations of levels of evidence. Information that is used is progression from [ Indiscernible ] to malignant [ Indiscernible ] as well from [ Indiscernible ] do [ Indiscernible ]. That type of information is used. And where progression is known to be a possibility the most prudent course is to assume [ Indiscernible ] have the potential to become malignant. This is page 14.
That was Dr. Melnick. Russ.
Russ Cattley. Since I brought it up, I'm not really comfortable with the evidence for progression in the present study, even looking at the males. I do think that Dr. Melnick and interest Flake have indicated they will provide additional evidence for the potential for these to progress in mice.
Okay. Um, so, we could take a motion to change the conclusions as stated by Dr. Cattley. Or further discussion? Or another motion.
This is John Mirsalis. I make that motion.
And then we vote on that? We'll change the wording and then you can -- we'll vote on that. Dr. Novak seconded.
Can I ask a question?
Yes.
Compare the changes to the mouse and the rat. Were the papillomas similar? I guess it would be the males that wasn't seen in the females? Or any other kind of evidence to suggest they would act differently?
I don't think there were any differents that were noted in the papillomas of the males and females.
Thank you.
Let's see.
We've lost that sentence --
Do undo.
[ Speaker Faint/Audio Unclear ].
Just undo.
[ Speaker Faint/Audio Unclear ]
Now control C should just copy.
But you cut it before you copied it, Charles.
Can I just see?
[ Speaker Faint/Audio Unclear ]
Where is the text you want to copy.
I might make a suggestion. If the subcommittee has comments like this on other chemicals, write down a suggested change in the conclusions for, um, Charles. Did you have something, Russ? It might just facilitate things.
We'll just repaste that. Then we'll -- okay, have that for the male. [ Speaker Faint/Audio Unclear ]. Is it some or clear evidence for the female?
Clear.
Okay.
We would entertain any [ Indiscernible ] jokes at this point. [ Laughter ]
I have a question, in regard to my comment about the kidney findings in rats, is there a reason why it wasn't brought forward to the summary? And if it should or should not be included in the nonneoplastic effects.
Okay, has everybody read the modified conclusion?
We can add that to the summary. Again, it's a high background rate, the numbers that increased were not as -- you're dealing with a high back ground rate in the -- [ Speaker Faint/Audio Unclear ] but it's worth while bringing forward to the summary.
Okay. I would like to call for a vote on this conclusion. All those in favor of accepting, or more discussion -- sorry. Go ahead and call for the vote? Any discussion? All those in favor of the conclusion as stated? Raise your hand. All those opposed? One. Okay, thank you. Pardon me? Oh, Dr. [ Indiscernible ] would you state why were opposed?
I was okay with the original wording.
Okay, motion carries. So we're ready to move on to the next chemical. Which is Bromochloracetic acid. And Dr. Melnick will again give our report.
Okay, thanks. Good morning, again. This is another disinfection biproduct from water chlorination, the compound is Bromochloracetic acid. I'll be discussing the drinking water studies of this compound in fish or F-344/N rats and B6C3F1 mice. It will exist as the Bromochloracetic acid acetate this is a drinking water disinfection biproduct. Similar to Dibromoacetonitrile. It's formed by the reaction of chlorine with naturally occurring organic matter. Concentrations were up to 19 micrograms per liter. The tox studies, measures showed that levels of not greater than 6.5 micrograms per liter. EPA has a rule and halo [ Indiscernible ] acids are included. The drinking water concentration for the sum of [ Indiscernible ], [ Indiscernible ], [ Indiscernible ], and [ Indiscernible ] should not exceed 60 micrograms per liter. As you can see Bromochloracetic acid is not included in this group. But I suspect the next time they review it will be. EPA nominated to the national toxicology program this. This is because of the widespread human exposure. And there had already been published studies demonstrating it's course know Jenic to the liver of rats and mice. I'll be talking about the two week, three month and two year studies. The water concentration selected for the two year study ranged from 62.5 to 1000-milligrams per liter. This concentration the high is similar to exposure concentrations that have been used in the chronic and toxicity studies in [ Indiscernible ] and [ Indiscernible ]. In the two week study there were increases in the relative liver weights. About 10%. There were increases in absolute and relative kidney weights in male. No gross exposure lesions identified. The same exposure concentrations were used in the 3 month study. There were significant increases in liver weights and kidney weights. The liver weights in rats were increased at the 1000-kilogram per liter group. There was increases incidences of [ Indiscernible ] in the liver males and males exposed to the 1000-milligrams per liter. I believe all animals at that dose showed the [ Indiscernible ]. So the concentration selected for the two year study included the 1000-milligrams per liter and half concentrations and half again as the mid and low dose exposures. No effects on survival. There were decreases in body weight at the end of the exposure period at about 10 to 12% in males and about 10% in females. The average daily dose can be calculated from the concentration in the water and the change in the body weight. You can see they range from 10 to 50-milligrams per kilogram, a finding of particular interest is the observation of increased neoplasms of the large inTess tin. There's been several studies on disinfection biproducts. The most frequent observed affect is with bladder cancer. There have also been reports of associations with rectal cancer. In this study we found increased incidences, a trend for males for theed a know mas in the colon or rectum. And in females a significant trend and significant incidence. The back ground rate in fish or rats is very low. It's a rare finding. Of the 1200 male rats in the database, two showed this particular lesion in males and 2 in 1100 female rats. So the findings of these two lesions exceeded the historical control. A -- similar to what I mentioned with other, with the Dibromoacetonitrile, there does not seem to be a sex specificity for colon cancers when observed in rats. When we see them this males we tend to see in females. The correlation is about 90%. And in studies which have shown colorectal neoplasms most frequently there's the observation of carsinoma indicating the potential for progression of these lesions. So these findings when looking at them together show these are clearly related to the exposure to Bromochloracetic acid in rats, inducing large inTess tinned a know mas. This is a section of a large inTess tin. This is the normal structure. This is theed a know ma growing into the lieu minute. You can see here the glands that are arranged in a lineal parallel pattern in the normal. It's totally disorganized in the adenoma. We don't see the gob let cells. These are inmature cells. And the darker bay sow feela. This is one of the adenomas from this BCA exposed animal.
Two other lesions worth noting was there was an increase in mesoTheo ma. Each of the dose groups showed a rate higher than the historical rate. And the incidence in the 500-milligram group was significant. As I mentioned, mesotheleo ma is a sight associated with Dibromoacetonitrile. Interesting finding in the ma'amry gland. There was no effect on the incidence with exposure. However, in the treatment groups there's a significant increase from the incidence of multiple fiber adenomas in the mid and high dose groups. Looking at the data on those, if we looked at the number of fiber adenomas. Molt policety is part of the evidence used in making an evaluation. This response to us is demonstrates an exposure-related response.
A, in this study we also saw a [ Indiscernible ] adenomas low numbers in male rats as well as in female rats. However, in rats the [ Indiscernible ] adenomas are uncommon. Occurring at a rate of about 1%. In association with the adenomas there were increased in [ Indiscernible ] in male rats and [ Indiscernible ] and [ Indiscernible ] in female rats. We consider this to be of a less certain finding. I point this out as an effect that may have been related to exposure to Bromochloracetic acid. [ Indiscernible ] were increased in the middose group of male rats, it exceeded the historical control rate of 8%, this would be 18%. However, it was not increased in the high dose group. And no effect in female rats. This to us, again, is another uncertain finding. In mice our two week study was at concentrations from 62.5 to a thousand migs per liter. There were no exposure related clinical signs. No chemical differences in organ rates. The same exposure concentrations were used in a 3 month study. There was it decreased body weight gain of females at 250-milligram group and higher. There were significant increases in liver weight of males and females. The increases were as much as 20 to 30% higher in the high dose group compared to controls. There were no [ Indiscernible ] effects in this study, however there were observed increased incidences of [ Indiscernible ] in the spleen and these were not exposure related. But they were observed in several of the dose groups of males and females. The two year study was conducted at exposure concentrations of 1000, 500 and 250migs per liter. There was a decrease in survival in the high dose male mice. This was attributed to [ Indiscernible ]. There were decreases in body weight in ranges of 5 to 12% in males and about 10% in females. Here are the average daily doses throughout the two years. The main finding within this study was effects on the liver. If you recall, in the previous slide the liver was an identified target organ. There were increases in [ Indiscernible ] adenomas or carsinomas combined. There were increases in molt policety of [ Indiscernible ]. And the combination of [ Indiscernible ] and adenomas, carsinomas or [ Indiscernible ] show high significance in this particular study. Included also as reported before, increased sight playsic [ Indiscernible ]. Similar to the males, the females showed the increased incidences of [ Indiscernible ] or carsinomas. I think there were too many to put on this particular slide. As well, as increased sighto playsic [ Indiscernible ] and [ Indiscernible ]. Our conclusions from this part of the study, clear evidence of cars know Jenty in male rats. Based on the increase incidence of the large intestine [ Indiscernible ]. The increase in [ Indiscernible ] adenomas, which was small, we considered it may have been related. And the pancreatic [ Indiscernible ] may have been related. Would we consider to be ee equivalentcal. In female rats the evidence was clear for large intestine adenomas. And as I mention, there is evidence from previous NTP studies of the progression of adenomas to carsinomas, we're all aware of that, as well in humans. Ma'amry gland, we considered to be exposure related. The liver adenomas may have been related to [ Indiscernible ]. What I didn't show, we also observed increases incidents of [ Indiscernible ] in the lung of female rats. In mice, the level of evidence of cars know Jenist was seen to be clear. In female mice the increase was clear based on the inyeased incidences of [ Indiscernible ] and carsinomas. The nonneoplastic effects included the liver, [ Indiscernible ] in male mice as well as in female mice and the [ Indiscernible ] in female mice.
Now the [ Indiscernible ] are an interesting group of compounds. They look simple when you just take a look at their structure. But their metabolism occurred by an enzyme [ Indiscernible ] which causes the initial displace of a [ Indiscernible ]. The first stable product of this pathway is [ Indiscernible ], which can undergo further transformation seen here. In studies where either, any of the three have been administered to rats or mice, what you find is after several days of exposure the clearance of the parent compound is reduced, as much as three to five fold decrease in the clearance of the parent compound. What is shown to be the case is that in this metabolism forming the [ Indiscernible ] that this particular compound can bind covai lantly with [ Indiscernible ] causing this suicidal inhibition of the enzyme. It becomes a consequence of the destruction of the enzyme and its reisn't sis. -- reisn't sis. This is an observation which is also made with people. Because [ Indiscernible ] has been used therapyically for treatment of lactic acid removal. But in any case, the intermediate, when it's formed can bind covai lently to disor release to form [ Indiscernible ]. It depends on how much stays bound, which influences the removal of the [ Indiscernible ]. So, trying to understand what might be happening in the cars know Jenist of [ Indiscernible ], we can note there are several common sites. The mesoTheo mas in rats and the mouse liver tumors with all three compounds. NTP had sponsored some research which demonstrated DNA [ Indiscernible ] in the livers of mice exposed to [ Indiscernible ] and [ Indiscernible ]. This may also have some role in the cars know Jenist of this compound. The compound [ Indiscernible ] is also mute Jenic. Another pathway of interest is that the [ Indiscernible ] has also been shown to be the same enzyme, with a different name, [ Indiscernible ], which is the [ Indiscernible ] enzyme in [ Indiscernible ] metabolism degradation. By suicidal inhibition of this enzyme it's possible that the intermediates of [ Indiscernible ] are also accumulating one of these [ Indiscernible ]. As I mentioned, based on human studies the clearance of [ Indiscernible ] is observed in adults and in children. So, we are in the process of working on developing a physiological model of [ Indiscernible ]'s position. During the course of these studied we collect add fair amount of time course data on [ Indiscernible ]s. Our strategy is to develop a model to simulate these data. The purpose of that is to see if we can estimate values for a variety of dose metrics that might be, a, as occurred during chronic exposure and use them for dose response analysis. Some of the dose metrics we're considering are tissue concentrations of the parent compound, tissue compounds of [ Indiscernible ], liver levels of [ Indiscernible ] activity. There's others that also could be considered. The purpose of that is to see if we can identify dose metrics that might be associated with Bromochloracetic acid or [ Indiscernible ]. We feel this might be pertinent to human studies. So with that, I thank you for your time.
Thank you, Dr. Melnick. Any questions? Yes?
The level in water was 6.5, could you clarify, is that out of the tap? Or is that what the control animals --
Both.
The reverse osmosis water didn't take that out? I'm spiesed to see -- surprised to see that. It's just, it's a con founding variable.
Yeah. We use tap water. It wasn't -- reverse osmosis water for the controls.
Correct. The typical control water is tap water at the laboratories.
Dr. Crump.
Kenny Crump. I had a question about the female rats. There was a consistent dose response for, um, for skin. I think my bro mas went from 112 and 6. I'm just wondering why that wasn't included, or commented on in the report.
I would have to look at the data.
We should ask those questions before we have heard the reviews.
Okay.
Let's move forward. Dr. Soper is the primary reviewer.
I thought the study was nicely done. I agreed with all of the conclusions. I spent some time considering whether it might be more appropriate to classify the [ Indiscernible ] in the male and female rats as being ee equivalentcal evidence. My reason for focusing on that is the [ Indiscernible ] adenoma is a target organ based on observing [ Indiscernible ] and the related compound DCA. For the female rats we had three animals with [ Indiscernible ] adenomas all in the top dose group, that was statically significant. It would be helpful to have a table for the female rats, add that to the report. In the male rats, we did not get statical significance. That was lanchly because we had two animals at a 50. The highest incidence that we have at any prior historical control is about 1 out of 50. There were two dose groups outside of the historical information. I was curious what some of the experts would think about those findings. I thought the study was nicely done.
Okay, thank you. Dr. Melnick, do you want to respond, or wait? We'll go to the reviews and come back.
Dr. Bunton.
Yes, I would agree. I felt the study was nicely done. I did have mainly editorial comments. I won't go through all here. I did want to bring up one. That was in the conclusion, I wasn't sure what you were saying about the incidence of the [ Indiscernible ] adenomas as being some evidence. You had, it was in, I think on page 92, you mentioned how they are the, there was an increase in foecy and the fact that the tumors are rare made you believe there is some evidence. I didn't know if you were associating the findings, or if it was all of the findings together is what led you to that conclusion. So I felt just that we needed more clarification on that.
Okay, thank you. Dr. Novak.
I think the study was well constructed and designed and carried out. I'm in agreement with the conclusions. Again, my concern is just the upper concentrations that were used. That's been noted already. Um, the only other question I would have is were there any other clinical chemistry [ Indiscernible ] to measure the [ Indiscernible ] in the animals in the study. Was there an effect on the lipid profile? An indication that an metabolic disorder might be going on? I didn't remember seeing anything, it's just a question.
Okay, thank you. Dr. Melnick, would you like to respond?
It seems there were two questions related to the [ Indiscernible ] neoplasms in rats. What we were calling the evidence. If it's not clear that we were calling this ee equivalent quail. We will try to work on our language on that. But this, when we use the word may have been related implies an ee equivalentcal response. I'm not sure if from I heard from Dr. Soper you were thinking this was higher level than it -- than may have been related or ee equivalentcal, or whether ee equivalentcal was appropriate.
Some evidence as a response.
A -- largely I think it's based on that finding of for male rats, the incidences were not significant in any of the dose groups. The trend wasn't significant. It's largely based on that comparison to the historic yale database. The only positive affect is the trend in female rats. Although the foecy are potentially precursor lesions, there's not neoplasms. Based on that we feel that the evidence is appropriately uncertain at this point. Perhaps longer time might have shown something different. Based on the two year expose the conclusions seem to fit strongest with ee equivalentcal. I think it -- a -- I don't know -- it's about 1% also in all rats of exposure. By all rats of exposure for male rats we've seen 0 to 4% in male rats. And in female rats there was one study that had several more than 2 animals with a [ Indiscernible ] adenoma, but not by the drinking water route. I don't know which route it was. The evidence to us didn't seem strong enough to call this some evidence at this point.
We didn't see [ Indiscernible ] with [ Indiscernible ]. But they were reported in a study in the literature with [ Indiscernible ]. So it's one which is sort of borderline in our view. Uncertainty is where we're at. [ Laughter ]
Okay, thank you. Any other comments from the committee?
We did not do lipid profiling in the two year study. Our clinical chemistries are limited to our 90 day study. I don't recall any data that indicated there was anything.
I saw the effects that you have on the liver. I was wonder if there were associated metabolic effects to parallel that. That was the reason for the question.
Okay. Yes? Dr. Howard.
Please bear with me. I'm a little confused, not confused. I want you to help me sort thl out. In the male rats, you call this clear. If you look at the [ Indiscernible ] in statistics it shows that only the middle dose, with no trend test on that. If you look at the large intestine adenomas for the males there's no [ Indiscernible ] comparison significance, yet it does have a trend test. I greatly appreciate your evidence at the end of the talk about the [ Indiscernible ] considerations of how this may occur. Looking at boat of those are not real strong. You feel together they go into clear evidence, correct? I'm having trouble with that. There's not both trend and paralyzed comparison evidence with those two.
Yeah. I think the, when dealing with uncommon or rare tumors the [ Indiscernible ] database is important. For mesoTheo mas I believe there's are in the range of 3% in all of the dose groups that were included there was elevations with significance at the middose group. When dealing with ten or 6 -- I don't think these numbers are that different than each other to look for a real close dose response. What we see is elevation with the mesoTheo mas. On the large intestine neoplasms these are rare. These, like I said, occur in 2 out of 1200. The findings within the dose groups here are finding 2 out of 50, even is unusual relative to controls. Seeing 4 out of 50 in another dose group, this is just something which is well beyond expectation for anything anything other than a chemical-related response.
I understand that. I truly appreciate that. I will probably get a beer bought for me by saying that we're driven by statistics. I'm just a little, a little trigger going off, are we, it's just a concern. Are we overemphasizing that? It's not showing statistical significant.
But nor rare tumors we rely on our historical database. In the previous study we saw only 2 in the doats group. -- dose group. We felt that was related to the exposure to [ Indiscernible ].
I'm more comfortable with the females.
Also, when I mention we don't see sex specificity more large intestine neoplasms. When a chemical induces in one sex it tends to also in the second. We see a species difference. But we don't see a sex difference.
what is happening in the other, the other sex. If we saw it in two species that also helps. If we see it in both sexes that's strong.
You may want to check and see, I didn't catch that. One other thing, in your talk where you talked about [ Indiscernible ] you gave the incidence per rat and showed statistical significance. I didn't see that in table 9. Or anywhere else, unless I missed it.
It's in the discussion. It's not something we typically include in our data tables. It's something that I looked into after having the data.
I would like to just ask that you consider putting it in a table.
Sure.
Thank you.
Dr. Crump.
A couple of comments. Just a question for clarification. Is it standard operating procedure to combine [ Indiscernible ] over all sites? In your analysis. Just curious about that. I will bring up again the question about the my bro mas in the skin of female rats, it was -- significant trend, 1, 1, 2, 6. It was never discussed in the report. The reason for that? Or is it a oversight?
We do combine for mesotheleo mas of the pair that knee yum. These are all sites. The incidence is for that particular location that we do combine. On the skin lesion, there was a significant trend. But not a significant elevation in any of the dose group. The trend is driven by the high dose group. I would have to look at the historical group to see how that particular response compares.
The combination of [ Indiscernible ] and [ Indiscernible ] are somewhat common. Probably about 3% back ground rate. The roles here -- controls here are 2%. And our range can go up to mostly around 8 to 10%. Sometimes go up to 12% for combination. It's within what we've seen in historical controls. To have 12% in a group spontaneously.
I think that's worth a comment. A look in the report. There are cases where we would certainly in other situations call that ee equivalentcal evidence. I think it's worth some sort of comment in the report, at least.
We'll do that.
Okay. Any -- yes?
Russ Cattley. I think for the female rats the call of clear evidence is largely based on [ Indiscernible ]. I can appreciate the body of literature in other species, I wonder when the evidence for clear is called if we couldn't say for about the evidence in the report. I think that's why, if I'm understanding what you said, this is the reason this is considered clear evidence. The knowledge. We need to understand what data is being used to characterize that response. Part of that data comes from other studies.
Thank you.
Dr. Pinot.
Just a question. There was no progression. How typical is that for these intestinal tumors? I thought it interesting there was no evidence of hyperplay shas in the intestine. When you talk about the [ Indiscernible ] of the liver. The beginning of paragraph starts with a number of [ Indiscernible ] were considered [ Speaker Faint/Audio Unclear ]. It makes it sound like [ Indiscernible ] is a secondary change. I would clarify that paragraph. Write it so it doesn't sound like it's a secondary effect.
Okay. Thank you. Any comments from the audience? Public? Yes? Would you identify yourself?
[ Speaker Faint/Audio Unclear ].
Hold on.
[ Speaker Faint/Audio Unclear ].
Oop.
Okay. Ron Lawrenceson. I would like to ask Dr. Melnick if the ma'amry gland results were the only results that you had -- how would you have presented that? Where there was an increase in molt policety, but not incidence?
That would have been some evidence.
Let me suggest that we have a sampling problem here. The ma'amry glanldz in the rat is from stem to stern. You know, I think, probably what was reported there was by pal pages, I suppose. That brings implicitly size into the picture as well. Could you see them? Could you feel them? I don't believe typically an experiment does a thorough his toe path sectioning of the ma'amry glands. How do you get those numbers? Have you thoroughly looked at the ma'amry gland issue?
The ma'amry gland response was grossly detected. There were a number of tumors on each animal. We went back to the gross traceable lesions and counted. They were grossly des tebted. -- at the tekted.
I'm just bringing up a hype thetical. When you have a sampling problem, you should worry about how you got those numbers. This is one. Kenny I would just say to those skin FBI row adenomas is a similar thing. You have to be sure that you looked at the whole organ.
I had a comment. A question comes up about the process of our data review. I wanted to tell you how we do it. All of the data, when it comes out it is reviewed by the staff for potential treatment related findings. That's visually and with the flagged findings. We consider each one. Some of them we might decide are not bilollically relevant or important enough to bring forward because they don't fit within historical control ranges or they're not biologically relevant. We bring all of them into the results section. And determine which goes up to the abstract or not. So, um, with that said, the my bro mas we did consider that as a possible treatment related finding. Maybe we counted it and didn't leave it in the results decision. We could write in the review section our interpretation of that finding.
Okay. Any more comments? If not, um, can you display the conclusions?
Dr. Howard?
I'm curious again, why we're combining the male and female mice? There's a slight difference in the data that weighs the arguement.
They both had [ Indiscernible ] and carsinomas, [ Indiscernible ] were seen only in males and that's shown in parentheses.
Is this just conservation of words? [ Laughter ]
Okay. If someone would like to make a motion on the conclusions.
Keith Soper.
Jon Mirsalis, I second.
All in favor, please raise your hand. Looks to be unanimous. Motion carries. Um, Barbara would you like to start the next compound? Or time for a break? Let's take a break. Be back at 20 to 11:00.
There's a photograph, meet in the lobby please.
Session on morning break until 10:40 eastern.
Okay. I guess we are ready for our next report. Dr. Boudreau will be reporting on Aloe vera and total carcinogenesis.
Good morning. The page up does not work. So, enter?
The [ indiscernible ] plan, commonly referred to as Aloe vera has been used as a therapeutic dermatologic agent for more than 2,000 years. Because of its history and popularity, Aloe vera incorporated into a myriad of skin-care and cosmetics products. The Aloe vera plan has more than 75 potentially by active component. Evidence suggests that Aloe vera stimulates skin cell growth, enhances and eugenicists and light energize with ultraviolet radiation to induce skin cancer. The data toxicity of skin-care products and cosmetics is of a concern because of the large surface area of the body that is potentially exposed to sunlight. The National Cancer Institute nominated Aloe vera to the National toxicology Program for study based on its widespread oral and dermal exposure to humans and the general lack of toxicological information. So, the objective of these studies was to assess whether the topical application of Aloe vera planned components, alters the process of simulated cellular light and deuced total carcinogenesis in the [ indiscernible ] mouse. I might add that chronic dosed water studies with the Aloe vera plan components are on going. These will be presented at another date. The length of the Aloe vera plan is covered by a right. Just beneath the leaves, but within the parental tissue of the beet pulp R. Perry cyclic to Bills. These transport owl latex to the peripheral of the plan leaf. The aloe latex--Diaz are of he huge significance and to let the aloe species. The bulk of the leak is the leap pulp and is composed of large, then Wall cells that contain aloe del. Oneof the principal features of aloe gel is the presence of high molecular weight polysaccharides that are composed primarily of glucose [ indiscernible ] linked in a data one --Bond. Three test articles-Sorry, plan extracts, were used for these studies. Age of the plan extracts were obtained from fresh aloe [ indiscernible ] believes. Each extract, however, differed in either the portion of the leaf that it was extracted from or of the method of extraction. The it aloe gel test article is a [ indiscernible ] extract of the inner leaf Pulp. The whole leaf test article was the extract of the whole leaf with the lignified fibers removed. The whole leaf extract contained not only the aloe gel but the speed and 10 aloe latex as well put the decolorized whole leaf test article presented a cost-effective means of producing aloe gel by affording the process of hand filleting and the leafs. However, in doing so, it is treated with activated carbon which removes the aloe latex. It also removes some of the high molecular polysaccharides of the aloe gel. This extract actually differs from both the aloe gel and the leaf. Aloe [ indiscernible ]--of the aloe latex. This compound was selected as a positive control. The Crl:SKH-1 mouse model wasn't selected for study based on its long history of use and other carcinogenesis studies. Each of the test articles was used at two dose levels probably test articles were incorporated in water and incorporated into an oil and water in motion-Based Karim. The topical application of claims was to the dorsal scan of the mice. It extended from the nape of the neck to the base of the tail and midway along the sides of the animal. The source of the radiation in of this study came from filters, six and and a half [ indiscernible ]. Because the emission spectrum of these lands is very similar to that of natural sunlight, they are called simulated solar light. And the following slides, I will refer to that as a SSL. The topical application of the greens to the mice and the exposure of mice to the simulated solar light was conducted five days a week for a period of 40 weeks purpose was followed by a 12 week observation period. Mice that received no cream treatment but received increasing doses of simulated solar light and served as a positive control for the light. Animals that were treated with the control creams with the aloe extract cream or with the aloe in cream either received no light or the same middle toes of like that was also administered to the no cream animals. Survival and mortality in this study do not contain the usual definitions of the terms. Mice were removed from the study due to morbidity or to death. Most often, they were removed because a dorsal skin lesion had reached in the absence of a limit on size. In this study that was five mm. The exposure of mice to SSL resulted in a significant those dependent decrease in survival. This was expected. These and no Green animals serve as our positive controls for the experiment. At the same level of SSL, there was no effect on survival and animals that received the control cream compared with the no cream animals.
Similarly, at the same level of SSL, which again, is the middle dose of light, there was no effect of the Aloe vera text extracts' when compared with the controlled cream animals. The topical application of Aloe-emodin was associated with a significant decrease of survival and female my's only. Neither the exposure of to SSL or the topical applications of grain had any effect on the body weights of mice. Skin lesion data was collected and evaluated in the two phases during this study. During the study, during the in life phase of the study or the clinical phase when the animals were still alive, digital photographs of the mice, individual mice were collected weekly. Visual assessment of these photographs was used for the generation of the in-life skin lesion data. Assessments included skin lesion on set, incidents and numbers. Then, at the end of the study at necropsy, the mice were, again, digitally photographed, growth skin lesions were noted and collected four hits to pathological evaluation. These assessments included incidents in number on the non-neoplastic as well as new plastic skin lesions. We will begin, first, with the results from the in-life.
This side presents the skin lesion on said data. On set refers to the time point in the steady at which 50% of the animals and a particular treatment group have at least one unmeasurable skin lesion that was observed and is consistent with the information of SSL-Induced skin tumors. This is an example the Kurds that were generated from the skin lesion on said. This is for the animals that received no cream treatment and increasing doses of light to. As we go across the slide from the right to the left, doses of light are increased. When we find is that there is a [ indiscernible ] significant decrease in the median time to Ansett. This, again, was expected because the formation of skin lesions is a dose in time effected with SSL. At the same level of SSL, animals that were treated with the control cream did not differ from animals that were treated-Country debt, sorry, that received the note cream treatment ago at the same dose of SSL, animals that received the test articles did not differ from the control cream groups installation on sept. Incidents and multiplicity will be evaluated from this slide. Incident refers to the percentage of animals within a particular treatment group that have at least onedetectable skin lesion that was observed in-life and is similar to the legions induced by exposure to a simulated solar light skin tumors. So, as would be expected, in the animals that receive no cream treatment, as the dose of light was increased, there was a increase in the incidence of in-life-Determined skin lesions. I might add to this, however, that in this snow cream treatment group at both the middle and high dose of SSL, incidence approached 100%. At the same dose of SSL, the animals that were treated with the control cream group did not differ from those with the no cream treated animals, nor did any of the animals that received the test article claims differ from the control cream at the same level of SSL. Multiplicity in this study refers to the total number of skin lesions per animal within and at risk treatment group to go in other words, the total number of skin lesions per treatment divided by the total number of animals in the treatment group. As would be expected, as the dose of SSL increased in the animals that received no cream treatment, there was a concomitant increase in the multiplicity of in-life treatment. At the same level of SSL, the animals that were treated with the control cream did not differ from those that received no cream to in. Nor did the animals that receive the test article claims differ from control cream animals. We will now turn our attention to the history pathological at Data. The exposure of Crl:SKH-1 lies entombed simulated solar light is associated with the diffusive bidding of the epidermis as shown in-This was an normal scan. This is referred to as women sell hyperplasia. In this study it the study was documented. They are not showing up as well on this presentation as they did before. The exposure of Crl:SKH-1 myself to a simulated solar light is also associated with the nodular the thickening in the epidermis, which is usually detected by growth examination. This is Sean and C. This is characterized by this plastic changes and really basil disorganization, as is shown here. This condition was evaluated based on incidents as well as multiplicity in these studies. As would be expected, as the dose of light was increased, there was a concomitant increase in next the incidence of squamous cell hyperplasia. Mice that received no cream a treatment did not differ from animals that received the control cream either in incidence or multiplicity. The topical applications of the aloe test articles in female mice showed a trend toward a decrease in the incidence and multiplicity of squamous hyperplasia, in particular in the multiplicity of focal atypical hyperplasia. The exposure of Crl:SKH-1 mice to simulated solar light forms a dose in time dependent skin lesion proliferation. This proliferation forms a continuum of displastic and pull their Frank changes which forms three types of squamous cell neoplasms. What is shown here is the [ indiscernible ] papilloma which is a solitary focus with barbarized projections, --The photo micrograph of in A shows a [ indiscernible ] papilloma with its stock and mushroom like appearance, where as B shows a example of [ indiscernible ] which is more flattened and does not have a stalk. Papilloma's progress but exposure to light in to squamous cell. These are small, discreet models that rise above the skin surface. They have very sharply demarked borders which often compress the darkness. They are composed of sheets of epithelial cells that's lack orderly arrangement. Again, I am sorry about this broke it showed up clearer before. This is an example of squamous cell carcinoma and is composed of downward projecting sheets and the courts of [ indiscernible ] neoplastic cells. They tend to be a larger type of neoplasm. [ indiscernible ] appears also raided and in greater form. We will look at this as the histopathological data to go in mice that received no cream treatment and increasing doses of simulated cellular light, we saw an increase in the incidence of neoplasms by type, as well this is a combination, some combination of all neoplasms. We saw an increased dose trend. I might add that this reached 100% in animals at both the metal dose and High dose of light. At the same level of SSL, animals that were treated with the control cream did not have an effect on the incidence of neoplasms, either by type or combination. The incidence of squamous cell papilloma is worth significantly increased in female mice that were treated with the aloe gel and exposed to simulated sold their like again at the middle dose of light. There were no significant differences in the squamous cell carcinoma [ indiscernible ]. Nor were there is significant differences compared with controlled animals in the combined, some combination of all squamous neoplasms--Incidence approached 100% and all groups that received the middle dose of like. As observed with the aloe gel cream treatment, mice that received the whole leaf extract and exposure to simulated solar light showed an increase incidence and squamous cell papilloma as. That was observed in males and females alike. There were no differences and carcinoma [ indiscernible ] or carcinomas in some combination of all neoplasms. Similarly, as with the aloe gel and whole leaf, there was a significant increase in the incidence of squamous and cell papillomas in female mice that were treated with the decolorized whole leaves creams and exposed to a simulated solar like. There was no effect of decolorized whole leaf green treatment on the incidence of squamous cell carcinoma [ indiscernible ]. However, there was a decrease in the incidence of squamous cell carcinoma in female mines. This might be partially related to the fact that in this study, animals were removed when the skin lesions reached five mm and squamous teefourteen carcinomas tend to be a larger neoplasm, since the animals were removed early, they might not have had time to develop. In animals that were treated with the Aloe-emodin to creams and exposed to the SSL, there was an increased incidence of squamous cell papillon as in the female mice. There was no effect on Aloe-emodin claim treatment on the--Carcinoma or in India some combination of all neoplasms. Multiplicity, again, it refers to the number of neoplasms pronounced in the at risk treatment group to go in this and the following slides the dapper dagger shown in red preferred to significant dose trends while the times refers to significant [ indiscernible ] comparisons. In animals that received no cream treatment, there weren't significant dose trend increases in the multiplicity of squamous cell neoplasms, regardless of type or combination in male and female mice. In terms of their wise comparisons with mice that receive no cream treatment and no exposure to a simulated solar light, significant differences were observed for all neoplasm types and combination with the exception of carcinomas. Again, this might have been due to the early removal of the animals based on skin lesion sides. --Size. The it multiplicity of squamous cell papillomas and the combination of all neoplasms showed significant dose trend increases in female mice that were treated with the aloe gel cranes and exposed to simulated solar light and and pear-Wise comparison tests, but the three and 6% dose levels of the aloe gel were significant compared with controlled cream animals. Similar effects were not observed with a males. In female mice that were treated with the whole leaf cranes and exposed to simulated solar might as well in the male mice, we saw a significant dose trend increases and the multiplicity of papillomas and in male mice we saw a significant increase in the combination of all neoplasm types. In pair-wise test, their work--Compared to the control cream mice. At these 6% dose level as well as in pair-wise comparison tests in the combination of all neoplasms and all male mice. There was a significant dose trend increase in the multiplicity of squamous cell Pat,s and in the combination of all neoplasms in female mice that were treated with the decolorized whole cream and exposed to solar like an ink pair-wise comparisons, both three and 6% doses of the decolorized whole leaf were significant when compared with control animals. As with the Aloe vera plan extracts, the topical treatment of aloe claims showed a significant dose trend increase in the multiplicity of squamous cell papillomas and in the some combination of all neoplasms in female mice and add the 6% dose level and comparison test of control animals, the multiplicity of squamous teeforteen papillomas carcinoma as well as the some combination of all neoplasms was significant. So, in conclusion, the topical treatment of mice with the aloe gel cranes and the exposure to it simulated solar light had little effect on the incidence, multiplicity and onset of in-life determined skin lesions. Similarly, there was no effect of aloe gel cream treatment on the incidence of squamous cell neoplasms. Of course, this was close to 100% among all treatment groups. There was an increase in the multiplicity of squamous cell papillomas that was detected in female mice. There was no effect on the in-life incidence multiplicity of in-life detected skin lesions or on the incidence of squamous cell and neoplasms and mice that received the whole leaf cream treatments and exposed to simulated solar light. As with the aloe gel, there was an increase multiplicity of squamous cell neoplasms in female and male mice. There were no significant differences on at the in-life determined--Of skin lesions or on the incidence of squamous cell and neoplasms in mice that received the decolorized whole leaf green treatment and exposure to simulated solar like. As with the other test articles, there was an increase and the multiplicity of squamous neoplasms in male and female mice. The it treatment of mice with the Aloe-emodin screens, which was our purported positive control and exposure of mice to simulated solar light had no effect on the in-life determined skin lesion on cent incidence or multiplicity or on the incidence of squamous neoplasm perhaps brother was an increase in the multiplicity of squamous neoplasms in female. That concludes.
Thank you, Dr. Boudreau. I think .
This is a really complicated design and results and is kind of hard to get your hands around. I would like you to comment on the difference between the results for the multiplicity analyses for the specific data and the pathological data, the results are very different. Is a nothing with using the in-life data but do with the pathological data. If I understand the analysis, which I am not assured that I do, I would think that the in-life data would take into account the time in which these regions occur and the pathological analysis would not. We should give you greater power for the in-life analysis than for the pathological data. I was curious about that.
The in-life does give you on said, which, of course, we cannot determine histopathological. Animation on set is different. The incidence and both the in-life and the at histopathological approached 100% in both cases broke and they were not different. In terms of multiplicity, though, we were not able to resolve the differences between the histopathological to murder counts or neoplasm accounts and the skin lesions that were observed on digital photographs. We thought they should be similar, at least show similar trends. We did not find that. We are attempting to resolve these matters.
Dr. Howard?
I just wanted to point out-Let me get my glasses on, on page 121,--For those that wanted to see them and they were not on the screen. To supporting what Mary said is that we are counting bombs on the in-life. They are segregated at histopathological into [ indiscernible ]. We are counting funds that are consistent with the formation of a legion to go so, there are really two different things that are being counted. Oneis looking at a tumor type and one is looking at a bought.
In addition, one pump might be two different neoplasms or more.
Although the multiplicity of tumors increased, the multiplicity of non-neoplastic decrease. If you add the some of them, it would probably be pretty equal to the in-life legions. That is the way that I am interpreting it.
You presumably have the data that you can correlate the bumps with the Legion. You are saying that they do not correlate?
No. We are in the process of attempting to correlate the data. We have digital photographs from both. The trace a gross lesions are studied by the that biologist at the time of necropsy. The in-life bumps that we are counting-A committee examined all of these photographs and counted the lesions on these Ken. We, technically, should be able to trace trace Leeson's with the number of bumps. We have to examine the pathology, digital photographs in order to do that. We have those now. We are in the process of being able to analyze this correlation. That was not part of the steady. The correlation is not part of the study data.
Okay. Thank you. So, people move on to the reviewers. Dr. Crump?
First of all, I think I agree with the conclusions of this report. My comments are pretty much editorial. I do have a lot of editorial comments about this report. This is, obviously, much more complicated than the 2-Year design. Unique features include the dual exposures to light and to the cream. You have the in-life data and the pathological data. To have the multiplicity of verse is the incidence. There are a lot of issues that you do not typically have with the M2-Year-Standard and 2-Year standard to go to the authors have done a great job in synthesizing all of this data and laying it all out for us to look at in terms of tables and figures. The 2-If you're bioassay, we have a standard template and still comfortable with that part of the analyses are standard. That is not the case with these. I think we need to move in that direction. I did have a lot of editorial questions, particularly about the statistical analysis. I will not go into detail on these. I have written comments that I have provided. Just to give you a couple of ideas of what I am talking about, first of all, I am talking about the multiplicity analysis and multiplicity date the. Riding the incidence data are fairly standard. There are no references in the report that I could find to the statistical methods that were used with these data. I do not know-I feel pretty confident that they are all okay. I do not quite understand what was done. There are notation and terminology used in the reports that are not explained. I do not understand what the terminology is. Another example, it says the same method of analysis was applied to the in-life data as to the terminal data without comment to go it seems to me those data are different and you must have time of onset for the in-life data. There is no statement that those data were analyzed differently. So, I would really like to see a much better job done of explaining exactly what was done in the statistical analysis and lay it out very clearly. I think what we should aim for is a given the explanation and given the data, someone should be able to reproduce the analyses. I think that is true with the two-year bioassay that we have been using. We need to move towards that with this data as well. Regarding the conclusions, as I said, I think I am generally in agreement with them. As stated, they are very difficult to follow. I need the mistake, I think of reading the conclusions first before I read the report. I was sitting there scratching my head trying to figure out what the conclusions are not contradictory. Then, I realized that the key phrase there is in-life versus one sentence says in-life and the other does not have in-life at the that is the key difference. I think there are some ways that we can simplify these analyses and make them more clear. We can just say right off-Right at the front is that nothing was significant using the in-life data and not have to repeat that every time. I guess I am also a little bit not an easy with the distinguishing between incidence and multiplicity put up there is no evidence on incidence but there is evidence on multiplicity. Basically, my thinking is multiplicity is just a more general way of looking at incidence. The role critical question is, did you get an increase in these lesions? I do not really think we need to get into that Lovell of detail in the summary conclusions. I think that would also simplify the conclusions. Actually, I tried my own hand at wordsmithing. I will hold off on that and see what is presented here. That is all. Thank you.
Thank you. Dr. Cattley?
Dr. Cattley: I have some comments about information that I thought would be useful to clarify the findings and gain perspective on them. I do not have any question about the actual data itself. Let me just mention that you did talk about the positive control Aloe-emodin. I might have missed it, but I was curious as to how much Aloe-emodin was present in the other three treatments? It seems like you are left with a result where your positive control did not exactly to what some of the other treatments. I do not know if that is due to the amount-Whether the amount of Aloe-emodin might be different between these things or not. I would expect that there might have been less in the other one's. I have no way of relating those doses with what I think you presented as the active ingredient. The other thing is, I was interested in some of the literature that you reviewed in many of the studies used, rather than using simulated solar light, and you used something called UVB rate of exposure. I am interested in the SSL in the current study and UVB in the scientific literature. Did have a reference to a study of Strickland and colleagues that used UV and Aloe-emodin. It produced melanomas. I am baffled by that difference in tumor response. Maybe you have some ideas about why that is. Since you raised the study, I think it would be good to close the loop as to what the difference is, or maybe it is and unexplainable differences. I feel like I might have missed reading, but I was not sure about the study design and why there was a 12 week recovery period after the end of treatment. I think it would help the report if the readers understood what the rationale was for that. I could guess, and it might be there, and I might have missed it. Finally, when it comes to the conclusions, this is a very different study design. I guess I am not sure how we applied some of the criteria that have been used in and the two-year studies. From my standpoint, it would be helpful if the conclusions-I do agree with the comment about the in-life not contributing their much to how we would interpret the it steady at this point. From my standpoint, it would be of interest to delineate the benign from the moment and turn malignant neoplastic in these groups. For the concluding statement when you talk about the significant effect on multiplicity, you are really looking at a combination of all neoplasms. I would be interested in knowing for each active ingredients, which the differentiation was between the response of the papillomas versus the other two malignant tides of lesions, the [ indiscernible ] and squamous cell carcinoma. I think that is it for me. Thank you.
Thank you. Dr. Bunton?
Some of my comments have already been mentioned by Dr. Crump and Dr. Cattley. I will just reiterate them as I have them listed here. I did have the same question about what to appeared to be a recovery Group and using a title of a one-year study when the animals were actually exposed four 40 weeks and there was this 12 week period that I was assuming was a recovery, but there is no mention of it that I could see or any explanation of why that occurred or what the results of that were. I think that would be worthwhile to bring forward. I also had some of the same comments about the conclusions. To me, as they are written, they are not really conclusions. They are stating, again, what happened, what the results are. You have not taken the next act like we have done with these other reports and saying what it means. I do not know if that is just the way this is going to be done to. I think we should discuss how we should handle it in this case. There were other editorial things that I did not understand, such as the tables that included whole areas that said, systems examined with no neoplasms observed. Every system was listed. Then, you have not done histopathological. I was not clear about what exactly had happened. There was an area that said histopathology had been stopped, but no reason. Of like to hear more about why. The part of it like to make with that is that this is a large study. If, in the future, there would be any kind of a plan to assess, which is a systemic effect, a toxic effect due to these compounds, you have that data set, you have those tissue samples. I would like to see those used, rather than repeating and doing another steady progress that is an overall impression of what was used in this study as big and complicated as it is. I would like to avoid or at least have considered, if it needs to be taken up again in the future that this information might exist in the tissues that you have from these animals. I did have a question about testing all of the different components, but I think I figured it out afterwards as far as the differences between them all. You also clarified it here. I appreciate that. I think that is about it that is necessary to bring up.
I am going to try to cover the points one at a time. In terms of Dr. Crump's comments, even though they are mostly editorial, we have discussed this, and we will expand the description and the report to provide a better description of the statistical methods that were used, in particular, for the [ indiscernible ] regression model, I believe what you are referring to. In terms of the Aloe-emodin, we do have that data. We will incorporate it into the report to. This was in the characterization. I just fail to include that in the report. I will be happy to do that. That would also include the amounts in the various test articles. I believe you mentioned in terms of the conclusions, that you would like to see the different skin lesion types separated as well as the combination. I believe we can look into that and see if we can include all of that in the conclusions. Then, in terms of the 12th week period following the 40-Week exposure, it was actually an observation period. It was not to recover. This was the induction of skin tumors, a time and those response time to the light to. You do not expose an animal to light and see the tumor develop immediately. This 12 week period was to observe the development of skin lesions on the animals before and sacrificing [ indiscernible ]. In terms of the systems that were mentioned that no histopathology was conducted on, that is a formatting, a template formatting issue that we will try to address. It is automatically produced. We will try to see if there is a way to eliminate that. In terms of the tissues and slides and cassettes, all of that is archived by ID, by treatment. Those tissues, slides, the cassettes that were used on the study, we have those available for any further studies on these compounds.
Before you move on-during that Wellfleet observation. , then, did you see any incidence of regression?
No. We did not seek regression. I am not sure-I would have to go back and look. I am not sure that we saw any further progression either. Perhaps [ indiscernible ] would be able to.
[ AUDIO/SPEAKER NOT CLEAR].
This is Paul Howard. Dr. Crump, you mention that this was not a standard bioassay. You are correct or the this is not a traditional two-year bioassay. This [ indiscernible ] has been used since, I believe, the mid-1980s for drug nominations to the U.S. Food and Drug--This was developed in the mid 1980's and has been perfected to this design received today. Given a similar to those of solar light and then you had this observation period for 12 weeks purpose has been worked out with how many--It is a considerable number. What the NTP has added, those are typically only a in-life observations. We have added is a pathology consideration of these bumps. That is what the NTP has done is bring in the pathology to ask the question of traditional in-life, can we enhance that information with pathology? That is where this differs from what is considered to be a--You are adding pathology.
I believe that I addressed-the UVB rate. Yes, we are going to calculate the UVB rate of these lights and include that in the report. In terms of the Strickland study, there were numerous differences between the study. Besides the type of light, there was a broad band UVB. Ours is simulated solar light. There be Haeckel, they use ethanol and reused water in a cream base. The method of exposure, they exposed vertically and we exposed horizontal the. Our animals are in cages and our light comes from the simulated solar light directly to the front of the cage. The duration of exposures were also a different. It is a difficult to be able to compare the two studies in terms of why they developed melanomas, which, I believe they subsequently said were not really melanomas, but rather, I believe [ indiscernible ] came back and said they were actually deposits of the compounds in the skin at lesions. I feel certain that is what she said. I believe that is about it.
The difference between the Strickland study and Mary's and study is the light intensity. They are delivering three times a week about twice as much effect of light to. You have to remember the fact that UVB is very effective in producing European and tumors. We are mimicking some like. UVB is much less effective. We are giving human exposure--They are delivering a dose of light which it will be more wavelength by wavelength--Overall, they are delivering twice as much light that only three days a week as we discover five days a week. There is much more impact on the skin, if I can use that term.
Okay. Are there any other comments?
These are pretty minor comments. On the summary on page six, the discussion of the aloe gel agreement being discussed, the significant increase in multiplicities squamous lesion neoplasms but on page 82, there is a decrease in multiplicity of the non-neoplastic--It seems for consistency that that should also be mentioned here. Again, I do not think it is a big deal. It reflects the overall decrease in the neoplastic lesions. On the map on page 37, it says that growth skin tumor lesions core by the skin pathologist. --At this point with the grows exam,--Finally, the least minor point is just for figures on tumor multiplicity, the female comes first and then the male comes first. It--
Keep you on your toes broken any other comments from the audience? So if not, can be put the conclusions up on the board? On the screen? Let me ask if there are to be any motions to change the text, specifically?
I would like to hear what other people have to say. To me, maybe it is just me, but they are stating what happened in the study. What does that mean here? As it does with the other studies that we are reviewing, is there carcinogenic activity? It is just stating, again, what the results are, in my mind.
I would second that. With the categories of evidence that we used for the other bioassays, the summaries [ indiscernible ] apply here. Are there some other thing we should go by in order to give a bottom line is to what this all means? The same question.
So, I can comment. This is the second [ indiscernible ] study that we have reviewed. The first was quite Kollek [ indiscernible ] that were used as a defoliant, I guess. In that instance, Dr. Howard was the steady scientist for that one. We came up with some descriptive language that did not really fit the template that we have for the two-year studies. I do not think that we, in trying to fit this information into the template for a two-year study, have been successful in figuring out how they relate, either in the use of the words themselves or from the standpoint of the fact that we do not have a lot of experience in interpreting the outcomes of these. We do not have a sense, clear evidence, in this kind of study, what it really needs in terms of the NTP making a call on it. Paul might want to comment on how the committee dealt with the [ indiscernible ] studies. In that instance, there were actually no changes or protective effects. That is the other thing that you can obviously see in these studies is protected in facts. There were not any hints there that would lead us to see how to best utilize the criteria that we have for the two-year studies. We would like to move in that way. We would like to make these kind of study interpretations more consistent with the interpretations of the two-year studies. In fact, I know that Kenny said he took a shot every writing something. We have taken a shot at writing something. The panel might want to consider these other alternative suggestions at this point.
This is Paul Howard. On behalf of the Food and Drug Administration, and suitable does not defoliate women to go [ LAUGHING ].
I am sorry to go exfoliates.
Thank you. [ LAUGHING ] the go this is on the record ago actually, what Dr. Bucher is saying is in the--Report 524, the conclusion we made states taking into consideration the survival data and pathology results, glycolic acid did not--SSL is the benchmark. We are including it in that light, no pun intended, that it did alter. 84 in [indiscernible], it decreased the effectiveness of the light. --Of the simulated subtler and Chancellor of like. There is no mention of a son, it is stating the fact, the most obvious fact. Dr. Bucher, I do not know if you want to put up the one that you and the NTP staff constructed after you received your report. At more reflects that type of conclusion.
I would ask Dr. Crump, if there is alternate language that he would like to offer first?
Well, I do have alternate language that I put together. Do I want to offer it [ LAUGHING ]. Sure. I will offer. It does not really deal with the question that Dr. Bunton has raised. I think it does-I think it simplifies-I think it makes it more clear on the best way to do it. I am looking at it here on my screen.
While we are rethinking this, why don't we put the language that we have up and you can see if what you have written agrees or disagrees with what we have posted. Can you move these screen over a little, Charles, so that it includes.
Yeah, this is just part of it.
Yeah.
I kind of like the wording. If I wonder if the second sentence is really necessary. Does that add anything? We're talking about incidence here, right? Wait, no, we still have the [ Indiscernible ] life part. Okay.
[ Speaker Faint/Audio Unclear ].
Yeah. Okay. I think they're all saying the same thing. I think people can read that and they can figure out what is going on, so.
Can you go back, Charles, to the first one. Let people really have a chance to read through this. Dr. Bunton, does this address what you are getting at?
Yes. I think it does, certainly. I can appreciate the difficulty with this. This gets better at an actual conclusion based on the results that you found from this study.
Impliflt in this, we struggled with coming up with words to describe the response. The issue is related to whether you put your money on the traditional way in which these studied have been done and interpreted. But does not include the his toe pathology. In that case the words weak effect may not be sufficient. Or if you come down in the middle, which is basically where these come, I think, in that there is no increase in the traditional measures in the study, but an increase in the multiplicity -- therefore at this point this is weak evidence. Or evidence of a weak enhancing activity. But we're open to a complete discussion of this particular issue. This is key to us. We don't have a lot of experience with these studies.
Jon Mirsalis. I like this is a lot better. I read the conclusions and didn't know what to make of it. We're so used to reading this [ Indiscernible ]. These are such different studies, it would be misleading to put it into the conventional. When we talk about an NTP study, this clear, sum equivocal. If you try to apply that to a 40 week study with recovery, it gives maybe more strength to it than it deserves. I don't mean that in a negative way. These are different studies. We need different language to avoid using the conventional NTP language.
Kenny Crump. I think this is better than the first versions. I still have a problem with the second sentence. And the last sentence. Particularly, there was no, did not increase the incidence. I think that's misleading. The reason it didn't is because the incidence was 100%. There was no power to increase the incidence. Pointing that out is giving a misleading direction, maybe, to some people. So, a, as I said earlier, I will prefer to present these results without distinguishing between multiplicity and incidence, they're two sides to the same coin. If you didn't see it with incidence, I think it's a little misleading.
Can I add to that? I think at the lower levels of light intensity the incidence was not topped out. You could have picked up something there, if there was a tendency to increase incidence.
You could say you got it without reference to whether you saw it with incidence or multiplicity. You would also see it with multiplicity there, you should. Is there some biological reason to look differently at incidence and multiplicity? I can't, I don't know of one -- but --
Dr. Howard?
Trying to think how to phrase this. One thing we do know is at a point in time incidence is related to dose of light. When incidence is Maxed out, I think also during the course of onset of tumors multiplicity is also dose related. I don't think they're necessarily connected. The first tumor you see happens to be the one that a biologically uncoupled from growth control. Whereas, I think they're not giving the same information. I think it's a different type of information. One is multiplicity is much more dose-related. And incidence is much more time [ Indiscernible ] dose related. I can't describe it. I wouldn't want to lump them together. In this case where you had near a hundred% incidence your power of deteblghting a difference is so diminished. I would agree with you. If we had used a lower dose of light, definitely incidence would have played into this. [ Speaker Faint/Audio Unclear ].
Any more discussion?
Is it possible to see the second page again?
Yes.
So would anyone like to suggest a change in verbage for the incidence versus multiplicity issue? [ Laughter ] Dr. Crump?
An idea. What my version is. See if you like it.
That's a great idea.
I can read it. I'll read the first part. Once you get the flow you can see. Males and females SKH-1 mice were treated topically with aloe jell creams. Data on skin lesions were [ Speaker Faint/Audio Unclear ] no effects of creams upon skin lesions were identified from data collected during [ Indiscernible ]. Next paragraph, all these paragraphs are the same. Squamous cell [ Indiscernible ] identified in [ Indiscernible ] were increased in female but not male mice treated with aloe jell cream and exposed to SSL. [ Speaker Faint/Audio Unclear ]. The statement is simplified. It doesn't say multiplicity or incidence. Just says that they were increased. And the other conclusions with the other creams are similar.
Yeah, I guess the only difference it gets away from making a conclusion. It's more a statement of the data. Is there more discussion?
I think with the statements up there, what I like it's saying there's a weak enhancing effect. It's okay to use as a different language with these types of study. On the photo carcinogenic activity, to me those are the keys. You are saying what the effect is, and specifically this is -- what you believe is a result of these data. At least in my mind. Versus restating that you have more tumors in a specific group, or increased multiplicity. I'll stop there -- you know what I'm saying. [ Laughter ]
Uh-huh.
You can't read this once. The way it's written. You have to read it twice. You get to the second sentence you feel that you are reading the opposite conclusion. You have to go back and see there's a distinction between multiplicity and incidence. That's always a cumbersome way to present things. I don't know what the solution is. It might to be drop the last sentence. I don't see how to get through this reading it once. If you read it cold.
Russ Cattley. I guess I would like to know that it was based on multiplicity in a conclusion statement. You know, there's ways to word Smith and make it readable. Just dropping the last sentence would also fix it.
This is Mike Pinot. I agree. I do agree with Kenny's comment. I already almost at a hundred%. It's meaningless here.
I agree.
This is Paul Howard. In a tradition assay onset is the most important term. Not incidence.
Because incidence does go to high levels, usually.
It doesn't Max out at the lower doses.
I would also say from modest experience in the field that multiplicity is an enpoint of -- end point of interest to researchers in that area. As any other carcinogenic end point.
Is there any support for the simple solution of dropping the second sentence and saying what we didn't find. But saying what we did find?
I think from a public health stand point [ Speaker Faint/Audio Unclear ] it also serves some utility to state a negative result. As we did in the previous study. That also needs to be weighed as carefully as a positive affect that was detected. That's just my -- I think sometimes negatives are just as important.
This is a different kind of negative. We saw it one analysis, but not in another. We're looking at the same end point. In that sense I wouldn't totally agree with you on that.
It seemed to me, if we use Dr. Crump's first paragraph. And into the second paragraph state the two positive findings, leave off the negative secondary findings, would that work? You summarize the in life data, which indicates latency and incidence as having no effect. If I got it right. The second paragraph was -- or we could add a second paragraph that would speak to the multiplicity and the conclusions there from. Would that work?
[ Speaker Faint/Audio Unclear ].
Yeah. You have one sentence to summarize the in life findings. That was really good to start off with.
No effect of [ Indiscernible ] upon skin lesions were identified from data collected during life.
You could maybe expand on that as to what data you get out of an in life study. In a second paragraph you could do the positive findings.
We could break for lunch and work on a combination statement over that hour.
Jon Mirsalis. I like Kenny's statement. You could probably say that no in life affects were observed. However, [ Indiscernible ] revealed increase in multiplicity -- period. We can argue about whether we're all callout incidence [ Indiscernible ]. But the point is, it's a good point, Paul makes a good point. You want to get in life we didn't see anything -- but, or however -- in a [ Indiscernible ] evaluation some multiplicity was seen. A single sentence that nothing in life but -- [ Indiscernible ] would give the best feel for what we are trying to convey.
Use the term weak? Weak enhancing then?
You could say however, weak enhancing effect was seen based on increased multiplicity in the his toe pathlogical evaluation, something like that.
John? Did you want to say something?
I think it's important to make sure we can agree on language so we can work on this.
[ Speaker Faint/Audio Unclear ].
Will you identify yourself?
Ron Melnick. You have an unidentified modifier. Which could be problematic in future studies. If future studies come you need to define that. What are the criteria more weak or heavier different level of evidence. I would uncomfortable with using the word weak without a definition of what you mean.
Ron always keeps us honest. [ Laughter ]
Okay, whoever will word Smith is -- we'll break for lunch.
What time back?
1:00.
Session on lunch break until 1:00 eastern time. Please standby for realtime captioned text.
As we are waiting, please read the version that is on the slide.
Good afternoon. I'd think we can resume the discussion on the revised conclusions of the Aloe vera study. If everybody on the committee has read the revised version, is there any discussion lacks Dr. Howard? The map on the last word of the paragraph, should it be a life or in-life?
I suggest during the in-life phase.
I am still thinking about Ron's comment before lunch about where do we mean by weak? I confess that I do not have a better word for it to. It gets to the old minimal, my, moderate Mark. What is weak? What is strong? Without a clear definition of what that means, this kind of ambiguous. On the other hand, the sentence is kind of ambiguous. I am a little uncomfortable with the word weak, unless I know what weak means.
When should never disagree with one's Supervisor. In all of the conclusions on the NTP studies, it has modifiers of strong, equivocal, clear. It is consistent with that.
It is consistent in the sense that we have a modifier. I am not sure that is consistent in any other way. If we leave it there, we will set a precedent of what we mean by weak. I am not sure that we have thought about this enough to really set that precedent.
Dr. Mirsalis: I do not recall the statistics on this. Was is it statistically significant effect? Do we say a statistically significant enhancement and not get into weak, strong, super strong, moderate, clear and as a statistically significant enhancement?
Can I make a suggestion? We had intended to quantify the defects in this study and even back to the alpha and beta hydroxy studies. Basing them on the SSL equivalent, that is how much the [ indiscernible ] compound represents as a change in the SSL activity. Would that be sufficient to quantify weak? That had been suggested originally in a [ indiscernible ] meeting. It was always a good idea. We have not put it into the report yet.
Dr. Howard: We can do that for Ansett because we do have multiple doses of like. Can we do that with multiplicity on the pact ology?
Sure. Can do it with any endpoint in the study. We measure it on all of them to go in a sense that is quantifying something against nothing. People will have a understanding of what a particular light level represents as a carcinogenic activity. Is it sufficient to resolve the issue of weak? -week-old I am not sure how that would translate here.
You have a certain level of activity on the, let's say, multiplicity. We have the scale. Oneof the reasons we did the no cream SSL group is to affect the compound to have a like equivalent to it. We would quantify the effect of Aloe vera, whole leaf, let's say, on a unit basis of how much like it would take to give the equivalent effect. If that is a seemingly minuscule amounts of light, then, probably, you would generally agree that that is a weak defect.
Dr. Crump: I think that is a good idea but is estimating potency, which goes beyond what NTP normally does. I could go with not having a quantifier and could go with statistically significant.
How about statistically significant? Does everyone buy into that?
I guess I would not use statistically significant therefore the enhancing effect. That is not what you measure the statistically significant. That was for the increase multiplicity of tumors. If you stay distich Lee on--Increase and the multiplicity of tumors, what was actually significantly increased, not the enhancing effect-That was the conclusion based off of that observation.
I will just add one more comment. This is Dr. Kerkvliet. There was a we enhancing effect. We are defining a weak enhancing effect. If you put one more modifier in there, you could qualify it. There appeared to be. There was a potential for, one more qualifier that takes it away from the defining weak. I'd like to just throw that out.
This is an expert panel to go this is your subject of expert opinion on weak. Do you agree that it is a weak defect. You are trying to get to define specifically what weak is. You are getting into a subjective interpreted call here and not the point of we of what we are asked to do based on the data.
I guess I would agree with this. And a lot of time you are going from an average of 6 to 8 or nine. To me, that is a weak defect.
So, with someone like to put a motion for to accept the conclusions as written?
Dr. Mirsalis. I will accepted as written.
Any more discussion? A second? Thank you, Dr. Novak. Shall we vote? All those in favor of the conclusions as written. It is a unanimous approval. So, moving on. We will now be discussing the Chromium picolinate monohydrate Report. Dr. Stout?
Good afternoon, everybody. I am a Dr. Stout. I will talk about the toxicology and carcinogenesis studies of the Chromium picolinate monohydrate. Before I get into the study, I would like to talk a little bit about the form of chromium in this compound. Chromium picolinate monohydrate is a dietary supplement that contains chromium and the unsettled state. To my recall that at the last technical report meeting we talked about the [ indiscernible ] which have the chromium and a different [ indiscernible ] state. Is an essential element. It is found in many foods and is involved in lipid metabolism. It is taken for weight loss and the entire diabetic effects. The oral absorption of this metal is poor. [ indiscernible ] is thought to increase the tissue absorption. It is at present in a [ indiscernible ] form. The typical daily doses contain approximately 2 00 to 600 micrograms of [ indiscernible ] chromium which comes out about 3 to 9 micrograms of chromium three per kilogram per day for a normal individual. So, chromium picolinate was nominated by the National Cancer Institute and a private individual because of the potential for widespread consumer exposure through its use as a supplement. The monohydrate form was selected as the test article because that was what was commercially available and feed was selected as the route because humans and just chromium picolinate and supplement's. I have the structure at the bottom right corner. As you will see the [ indiscernible ], and insulated by three molecules of a and associated with one molecule of water. The NTP conducted a pre year and its--And beat in both female and female F-344/N fish and B6C3F1 Weiss. The concentrations 43 month studies went from 0 to 50,000 parts per million. That top dose of the 50,000 or 5% is considered to be a limit doughs and feed studies. So, in male and female rats and mice, there were no biologically significant changes in any of the end points that we measured. These included survival, body weight, speed conception, hematology, clinical chemistry, where and which, histopathology or the sperm apology and vaginal cytology parameters. The lack of a body weight effect is of no--People in just this supplement in part for weight loss defects. So, the NTP conducted genetic toxicity studies as well. Part of these studies were part of the three month studies. In vitro--Were conducted in salmonella, on both Chromium picolinate monohydrate and a compound characterized as not the model hydrate form. All of the strengths tested in the presence and absence of the metabolizing fraction. There was no evidence of Munich tenacity. [ indiscernible ] were also conducted in mice as part of the 13 weeks a study produced these results were negative and male mice and equivocal and female mice. An additional study was conducted--Three times one they are part. These results were negative as well. Following the three month studies, two-year studies were conducted. The exposure concentrations were the three highest concentrations from the three month study is. No defect was observed. Again, there were no biologically significant changes in the survival, body weight, feed consumption common incidence as of non-neoplastic reasons--Or in male or female mice. Again, I would note the lack of a body weight in effect in terms of what the compound is consumed for. So, in male rats, we did observe an increased incidence of t he--At the 10,000 doses, which we considered that might have been related to exposure to the compound. I have these data listed on a table. Deeply to show gland is the male accessory sex glands and program--Is the corresponding gland. Adjutancy in both male and females, there was no evidence of hyperplasia. In female, there were no increases in neoplasms. And males at the meadows and we have an increase that was statistically significant and exceeded the historical control range when which went up to about 10% for the studies and studies of all [ indiscernible ]. We considered that to be an equivocal finding. In order to support the toxicity and carcinogenesis studies, w e--and excretion studies as well as tissue distribution studies to look at the distribution of the compound. For the [ indiscernible ] studies, rats and mice were given a single dose of a--Of Chromium picolinate monohydrate and urine was collected for 48 hours. Chromium and--Each part of the complex could be determined separately. In looking at, and excretion, a common expression in the species was must greater than The urinary excretion, while in the case of the radiolabeled the excretion was similar to the urine PCs and most of the urinary excretion was in the form of [ indiscernible ] which was proposed to be the reaction of a [indiscernible]. These data suggest there was--That led to the conclusion that most of the acid was not bound to the during absorption--Is thought to be very well and, also suggests that this was a study of not only chromium picolinate and picolinic acid acid, separately. As part of the two-year studies, coming to shoot distribution was analyzed. Additional groups of male rats and female mice were treated the same as in study animals and were removed from a study on days of four, 11 and 180. Adding in a 48 hour watch out three in to remove unobserved chromium were common during was collected on basics, 13 and 182. In addition to urine and feces, erythrocytes, plasma, liver, kidney,--Were collected as well for a total chromium Analysis. So, in terms of looking at the results of these studies in both rats and mice, increases in total chromium were most apparent and kidney, liver and plasma and these concentrations were highest and right kidney and mice Oliver. In these tissues, primarily, but there was also evidence and other tissues, you saw when looking at both tissue concentration against exposure concentration or exposure to ration, you saw a plateau of total chromium concentration. On both of these Graves, either the exposure to ration goes from day six, 13 or 182. Of the doses go from control of two 50,000. You can see the leveling off of tissue chromium, which suggested to us that the IRA come in concentrations could not be achieved. These results also did indicate that there was systemic exposure to [ indiscernible ] chromium, despite its low absorption. So, the conclusion of the two-year studies on Chromium picolinate monohydrate, there was evidence of carcinogenic activity and Mail F-344/N rats in precocial planned adenoma. There was no evidence in female rats or male or female mice.
Thank you, Vt.. That was very nice. Are there any specific questions before we go into the reviews? If not, Dr. Cattley?
Dr. Cattley: This is a fairly straightforward study, survival and body weight gain was a reasonable. I think it was a very adequate test of the carcinogenicity of this model. At the level of treatment, certainly, was achieved at a very high rate and definitely covered the range of concern that probably lead to its nomination for evaluation. I think that the findings are adequately dealt with in terms of your explanation and also the interpretation put go I have one slight commented in the discussion where there is a mention of explaining a lack of exposure concentration response relationship for the proposal of land adenomas. If it is an equivocal finding, I would suggest we do not talk about an exposure response relationship on it. Other than that, I would agree with your conclusions.
Dr. Mirsalis?
Dr. Mirsalis: I would echo most of those comments. It was a well written report and straightforward. I agree with most of the conclusions with one minor point. I would say that the correlation in the chromium tissue levels and the results was very nicely done. Kudos to the people who designed and conducted the studies. I think that adds a lot to the report. Just a couple of someone in a picky things. We offer our outside experiences to those proposed last year our institution had its triannual [ indiscernible ] at inspection and a FDA inspection applicable points that I will bring up relative to that purpose might just be a point of clarification. I notice that we do carcinogenicity studies, we will spell out two or three animals per cage and the 13 weeks study is five animals per cage. It is a note to the people during the studies, is that keeping a five per cage four 13 weeks within the current NRC guidelines. We are getting hammered-We have to start pulling them down to three or two per cage when they reach a certain weight. We are doing it for the carcinogenicity studies, I would look at it for that and all studies whether or not-We are in fact, monitoring weight and when you reach that capacity, which is spelled out pretty clearly in the [ indiscernible ] regulations, that you are pulling down the number of animals per club that is something that struck me, I am sure I have read these reports 50 times before but after getting dinged on it by [ indiscernible ], it is now on my mind. Another thing, related to feed, this is a common form of reports but generally on this, this is a feed study, but you do not sit with the feed as. If you go back into appendix K, you can say that is [ indiscernible ]. Is a dispelling it out in the body of the report when the safety, especially when it is a dosed study. And other trivial point. I know that we officially claim these studies are all under FDA [ indiscernible ]. FDA is now mandating that you do independent verification of the impurities of feed. Simply getting a certified feet with a Certificate of Analysis is no longer adequate under FDA guidelines. You have to do an independent assessment provided another one we learned the hard way last year produced of this has been brought on by the Chinese but the debacle of a couple of years ago put that is a requirement that I guarantee no one is complying with including any of your contractors. We got a 483 on it to go anyway, that is just something to bring a three-year program, go back to your contractors on a feed. You have to do periodically analysis, even if you are buying and analyzed Chou. They say they no longer trust the vendors and their analysis. Another, again, this is what my staff calls a Johnism would like to see a number-I will bring this up on body weights. It would be easy to say increased 8% over control instead of saying increased predictably 3% with a type variants. Again, that is something else. My one subject of a point is about the micra nucleus of results. We are calling the male negative and female equivocal. I did a little quick analysis of all the reports we are covering in these two days. The range of Micron nucleus for the seas and mice is part five to 3.9 per thousand. One to 4 comment with the industry gets in the Reports. That is fine. In the male, the council was to play eight and today was 2.9 to [ indiscernible ]. We call it not significant role in the female the control was 2.1, a little while and the range was one printery to 3.4, all the [ indiscernible ] were negative to go now, all the sudden, it is a equivocal did not toxic agent. Certainly, if you sent is that as a plan compound to a testing laboratory, this would not be as-Well within the historical ranges. If that control instead been to .1 had been 2.4, which is about three micra nuclei out of 10 animals scored, that equivocal would have gone away. I question equivocal put my opinion on it would be that it would be a clear negative because it is so well within the historic ranges. I understand you are erring on the very conservative side for risk identification. I do not think the data hold up to the difference in the males between the controls and the highest response was greater than the females yet you called the male negative. Other than that, I thought it was a very good report.
Thank you Dr. Novak?
Dr. Novak: I think this was an excellent study with the total analysis and the data and everything. I support the conclusions. I have no other further comments.
Great. [indiscernible], which like to respond?
I would like to divide the reviewers for their comments. In response to Dr. Cattley a comment about responsible, delete that from the text, in response to the comments on housing, we went back and looked. Our five per cage does meet the [ indiscernible ] requirements.
Just to clarify, it is based on total body weight in the cages broke you tend to use of Fishers, which stand to stay small logger. You very, very quickly within two or 36 into the study by the time they are about nine weeks of age, will not need it. It is not stray animal number, it has to do with what the weight of the animal is. If you are checking and are fine, I am good. I just wanted to call it to your attention to check it.
Thank you. In response to the comment regarding the diet, we do analyze each lot of seat independently. There is an independent analysis per vote during the course of each study, an animal will receive several lots of the. We go beyond the periodic analysis that is recommended and the GOPs. Speaking of the comment about micro nucleus, we do agree that this response is not impressive. We considered a response equivocal if there is a significant trend or a single group is elevated. It does meet our criteria for an equivocal response. Again, we agree that is not tremendously an impressive finding. We will at the detail on the increases in Oregon leads to the report as well. --Organ weights to the report as well.
Thank you. Either any other comments from the committee? Yes, Dr. Howard, please.
Dr. Howard: I have a minor one. On page 17 you refer to the typical daily dose of Chromium picolinate monohydrate to--Of a fight to have a reference at and see if that is a dietary supplement or normal diet. You do say recent reviews--Just please for clarification point out where you got that information and if it does to refer to regular diet or dietary supplement. Thank you.
Any other comments? They're being none, which is like to display the conclusions? --Would you like to display the conclusions? If I could have a motion on the conclusions.
Dr. Cattley so moved.
Dr. Mirsalis has second.
Any discussion lacks all those in favor? --Any discussion? Opposed? It is unanimous. Let's move on to the next chemical, one, 2-Dibromo-2, 4-Dicyanobutane. Dr. Dunnick?
Good afternoon. We did toxicology and carcinogenesis studies of Dibromo to [ indiscernible ] dicyanobutane. I served as a study it scientist and Dr. Herbert did the pathology. This chemical was nominated for study by the NIEHS because of its widespread use in over-the-counter healthcare products and because of the lack of toxicology and carcinogenesis studies reported in the literature. The demo group of administration was to mimic the human exposure. The chemical was not mutagenic in salmonella or in other tests for Gina toxicity. The study design for this chemical was that the chemical was administered by the turmoil administration to male to a female in which the doses were from zero, up 37.52600 milligrams and rats and--In these studies we administered the chemical at a constant concentration varying the dose based on body weight per go toward rats, 0.5 milliliters per kilogram of weight and an mice 2 milligrams per body weight. Intestine the studies showed quite a bit of skin toxicity. In subsequent studies in the 13th week studies, the high dose was 18 milligrams per kilogram and rats and mice and the to your studies, the high dose is 18 milligrams per kilogram and rats and six mg per kg and mice. In the 14 day and studies, the primary finding was skin toxicity, as I just mentioned in all dose groups of rats and mice. The body weight presented here as a percent of control body weight was generally similar to that of controls and not statistically different by pair-wise comparison to the control group to go up the high dose with 18 milligrams per kilogram per go the survival was good and all dose grips. Oneanimal did die early in a female rap group and were not able to determine the cause of death provoked the body weight by a pair-wise comparison were similar in the treated groups to the control group. In male rats as presented in this finding there were a series of skin lesions including hyperplasia in the treated groups, [ indiscernible ] and some information. And female rats, the similar type of lesions were seen. At the high dose groups, while there were skin lesions, they were not considered to be severe enough to compromise a two-year study. The high dose was kept at 18 milligrams per kilogram for the two-year study in rats. Annette mice, skin lesions were, again, seen here presented for male mice including hyperplasia. However at the height dose, there were some Nicklauses and ulcers in the male mice. Thus, the high dose selected for the two-year study was 6 milligrams per kilogram. In female mice, we also have one or two animals with the necrosis as well as these other lesions of hyperplasia and some inflammation in the dermis as with male mice, a 6 milligrams per kilogram. Now, at turning to the two-year survival data in rats-Add to your body weight data and rats, the body weights were, in general, similar threat most of the study, a little decrease in some of the high dose and survival was a good. But, again, we saw the scan lesions that we have seen in the pre-Chronic studies including hyperplasia some hyperkeratosis and inflammation of the dermis and male rats as was found and female rats, the hyperplasia, particularly at the high dose. 82 of the animals presented with other findings such as the necrosis that we had seen and the inflammation in the dark as. Even though there were the series of skin lesions, there was no evidence for a carcinogenic response at the site of application or in other target organs and throughout the body. We did a full pathological examination. Survival was good interested groups of mice. And body weight were, in general, similar between treated groups and controls brought most of the studies. The primary target organ was the skin in mice common share presented for male mice, where there was hyperplasia and a few other animals presented with inflammation of the dermis and one or two ulcers. Again, no carcinogenic response in the skin or other organs in male mice. While there again boar's skin lesions and female mice, there was no evidence of a carcinogenic response. In conclusion, there was no evidence of a carcinogenic activity in male or female rats or mice. Dermal administration of Dibromo dicyanobutane--In both rats and mice.
Thank you, a very much. Are there any general questions before we move to the review? If there are none, Dr. Bradfield.
Dr. Bradfield: Overall about this was an inappropriate toxicology and carcinogenic study's purpose of the protocols and this is an exposure route all seemed rational. I really did not have any substantive issues with the report. I did notice one scientific criticism, which I think maybe had some fire my way. It seems to me that this time that NTP start to include transcription all profiling in their toxicology reports. It seems like a great example where I start to say to myself, did this compound get anywhere? I realize there are all of these sort of arguments about half life and absorption rates that are in the literature. Is started to make me think that I could get a lot of information out of this and all of the other studies I have reviewed if I had some information on profiling. I think it could be as sensitive, as powerful as any of the slides we are looking at that happen to be stained with [ indiscernible ]. I think we could stain them with [ indiscernible ] and get it some, I think, very powerful data that might drive some people's decisions. That is my one scientific criticism of the reports that I am reading is that I'd like to see profiling data on all of them.
Thank you. Dr. Mirsalis?
Dr. Mirsalis: This is another very nice report. If you keep reading very good reports without any errors in them, you do not need as. You ought to sneak in something every now and then to try to trip us up. I do not have any major criticisms, it is a couple of minor points per I put a note in the written comments about it being confused and between the varying the volume and concentration and not that I read the text it makes % to me. When I reviewed it, it did not. I do not know if it is the wording or not. Ignore my written comment on that. Again, another point I made on the previous report, when you refer to something been significant in the text, but to seek a number like the main body weight was significantly less, I would say, 8% or something like that. That would be helpful to tell the reader while you are reading it what the actual value as. Some of these are approaching the 10% mark, which is a fairly significant decline in body weight. The one Scientific comment is that I know in the past NTP has done something like PCNA--I would love to have seen self proliferation on this. I do not know if you have that data and did not put it in the report. I know you still have the blocks and could do PCNA. For something like this it could have been a useful and point to go in the written comments I gave I gave to comment on the [ indiscernible ] Miller paper where they did--and then did proliferation side-by-side with it. It makes for a nice story. I do not know if you have that data and did not put it in. That is an observation that I think would have read the report better. Otherwise, I put it was a very nice report. It was a very nice report and I agree with the conclusions.
Thank you. Dr. Bunton?
Dr. Bunton: I agree. It was a nicely written report. I was curious. There were comments that I have that I did add to this sheet. In your presentation you answered them all. So, I am not sure, maybe we have a psychic connection or something I did have a general question, though, about the negative trends that we are seeing in this study, and if you have any thoughts on that? And even as you went through-I believe and some of the non-neoplastic lesions they had a lower incidence at higher doses. So did some of the tumors.
Okay. Is that the end of your comments? Are you finished? Okay.
Dr. Dunnick?
Dr. Dunnick: Thank you I very much for your comments. I will add the comments of that were suggested. On the negative trends, the one that we did bring forth to the abstract was a decrease in mammary gland tumors and female rats where we saw 17 in the control, 19, eight and nine in the high dose. [ indiscernible ] we have a special statistic we do to make sure if this was related to any body weight per call this a decrease was not related to any body weight in fact. The eight and nine in the mid and high dose Group, a [ indiscernible ] was lower than that, seeing the historical control data that we have for this. Therefore, we do feel it might be chemically related. We do not have a mechanism that could explain this to decrease. We do know it this in the discussion. There were some decreases also observed in the pituitary gland tumors and female rats. These decreases that work in the historical range--We did not feel that this could be related to the chemical as also was the case for a liver tumors Annette mice. Their work, perhaps, some of decreasing in male mice by numerical analysis, 41 in the control and a 31 in the high dose. This 31 was within the historical range for liver, as I think you might have seen, there is some variability from a study to a study. We could not determine that this was clearly related. We do note of this in the discussion that these incidence, for some of these tumors are within the historical control range and these decrees incidence could not be clearly related to chemical Administration. We do want to do studies where ever warranted that further explain results, such as further analysis by Texaco --We appreciate your recommendations on this. We do have a NTP [ indiscernible ] faculty which [ indiscernible ] heads up. He has gathered a number of experts to assist him. There are many people here property expert in the field. We do bring projects forward for consideration for microarray and I will take your suggestions on this, more interest in this whole topic and Dr. Walker and his colleagues will discuss it further as we will your recommendation for PCNA staining.
Great. Are there any other comments? From the audience, any comments?
I have no one question for clarification. In the two week rats said there was a decrease in the thyroid gland weight I went to the end civil study to see if there was anything there. The thyroid was not wait in the three month study. It is typical that you do not weigh the the rights but they are weighed in the two week study is. I was curious as to why that was done like that?
Over the years we have had several target organs that we do away. We do additionally add organs to this. Midweek could have done that in the 13 weeks studies additionally. I guess we were not able to capture that data.
That is interesting. I did not realize that. I will have to go back and look at that. Concerning the issue of the routine microarray analysis, I do not know whether Nigel by to make a comment about this. I suspect we will have a chance to talk about this tomorrow. I would encourage you all, actually, to give this thought overnight to. We have considered throughout the years the rather routine collection of tissues during to your studies--Two-year studies for genetic analyses and for a number of technical reasons have not done not. The [ indiscernible ] faculties that Nigel had of has argued the point whether they should be a screening activity or hypophysis driven activity perked up as we have always come down on what we thought was a more prudent approach with a high profit this driven [ indiscernible ] that we would have studies specifically that were for that purpose. As technology moves along and you are able to do more with tissues that are fixed and things of that nature, I would encourage you all to give thought to it and give us advice as to what you think might be appropriate at this stage as we take this program forward.
Any other comments? If not, may we see the conclusions? I would entertain a motion on this conclusion.
Dr. Mirsalis, I move to accept.
Second? Dr. Bradfield? Of those in favor of the motion? It looks unanimous to me. Thank you, very much. Dr. Bucher is going to speak for a moment.
When we put together the schedule for this meeting, we had anticipated having one more report today and perhaps a three tomorrow probably put out a Federal Register announcement suggesting we would have three reports for tomorrow and we have some public comments on those reports. At this stage, I think all of those folks that have been emitting you and today with the knowledge you could not respond are going to be disappointed. I think you will have time to respond to your e-mail. We will have to take up the rest of the business in the morning, I believe. You can be more efficient wintertimes.
Thank you, John. Do you have closing comments for today's?
I do not have any comments. I need to know what time I can take you back to the Hotel. I will have to scurry around and see if we can get the bus. If you can hang around here, and as a novel so that I can let you know.
Barbara, did you want to say anything about dinner?
As far as dinner is concerned, the bus will pick you up at the hotel at 6:30. We will meet at 7:00 at [ indiscernible ] for those that are coming on their own, separate the part of the bus will take you back to the hotel around 9:00 or earlier depending on what time we are finished. Tomorrow morning, the bus will pick you up at 9:00 to take you here for the meeting to start at 8:30. No breakfast briefing tomorrow, just read into the meeting. Thank you.
Thank you. Thanks, everybody.
Day 2
Event Started: 2/28/2008 8:16:04 AM ETGood morning. I would like to call the meeting to order. We'll begin the day with introductions.
[ Indiscernible ].
Dave Malarkey.
John Peckham.
Grace kiss link.
[ Indiscernible ].
Tracy Bunton.
Mike Pino.
Mark [ Indiscernible ].
Paul Howard.
Kenny Crump.
Russ Cattley.
Chris Bradfield.
Keith Soper.
Mike Novak.
[ Indiscernible ]. John Bucher.
Mary wolf.
Susan Elmore.
Charles all Don.
[ Indiscernible ].
[ Indiscernible ].
Bill Iverson.
Ron Lawrenceson.
Robert [ Indiscernible ].
[ Indiscernible ].
Rick Irwin.
Angela king Herbert.
Bill Foster.
Matt Stout.
Cynthia Smith.
[ Indiscernible ].
[ Indiscernible ].
[ Indiscernible ].
Christine [ Indiscernible ].
Ron Herbert.
Greg [ Indiscernible ].
Okay. Thank you. I'm Nancy Kerkvliet. Dr. Shane, did you have announcements?
Good morning. Please make sure that you have signed in again today. Please use the microphone and identify yourself when you make a comment. It's being taped and video cast. If you are making comments from the public and you have not registered, please register outside if you need to make a comment today. The draft reviews are preliminary reviews and will not be included in the minutes. The board members must be verbalize any comments they wish to be stated. I will now read a conflict of interest statement. The members of the NTP board of scientific counselors technical review serve as individual scientists. Each member is to exercise judgment prior to any meeting as to whether a potential conflict of interest might exist relative to topics for discussion by the board. Should there be a potential conflict the member is to decline services is a reviewer for the relevant report and not to participate in the discussion or vote. There are no conflicts of interest today. Thank you.
Thank you, Barbara. The format for today is the same as yesterday. We'll have the study scientist and if necessary the pathologist make presentations. There will be questioned asked of them. Then the principal reviews will present their reviews and make recommendations. Then others may make comments and then continued discussion. The first chemical this morning is Estagole with Dr. Bristol.
Thank you. I'm here today to present the results of our 3 month toxicity studies of Estagole, which is a naturally occurring exon. It -- compound.
It occurs in plants. It's 95 to 97% of the oil from the buds of this plant. It occurs naturally in a variety of foods. It has commercial sources. The distilled from the oil of term tine -- terpintine. Its uses are derived from its odor and sweet taste. We call it a diettary chemical. It's in a variety of foods and beverages. It's in baked goods, for example. It's in a whole variety of personal care products. It's used in central and south America as a medicine. In the southern U.S. it's used as a spice. It has antimicrobial activity. I wanted to make a moment to look at structures here. In general these compounds are called [ Indiscernible ], in the botany world and whatnot they're referred to as [ Indiscernible ] compounds. They generally, the size chain double bond, its location defines two sets of analogs here. So with the analogs we have the saturated [ Indiscernible ] group in between the double bond of the propeen and the air mattic system. The double bond here is congregated with the fennel system. This could be called a beta methyl sty reen. [ Indiscernible ] then is one of the [ Indiscernible ] compounds like methyl [ Indiscernible ]. Later today we'll hear about Isoeugenol. Here I have shown the structure of [ Indiscernible ]. Safrol has this group. In the late 50s and through the 60s is when toxicity tests were run and it was realized that it was a rodent carcinogen. With regard to the toxicity, the metabolism of these [ Indiscernible ] compounds in particular has been studied. They form a [ Indiscernible ] at that [ Indiscernible ] carbon. That gets sulfated, which produces the active compound that leads to the [ Indiscernible ] in rodents and punitively in humans. NTP performed studies with methle [ Indiscernible ]. There was [ Indiscernible ] in the liver of male and female rats and in mice. Both Estagole, Estagole is known to form the [ Indiscernible ] compound. And it's also known to be [ Indiscernible ] in the [ Indiscernible ] mice assay that the millers used back in the 80s. The [ Indiscernible ] is inhibited by pretreatment with [ Indiscernible ] or by using [ Indiscernible ] that do not have the ability, they don't have the [ Indiscernible ] to convert the alcohol to the sulfate Esther. That's the theory.
In general the aloe Ben design compounds form DNA [ Indiscernible ]. So Estagole was selected by NIEHS. We've done [ Indiscernible ], then [ Indiscernible ] and Isoeugenol was underway. So Estagole was added to that because of its widespread human exposure and the literature knowledge about it being [ Indiscernible ] in mice, um -- some of the aloe benzine compounds have been listed in the report on carcinogens. They're anticipated to be carcinogenic in humans. We want to compare it with the results from methyl ugole.
We used the gavage route of administration. We wanted to design a studies and conduct them so it would be easy to compare the results. The dose levels ranged up to 600-milligrams per kilogram. Corn oil was the vehicle. Also, because special toxicity studies were done with the other compounds, we did them in this set of experiments. They were designed around one month exposure to look at stomach PH. Serum [ Indiscernible ] production and the histology that might occur at the stomach and [ Indiscernible ]. Which were the salmonella tests in vitro, negative. In vivo the test on bud smears taken from male and female mice and Estagole was negative for micronuclei in both sexes.
We saw after 3 months of exposure all of the animals exposed the high dose. There were decreases relative to controls in body weights. Down to 73% in the males and 87% in the females. With regard to the hematology affects, there was a pattern that reflected anemia [ Indiscernible ]. There were increeted plate let counts. There was an iron deficiency as sublghted by the total of iron binding capacity. There was decreased serum iron. In general the effects were more pronounced in males than females. The mice hematology was unremarkable. There were other liver affects in the rats. The organ weights increased in the two high dose groups of males. And down to the 75, or middose group of the females. For clinical chemistry there was increased [ Indiscernible ] and [ Indiscernible ] activity in the serum. And the [ Indiscernible ] concentrations were increased, indicating liver injury had occurred. Um, the most surprising finding in the study was neoplasia after three months of exposure. Two of the high dose male rats had multiple [ Indiscernible ] carsinoma. A third male rat had [ Indiscernible ] adenoma. The occurrence of these lesions was supported by the pattern of [ Indiscernible ] fibrosis here in the high dose. [ Indiscernible ] in all of the exposed groups. There was also [ Indiscernible ] down to the 75-milligram per kilogram group. The [ Indiscernible ] inflammation and infill trace of [ Indiscernible ] was also noticed in all of the exposed groups of males. There was a pattern of foecy in the higher dose groups, generally.
Um, and so we checked the historical controls for rats and saw there were no liver tumors whatsoever in the control rats from three month studies that involved 662 males and 677 females. That's by all routes and vehicles. And so these are, this is truly a rare occurrence here that we're looking at. With regard to the female rat liver, the pattern of nonneoplastic lesions was similar to the males. But it lacked 9 neoplastic response. And we had the [ Indiscernible ] and [ Indiscernible ] at all levels. And [ Indiscernible ] at the higher levels. [ Indiscernible ], [ Indiscernible ] occurred and we have this smattering of foecy. The denominator is ten animals in each group. Um, we did obe gland lar stomach effects in male and female rats. After one the [ Indiscernible ] were both increased in the high dose males and females. Gas trick glanl atrophy occurred at the two highest doses in males and females. In the three month exposed animals we also saw the gas trick gland atrophy down to one 50-milligram per kilogram level. These are slides that show what the atrophy looked like. So there was a group of lesions that involved reproductive and endocrine affects in the male rats. The test ease had decreased weights and had [ Indiscernible ] of the gem nail epithelium. These are in the two highest groups. And this collection of lesions indicates some potential there's reproductive toxicity of Estagole in male rats. Although that study has not been performed yet. Other nonneoplastic lesions that were occurred include [ Indiscernible ] of bone marrow, at the kidney there was weight increases, and renal tubal lesions at the higher dose levels, there was also Sal vair gland and [ Indiscernible ] of the two higher dose groups of males and females.
Turning to the results. All of the female mice died during the first week of the exposure. Whereas the survival of the males, there was no problem. There were dose related decreases in body weights, down to 79% in the males. And down to 82% in the survival females. Organ weights, the only significant change was the relative liver weight, which was increased from the 75-milligram per kilogram level up in males and females. So from this it appears that the females might have been more sensitive to the toxicity than the male mice. With regard to the nonneoplastic lesions, at the libber we have the [ Indiscernible ] again. Along with degeneration at the two higher dose groups in the males. The high dose females that died early all exhibited necrosis.
Other lesions in mice included at the glad lar stomach [ Indiscernible ] and at the forestomach [ Indiscernible ]. In the highest dose groups there was olfactory epithelial degeneration in the nose. And the two highest groups of males and females. So to summarize this, from the literature we know there's ample evidence that Estagole is [ Indiscernible ] in mice. But this is the first evidence that it is also carns know Jenic in male rats. The toxic efforts of Estagole and methyl [ Indiscernible ] turned out they were similar. I have a slide at the end of the Isoeugenol talk that compares across the chemicals and columnlike fashion. Estagole and methyl [ Indiscernible ] are very much the same in their toxicity affects. The [ Indiscernible ] for male mice was [ Indiscernible ]. But it could not be determined for the other sex species in the experiment. Our conclusions from the three month study are that Estagole is [ Indiscernible ] to male F-344/N rats based on the occurs of [ Indiscernible ] carsinomas and he pattic cellular adenoma. Because they were exposed for three months these do not expose the full potential of Estagole. The combined evidence from this studdie and other previous studies make it highly likely that Estagole would be carcinogenic to additional sites in rats and mice after longer exposures. So that's, concludes my presentation. I would like to ask Dr. Malarkey to come up and show you slides about the his toe pathology that was seen for this study.
Thanks, Doug. Do you want to sit?
I'll wait.
Tell me if he does anything behind my head. Thank you. I would like to share with you the features of [ Indiscernible ] and [ Indiscernible ] in this study, the Estagole toxicity study. Of course this was a lesion in the rats. What is [ Indiscernible ]? This is an example here. There's a proliferation of bile duct. There's inflammatory cell reaction. You can see the blue dots, they represent inflammatory cells. Now this epithelial is atypical sometimes because it's piles up and you will see crescent shaped areas. These are usually small in portal areas. That's how they begin. They will progress and expand in the liver. They're [ Indiscernible ] to [ Indiscernible ]. Sometimes there's dilation with mucous accumulation in the center of these ducts. They can have gob let cells. This is from another study to demonstrate the gob let cell. It was not prominent in the Estagole stud Y. this is probably the source of the [ Indiscernible ] in the middle. You can see the duct here is partially lined. Over here there's a partial lining. It looks like a rupture might be going on here. Here's inflammation percolating through that collagen matrix. This is an example of a mild case from the Estagole study. You can see over here in this portal area, you can see the fibrosis of the imflamtory cells. Notice here the nod ewe lar appearance to the liver. This nod ewe lar appearance is due to hypertrophy. This is another case of a similar looking lesion. This nod ewe lar here. I wanted to show you what these bridge be areas look like, that's shown here, depicted here. These are he patio cites. Those are the oval cells of the liver. You've probably heard of them. Now you can see them. If you look closely you can see some, there's a formation of billary hyperplasia. It may be the stem cell in the liver. Um, that's, that's another liver lesion to show you that may be related to the [ Indiscernible ]. [ Indiscernible ], a common lesion usually found in the rat with treatment. It's rare in the mouse. But it has been reported in the mouse. It's not reported in 90 day studies, whether it's control animals or treated. We don't really see it. These were two year studies, some were reported at early as six months to begin to show [ Indiscernible ]. It's believed to be a [ Indiscernible ] lesion. It progresses on to [ Indiscernible ] carsinoma. In all of these studies there was an increased incidence of [ Indiscernible ]. What do the [ Indiscernible ] carsinomas look like? They were large masses am they had floored growth. The third bullet here, there's infrequent me tas 'tis has been demonstrated. [ Indiscernible ] had a low metastatic rate reported. But it appears to be infrequent. Here's [ Indiscernible ], this is one of the two cases in the Estagole study. You can see the flower arrangement. That's where the word florid comes from. If you look at higher magnification it's this atypical structure separated by a collagen matrix and [ Indiscernible ]. It's invading and replacing the liver. These are larger masses. This is the other case. The entire liver lobe has been pretty much replaced by the infiltrating and expanding [ Indiscernible ] carsinoma. At a higher magnification in that second case, again you see the atypical [ Indiscernible ] epithelium. There's invasion and replacement of the [ Indiscernible ], which is up here. That brings us to think about the origins of the [ Indiscernible ] in the Estagole study. Perhaps this is true in other studies as well. There's some evidence because of the histologic progression to the [ Indiscernible ] course know ma -- carsinoma. It may be arising from a stem cell, oval cell or billary cell. There may be progression right from this billary proliferation right to the [ Indiscernible ] carsinoma. So that's it for [ Indiscernible ] and [ Indiscernible ] carsinoma. Doug, you left.
Okay. Thank you. Any questions from the committee for -- yes, Dr. Howard.
Did you see any evidence of increased necrosis or apoptosis?
In the glad ewe lar lesions?
In the nonknee owe plastic?
In there was degeneration in the mouse that probably was involving ep toe sis. Or necrosis. Is that what you mean? There was in the mouse. Not as much in the rat.
Okay. We'll go to our first reviewer. Dr. Pino.
All right, Mike Pino. First of all as scientific criticisms, I don't really have any. Moving on to the results and information to be added or further discussed. Start with the clin path. [ Speaker Faint/Audio Unclear ]. I was just wondering, the finding was discounted even though it seems there could be a good explanation for why the white cells were elevated [ Speaker Faint/Audio Unclear ]. I guess before dismissing the white cell counts I would like to see the evidence for discounting that. And why it was discounted. The blood smear information. In discussion of the organ weights, it needs to be separated which effects were correctly related to the compound and what were secondary effects. The changes in the kidney, [ Indiscernible ], heart and lung was secondary. I think they should be split apart and discussed. Um, in discussing the histopath results only the statically significant findings were discussed. Whereas at the lower doses there were clearly evidence of an effect going on. I think that should be mentioned. For example, the [ Indiscernible ] foecy in the rat, in the female rats at 75-milligram had an incidence of 3 out of 10. None in of the controls. To me in my mind that's compound related and should be mentioned. The same goes for some of the other findings. In the kidney and the bone marrow in the rat and the -- deJen raitive [ Indiscernible ] in the mouse. Um, the renal pigment, or the pigment in the kidney [ Indiscernible ] cells. What pigment was that? Could that have been related what was going on in the hematology data? In the mouse study, I have the same comment about the organ weight affects. The liver should be clearly identified. In the discussion, I think when discussing the liver affects at the [ Indiscernible ] inflammation and [ Indiscernible ] infiltrates in the liver it should be brought up they weren't seen with [ Indiscernible ]. I don't know if could be involved in the patho genesis or not. In the mettle [ Indiscernible ] study they did report [ Indiscernible ], which in the report says it was similar to [ Indiscernible ]. Maybe that should also be brought up in the discussion. There's similarity to the methyl [ Indiscernible ] study. With a conclusion, I do agree. Since this wasn't really a carcinogen sis study. You did see tumors, it was designed as a toxicity study. You need to say what the target organs were as part of the conclusion. So that's all.
Okay, thanks. Dr. Crump?
Kenny Crump. I don't have any comments on the conduct of the study. Seemed like it was well conducted. This is another one of those special cases that we're not quite used to dealing with. I did have some concerns, I guess, about the conclusions. I think the flat statement that Estagole is carcinogenic to male rats, we don't use that kind of terminology with a two year bioassay, I think it would be understood to be clear evidence. I think in a two year bioassay the evidence we have [ Speaker Faint/Audio Unclear ] it wouldn't be rated as clear evidence. It may be even equivocal. I would feel for comfortable if we had qualifiers in that statement. Saying it occurred in three animals in the high dose. It would give better understanding of what actually, what the evidence was in the study. And, um, I guess I don't agree with the last statement in the conclusions, either. It was, it was sort of speculation of what would happen if you did a longer study. I feel like fill solveically, it's okay to speculate, I'm not sure it's appropriate to include that kind of information in just a very short summary of what happened in this study. That's not what happened in this study, what you can conclude from this study. And another reason is I think when people read that they will well, this is what happens in a two year bioassay. We would see a lot more lesions, which may be the case. I think you also have to think about the fact, I don't think yeve even seen these in the two year bioassay, I think we've exceeded the NTD. You had 25% lower rate in the high dose animals, I think would not have used that dose. You wouldn't have even seen these lesions. So, I would feel more comfortable if we didn't have that kind of discussion in the short summary of the report. That's all, thank you.
Okay, thank you, Dr. Bradfield.
Profiling, here we've done [ Indiscernible ] on [ Indiscernible ]. I would have found it to have a global transcriptional profile, rather than a more old-fashioned approach. I come away confused how it fits into the greater picture of the flavoring agents. I wasn't sure what exactly was gained from this study. I wasn't sure why this experiment was done, other than a vague comment how this would fit into the -- exact wording, better characterize the toxicity. It all seems vague to me. It would be helpful how this information is going to be used in this larger picture, in these reports.
Is that it?
Yeah, sorry.
Dr. Peckham.
All right.
First I want to say thank you for the review information. We appreciate the thoroughness and insights that go into providing those kinds of comments. So first, with Dr. Pino's comments -- starting with the three month rat study the comment about page 25, um, adding the target tissues that were examined microscopically in all groups, we'll do that. With regard to the increased Luke sight counts, on page 35, in the original evaluation that Dr. [ Indiscernible ] did for the study, he noticed some unusual effects like that and went ahead and performed counsels analysis on a subset of the blood smears. That turned out to be informative. He's here to share the results of his counts with you. And also give a rationale for why the elevated Luke sight counts are considered to be [ Indiscernible ] in this study, Greg?
Hello. So there's always a short answer and a long one. I tend to always give the long. I'll try to be as briefs a possible.
So we did notice the apparent increase in Luke sight counts. And in conjunction with the fact there was a liver effect with an inflammation in the liver it would have suggested that the Luke sight counts were related to the information. We did discount them, or I discounted them, more pointedly, based on -- based on how the data looked. We see, this was noticed in the rats and in the mice. But more prominently in the rats. I'm showing you the male rats here. This is a substantial increase here. We don't normally see that. We look and see the increases here. We don't know how these are derived. This is instrument derived data. The blood goes through the instrument. The red cell count is counted first. Then a licing agent leaves the white cells to be counted by an [ Indiscernible ] method. The instrument used in this case was a dual. It counted by impedance for most of the variables but it also had an optical reading for the white cell counts. We don't use the [ Indiscernible ] for the instruments. We read a 100 cell dif. You end up with the counts for the different cell lines. Interestingly enough, the percentage, or the percentage of the Newt fills and lymphocytes were the same regardless of dose. If this is related to inflammation you would expect this to be higher. That raised the flag. Was this a real affect regarding the increased white counts? That's a pretty substantial increase. There are chemicals that are compounds that cause this. For example, an ep enough rin response. That's usually a short duration response, usually acute. This is happening at study termination, didn't necessarily make sense. Not to say there couldn't be a compound that could cause it by changing distribution within the circulation. So this stimulated the estimated counts. Now this method is semiquantitative at best. But should have been tell qualitative the different between a 6000 and 15,000 white count. I did the male rats and then the male mice, just to see if I could demonstrate this really high white count. It doesn't hold up. You could argue maybe Tallahassee increase in the rats, these are estimated counts. There's a lot of variability. The same thing was found in the mice. That leads to the question, why do we have this over doubling in the white count in the high dose rats, male and female?
Okay we have the documented [ Indiscernible ] anemia in this study. Caused a very markedly change of the profile of the blood. It's known, I have to lice the white cells. If the red cell membrane is changed so it's more resistent to the licing agent some of the cells will be left in the count to be counted. This instrument and other instruments have been shown if there's resistance to the licing agents you can have them counted as particles in the white cell channel. We have moverlogical changes. Maybe it's the microsights that are more resistent. It wouldn't take many. Red cells are in millions and the white cells are in thousands. So it wouldn't take many red to be resistent to cause the increase this white cells. Well, that was my speculation based on those, on those three points. Any questions?
I have a question on the slide before that. What is a target cell?
It looks like a target. Target cell, target cell, target cell. [ Laughter ]
Is that your name for it?
No, that's an accepted name. It's related to distribution of hemeo glow bin in the cell.
When you did your white cell counts by hand did you see red cells?
To review the method, I took the blood smears and I just did the control and high dose, that was the most dramatic. Looked at eweny layer fields of the [ Indiscernible ] on high power. This is high dry power, which is a 40X field. Did ten random fields and I counted the number of white per field. Took an average. There was an algorithim. Basically did it that way.
Okay. Any other questions? Okay, thank you.
Thanks.
So then we'll go back to Dr. Bristol.
Thank you. Proceeding on. Dr. Pino made the comment about the, when we discussed the organ weights to distinguish clearly which are treatment-related and which are not. And we will do this in our report. For each sex species. And a similar general comment, there were several lesions where we had not commented on the extent to which the lesion occurred in lower doses, where they were not statically significant. We will review all of the lesions and for biological significance to support a statement like that, and where appropriate it will be included. With regard to special stains done, the renal pigment in the males Dr. Malarkey and Dr. Peckham have arranged to do that, that will be done. In the mouse study, some of the general comments are already taken care of. And then, um -- let's see, in the discussion, yes, we will mention the [ Indiscernible ] infiltrates in rats. I noticed you just commented about the billary [ Indiscernible ] and the methyl [ Indiscernible ]. I recall reading that session, I agree. We'll review that and try to incorporate that, that's a good point. Also, your comment about the conclusions. We can incorporate that into the conclusion statement. And I think that Dr. [ Indiscernible ] addressed your comment about the [ Indiscernible ] counts as well. Now for Dr. Crump's comments, with regard to our flat statement that Estagole is carcinogenic in the conclusion statement, we feel that's consistent with statements made for studies that had similar indications of carcinogenic effect in other toxicity reports. Um, specially where we do not plan to carry out followon with a full blown carcinogenicity study. The liver tumors in the male rat, we feel are, they are the result of Estagole exposure. Based on the striking similarity with the methle [ Indiscernible ] study, that's why we projected in the two year study we would see those results. With regard to the dosage used in the [ Indiscernible ], they were appropriate for the subchronic study. The 600-milligram per kilogram dose would not be used in the chronic study. One of the lower dose levels would be used. In the, at the 600 level, after three months there were 3 out of 10, the denominator was 10 for that group. That's very unusual and striking evidence. In the methyl [ Indiscernible ] study the highest dose was a 1000-milligrams. We have a similar situation. The high dose in methyl diswould not be appropriate for the chronic. And the lower doses in the chronic produced the full spectrum of [ Indiscernible ] in male and female mice. That is expected for Estagole as well. Um, one other point about our conclusion, about Estagole being carcinogenic in male rats is that regulatory groups, for example, California EPA in 1999, have said that Estagole is a carcinogen. Um, now for Dr. Bradfield's comments, the comment about global transcriptional profiles being generated with each study like this is interesting. And, um, the NTP does have a [ Indiscernible ] project in place. And it looks, in fact there's a project that looks at these [ Indiscernible ] compounds in particular with the goal of identifying patterns that can be used to correlate the liver carcinogen sis affects. This afternoon there's a seminar at 2:30 down in F193 that deals with that general topic. If you are interested, I brought along the announcement with me. I can show that to you. We have that as an ongoing study. It's just one indication of where this goes. With regard to the information that should be added, I alluded to the slide at the end of my Isoeugenol presentation that will summarize the similarities and differences. In a follow-up journal article we would expand on that to bring in more information from the literature so that's an indication of where this information will go besides the technical report here. The intoxicate cogenomics study on this group of compounds, I think, goes in the direction that you are projecting. That's it. I think.
Any more discussion from the committee?
Jon Mirsalis. Two comments. One is echoing what Kenny Crump would say. I would strongly object to the statement we will probably get a different [ Indiscernible ]. There are drugs that produce tumors fairly early. You take it out two years and you see the same ones. I don't think we should be trying to second guess what we might get. The other comment, you reported the gene tox results. About two decades NTP cut the list down to [ Indiscernible ]. For those two tests are poor at predicting liver cars know Jens. A long list are missed in those two assays. I would like to see a inveeo liver. Something, invivo liver is the way to pick up these compounds. In the absence of that data you have to make the statement that you haven't really assessed the [ Indiscernible ]. I think these are probably good, very nice Gino toxic carcinogens, based on their [ Indiscernible ].
Thank you. Anytime discussion? Dr. Howard?
I agree with the comment from Kenny and from Jon. If we look at the studies done in the past there's essentially one study by gavage. It was by the millers in the 80s. Would probably be considered inadequate for risk assessment. The rest of the studies in your report are subcutaneous or [ Indiscernible ] injection. The thing I'm concerned about is, is where do we go from here? I gather there are no plans to go forward on a two year bioassay study. Two out of 10 animals having tumors, or 3 out of 10. The hallmark of the NTP is a rigorous number of animals. And if ten animals per dose group were adequate for risk assessmentals we would have been doing that for the past couple of decades. I think this is an extremely adequate study for doing dose response and for setting up a two year study. I'm not sure it's adequate for risk assessment. If you look at the tumors, the carsinomas and adenomas as a measure of where the dose response lies, then you are somewhere between [ Indiscernible ] and [ Indiscernible ]. If you look, however, at what you are proposing then your left with no dose response. In the lowest dose you had a response. So you leave, you leave yourself juxtaposed between two conclusions. I question why a two year study was not proposed to move forward. The doses will be somewhere at 37-milligram per kilogram or lower. Which will drop your no observed level level orders of magnitude, perhaps.
Thank you. I wonder if you want to respond.
Sure. We've studied a number of these compounds. It was based on the similarity and the metabolism of this compound. And similar purported mechanism of [ Indiscernible ] and also seeing three tumors out of ten animals in the three month study at an early point. Other groups have also determined based on the literature and miller studies that it is considered to be a liver carcinogen in mice. This study now demonstrates it appears to be in male rats.
I want to respond to that one point. I agree with you as far as hazard identification. My issue is not with that. It's where the dose response lies.
Paul, I believe that Doug stated this was an NIEHS nomination's part of this class study. I would certainly would want to entertain from FDA if there's a serious request for dose response. You have to remember that Doug mentioned the genomic studies with this particular class of compounds. What we're trying to do as a program is move beyond doing a two year study on all compounds. When we have responses like this in three months. With we see this three months, we generally, would like to avoid what we think is sort of a redundant study and taking it forward with a 50 animal, two year bioassay.
Yes?
Paul Howard. I totally appreciate that, John. I understand why spend another several million dollars. I did not get a response from our risk assessment group. I will bring that back up to them. See if we will move forward. I want to encourage. I'm not criticizing the study. I'm looking at the adequacy of where we go from here. Since the pathway is most likely what you are talking about, then biomarkers might, I think biomarkers of exposure might be a more appropriate, or equally appropriate way of going, as well.
Dr. Crump?
Kenny Crump. Want to add to our comment, or what Paul has said. Often we may be, we overestimate the information that risk assessors use from these studies to come up with numbers and risk assessments. Basically it's some rough indication of the doses at which you saw some tumors. This chemical is known to be carcinogenic in mice. I'm not familiar with those studies. I'm guessing there's enough there for regulators to do what they need to do. I certainly wouldn't take the evidence that we have here as a necessity to go on and do a two year bioassay in rats. I feel just given that it's carcinogenic in mice and the information that you have that can make that call, that is what a regulator would use. I would basically doubt if anything you got out of a two year bioassay would change the quantitative results. Very crude approach that is being used.
Any other discussion?
I guess, Jon Mirsalis. Maybe a compromise position would to be propose rat carcinogenicity studies. FDA largely ignores now the presence of mouse live ever tumors as evidence by the very, very large number of mouse liver carcinogens. Rat tumors it makes you take note more. A compromise position, there's cost and animal use here. I agree with John's position. Where do you draw the line? A compromise might be one rat two year study to find an appropriate lower dose. Paul is right you might find a know eel that [ Indiscernible ]. If you did that.
Um, just to comment a little bit on the mouse liver tumor issue. I think that you are correct with regard to the two year mouse liver responds we see in many, many studies. I'm not sure that the protocol that is used by the millers is one that could be thought of generating the typical mouse liver tumors.
And just to at to that. In the methle [ Indiscernible ] we saw other target organs. There's a similar mechanism that we're seeing here in this 90 day study of Estagole. I wouldn't want to eliminate the mice if it would provide other information, about additional target tissues.
I would like to ask a question about structure activity relationships and how much data do you have on that for shorter term end points and you could make good predictions for long-term?
I've been in the literature in detail. I come away from that with some skepticism. It might be best illustrated by talking about methle [ Indiscernible ], [ Indiscernible ] and Isoeugenol. Those three, are similar in structure, are different with respect to the carcinogenic response that we see in a full bioassay. The [ Indiscernible ] study and studies might help us with that regard. But, um, it gets complicated, like satisfy roll has a [ Indiscernible ] group on the [ Indiscernible ] and the related compound says mol which has the [ Indiscernible ] of [ Indiscernible ], gives [ Indiscernible ] tumors which are rare. Is somehow a related effect or not? There are similar patterns for metabolism, but with Isoeugenol there was a study done using the uniformly ring labeled compound and using HPLC and radio chemical detection they identified at least 40 different peaks, or me tab lights that appeared in the urine. That's complicated. These studies that are done all produce information, I find it hard to put it together in a comprehensive way that I would be able to use it with confidence to make predictions. So, I like to rely more on the actual whole animal studies.
Okay, thank you. Any more comments? Any comments from the audience? Yes?
Is this on? Yes? No?
Yes.
Ron Lawrenceson from the center for food safety at FDA. I hardly know where to begin. Are we being taped? [ Laughter ] I'm here as a scientist to judge the veer asty of the performance and the interpretation of these studies and so are. If any of you want to, after you leave this room, join some vigil anti-group to remove all of the spices and herbs that we use every day, you are welcome to do that. I think that's still a civil right. But personally I wouldn't recommend it. So what do these studies give us? What they give us is some reflection of the carcinogenic load for humans, okay? That is attained by every day activity, namely, the way we eat. I would personally love to separate out all of these flavorrants, I'm not sure how you would do it and do a risk assessment to get some idea of what this is. This is not a trivial task. If I didn't have anything else to do I might take it up on the weekends, but I do have other things to do. But I certainly can't use a study like this Estagole study, which seems to be, even though the doses are fairly high, I believe that everybody is right, we would expect that a few ors of magnitude lower we would see results. And you know, these results are also [ Indiscernible ] by profound toxicities that make it very difficult to interpret what this means from a risk point of view. So while we didn't request these studies, this is a local nomination, um, the value in the studies of all of these safrol compounds [ Speaker Faint/Audio Unclear ]. I'll try to answer any other questions. We're here to assess the voir'sty of the performance of the studies. And the interpretations that are made here.
Thank you. I'm a little confused as to your point.
The scientific truth of the study. You are here to assess the science.
Of this study though?
All of them. Not the risk management. The risk management is what we do to deal with this issue. That is not issue. It's not my issue. Even though I give advice on it. You may feel strongly about it. Okay? In this room that's not issue. Outside this room it is your issue.
Okay. Thank you. Any other comments? Discussion? If not, display the conclusions.
Okay, um, any discussion about the, um, I guess maybe I should say is there a motion on the conclusions?
I guess I, Mike Pino, I would agree with Dr. Crump. Strike the last sentence. About speculation. I would also add wording maybe similar to page 55, about the target organs, what organs those were seen in.
Dr. Crump?
Kenny Crump. I would support removing the third sentence. I would also like to see the first sentence be a little bit more specific. I would suggest wording, Estagole showed carcinogenic activity to male rats based on the occurrence of two [ Indiscernible ] carsinomas and one [ Indiscernible ] in the liver of three male rats. And I would also suggest that the second sentence be modified, not only to show it was three months, but it was at doses higher than a normal two year bioassay. Because rats and mice were exposed only three months and at higher doses than a full life bioassay.
Yeah, I would add that you might want to say 3 out of 10. And Dr. Howard?
I agree with the suggestions. I don't think we want to say these are doses are higher than what would be used in [ Indiscernible ], because some have used 50,000 parts per million. These are higher doses since there was an occurrence of [ Indiscernible ] in all dose groups. This is really, I think, the point is these are high doses because of the [ Indiscernible ] that was noted. Not just because they are high doses, knew mayorically.
I didn't mean high doses, I meant high relative to the toxicity that you see here.
Dr. [ Indiscernible ]?
I wanted to clarify the intention of the program with regard to the third sentence. That basically is we didn't want to, by the virtue of the response in the liver only we didn't want to give the impression this is the only organ that could respond. Based on the overall similarity with the responses to methyl [ Indiscernible ]. That was our intention. If that didn't come across, then we need to -- fix that.
Mike Pino. The second sentence seems to cover that though.
Yeah, I think so.
Dr. Howard?
I agree with that, Michael. John, the problem I have with that is for this particular compound the biomarkers of exposure were not measured. We don't know dose response there. The carcinogenicity was only in mice in other studies. I understand structure-related exons -- compounds being brought into this. It's not showing a common intermediate. Unless that's really brought into the discussion, a really good multiple compound comparison where a reader can see where you are drawing a conclusion, I'm not comfortable with that last sentence.
I was referring to the issues related to the gas trick PH and other measurements we did in this study that were related directly to the methyl [ Indiscernible ].
Jon Mirsalis. This makes it sound like you, it's similar to the other analogs, when in fact your studies show it as negative. I don't believe that's the right answer. There's so much confusion about what we know and don't know. Comparing studies that are done by totally different mechanisms. I just, I think the whole, I think Mike is correct. The second sentence addresses it. Period. I don't think we, this group should start to predict what we might see based on other studies done a long ago by different methods. And try to determine what this is going to do. I think the last sentence really needs to go.
So is there anybody that would like to wordsmith it?
[ Speaker Faint/Audio Unclear ].
This might be a good point for me to mention, with regard to the [ Indiscernible ] toxicity of Estagole, we did the design in the study was our standard design for this three month study. And the DNA, in vivo studies have not been done for Estagole, as far as I'm aware. If they were done I think most people would feel that would bolster the analogy. It's easy to accept that kind of correlation for the [ Indiscernible ]. We need to beef that up in our discussion, like one of the comments suggested earlier.
I would add this statement is perfectly okay to put in the discussion section of the report. I see no problem with putting it there.
Okay. Did you want to -- Kenny, did you want to change the first sentence?
My recommendation, Estagole showed carcinogenic activity in male rats based on the occurrence of two [ Indiscernible ] sar comas and one [ Indiscernible ] carsinoma. And three rats, three male rats, in the liver of three male rats, um, in the high dose group.
Okay, Paul.
Paul Howard. Do you want to put a denominator on that?
I meant to say 3 out of 10. 3 out of 10 male rats in the high dose group.
Okay. Um, so do we really need to change the first few words? Just insert the number of animals. Or do you not like the phrase?
I don't like that very much. I think it carries a lot of bag and with it. I would prefer something like showed carcinogenic activity.
Okay. The rest of the committee good with that? Okay.
Okay.
Yes.
[ Speaker Faint/Audio Unclear ].
Okay. If the committee is happy we could take a 15 minute break while they wordsmith it. We'll come back and approve the conclusions. That should save a little time. How about we take a 15 minute break? Your chair needs it. [ Laughter ]
Session on break until 10:15 eastern.
If we could come back to order.
If you would like to read the revised conclusions and offer any comments.
Jon, Mirsalis, I move we we accept these.
A second?
Keith Soper.
Any discussion?
I would like to ask for clarification about the definition here of the NTD. The NTD in this three month study, or as is would relate to a two year. In my mine they don't [ Indiscernible ] based on duration of the study. So I'm just wondering what NTD, what you were trying to represent here.
Um, I was trying, thinking it was trying to represent the fact that the doses are higher than a typical use in a normal two year bioassay. NTD is what people think of when they think of the NTD, so a -- I feel there needs to be something that indicates the doses were higher. If there's better language, we could go to it.
Tracy Bunton. I think what now, as it's written it's a confusing. A couple of ideas. What is highlighted now belongs in the first sentence. Or at least as I understand what the two concepts are. And the original statement that was the second sentence, or whatever will remain in tact. Now it confuses, a little bit, what people are trying to say.
This is Mike Pino. If you put in the liver of F-344/N male rats at a dose that exceeded the [ Indiscernible ] instead of high dose group. Leave the rest of that second sentence the same. Would that take care of it?
I would take issue with the concept that we've exceeded the NTD. [ Speaker Faint/Audio Unclear ] where the accepted practice for selection of doses has taken doses that would be double that, one would pick for a two year study because of the trunk Kateed nature of the study. And the fact that the NTD was originally put forth so you could maintain a sufficient number of numbers to the end of the study to attain statical power. In this case we're talking about a three month exposure. None of those things make sense in a traditional way. It's a confusing, the issue to introduce that concept into this report. This is a statement of what happened in this three month study.
Paul Howard. I agree with that. The reader will go to the text and will see what doses were used.
Point of order. Do we need to withdraw the motion?
No, this is a discussion.
All right.
Nancy?
Excuse me. The motion -- was to accept it as written.
Jon Mirsalis, I'll make it easy, I'll withdraw the motion.
I would like to comment.
Dr. Bristol.
The data that we're reporting and the concept of NTD in the female mice there were deaths. You might say that, you know, that exceeded some NTD. But it is hard to apply the concept to this. In the male rats, were they sick? Was it effecting the power of the test? Those are the considerations we apply to assessing whether or not NTD was reached or exceeded in a chronic study. We're not talking about those here. In we did, the male rats, I don't think exceeded an NTD at this three month exposure duration.
Thank you. If we remove that phrase. Then I would entertain a motion to accept the conclusions.
I , I, maybe I'm arguing against the forces here that are going the other direction. The second sentence says exposed only three months. When people read that people will wonder what we get in a two year bioassay. It's legitimate to say this is study. And it used higher doses. I think those things are, are --
Jon Mirsalis. Because rats and mice were exposed for only three months and using doses higher than would be expected to be used in a two year study. Is that what you are trying to get at?
You're talking two different sides of the question. That doesn't make sense. If you want to qualify intoxicatist or whatever the problem is, do it in the first sentence about where you saw, or at what dose you saw the tumors. Then you say they were exposed for three months, you don't know what would happen at low doses -- or at any dose. You don't Novak what would happen.
I'm okay with it as is. The first sentence is under the conditions of these three month studies. That has a lot wrapped into it. I'm trying to address Kenny's concerns. At some point you have to read the report to see what was done, so --
This is Mike Pino. I agree, based on that first phrase. I make a motion that we accept the conclusion as as.
Jon Mirsalis. I second.
They don't have bone marrow. Would you considered aking those in there? -- adding those in there?
Short answer is yes.
[ Speaker Faint/Audio Unclear ].
Would you mine giving us the latitude to put the correct nonneoplastic effects in the second paragraph and just give that to us? [ Laughter ]
Yes.
That's part of the motion, to accept this revised conclusion.
Okay, I make the motion that we accept it, with the caveat that the nonneoplastic lesions will be added.
Jon Mirsalis, I second.
Okay. All those in favor? Of accepting the conclusion. Looks unanimous to me. Thank you.
All right, let's move on to Isoeugenol. Dr. Bristol.
While I'm getting ready, one thing I thought of that I didn't mention earlier is that nyingle walker is, he heads up our project. He's here at the meetings too. If someone wants to ask questions about that project, he'd be happy to answer. Turning now to Isoeugenol. This has widespread natural occurrence. Um, and the essential oils from plants and seeds and particularly [ Indiscernible ] provides Isoeugenol extracts that are used in the world's most expensive perfume, joy. Another source is nutmeg. And back in the 16th century European nations went to war trying to control the source and supply of nutmeg. So, it's had an important effect on history. It's used as a food additive. It enhances the sweet spice and clove flavor in foods and in gums, beverages and so on. It's a fragrance in many personal care products, like detergents used in the house. More recently it's been used in aqua culture to sedate fish in the rearing and harvesting of them. That's a growing food source. The FDA has given temporary approval for that source. With regard to isn't sis -- it's catalyzed by heating with either disor [ Indiscernible ] compounds. It produces the double bond shifts and the congregation shifts from being no congregation there to being congregated. This is more [ Indiscernible ] stable. You have to heat it up with some catalysts and bingo you are there. I've shown what we consider to be the parent compound for the aloe. The [ Indiscernible ] on the air mattic ring seem to have less of an effect on the activity of these lower molecular weight [ Indiscernible ] compounds than on the ones that as you add, um, you know [ Indiscernible ] or [ Indiscernible ] groups to the ring the effects, some of the analogies drop off. But now we're talking about Isoeugenol, which has that rop
Based on -- the general lack of information about 2. It's surprising how, seems like it's been overlooked, by and large by people who have done studies back in the 80s or 60s.
So the studies that NTP did include the three month toxicity studies, along with the kind used for -- andestrigol. The double -- how it's used, and how it occurs in nature. We use dose levels up to 600-milligrams per kilogram, based on information frommest eugenol and -- the special toxicity studies were done in rats with one-mont excoz month exposure. We did the two year toxicity and car sin cars nighsity.
The E. Coli assays and -- were negative without the usual vaikz, didn't use special activation as is sometimes done, can produce positive results, but we don't know, done didn't do do it here, not in the literature.
The negative result in males, positive result in females.
With regard to -- dropped to about 90% of controls. The weight of the liver increased about 14% in the two highest groups of females. The clinical pathology was in general unremarkable. The only histo olg in male and female -- the top three doses and female's liver, cyto plaza mick -- how the special toxicity studies turned out. There was a small dose-related decrease, not increase, decrease in the stomach PH at 150-milligrams or higher, females only. The magnitude of decrease. The serum gas trine levels were unchanged in male and females, no stomach lesions. The P 450 levels were slight decrease and activity of the CYP 182 -- bodies of males, females, relative to controls decreased in the high-dose group down to about 90%, liver weight increased in the end males, two top doses by 14 and 22%. Clinical pathology again was unremarkable. Histopath owe logic lesions only in the nose this case. males, females, minimal to mild severity. Minimal to mild in [indiscernible].
So, here are the doses selected for the 2-year studies, same doses for rats and mice, up to 300-milligrams per kilogram. These were based on the changes in body weight. At the high dose group in the mice, and the male rats, and there was concern that because ole faction is important in feeding behavior the higher dose might effect the body weight gain and the general health of the animals during the 2-year study, so they got below that effect or at least to where we thought it would not be a significant effect in effect in the study. No effect on survival, and in body weight, in fact the exposed animals had body weights that were approximately equal to E even greater than the controls, especially in the male rats here. At the end of the study they were about 10% greater than the controls, and the females, you can see not much effect at all at the 300 per -- kilogram level.
The male rats, after two years there were thigh mommas, [indiscernible], mammary gland carcinoma -- both of these lesions are rare in occurrence compared to historical database. It's small for the studies, not observed, all routes of exposure, for the thyme thy momma -- and the mammary, seen three times out of 1100, those are very e very low rates. And the assay -- rates for the trend.
Turning now to the mice, the survival rates in the males, high-dose males versus control, the decreases there were significant, and the trend with those was also significant. We have a survival effect that appears to be treatment related in the male mice. There was no effect on the survival of the females. With regard to the weight decreases in the males, they decreased to about 90% of the controls. In the females to about 85% of the controls. By the usual estimates of NTD, those did not exceed NTD. The neo plastic and nonneo plastic lesions in together liver for the male mice -- all that was there was hep at cellular add noma, carcinoma or combined Aden only a or cars carcinoma, all significant, and also the trend was significant. The only other nonneo plastic results for these neoplasias for Aden only a, ma, using the -- the rate is 50% using routes, and 48%, up to as high as 84% 84. For carcinoma all routes, the rates up to 38%.
With regard to the combined incidence of adden only a or carcinoma significant events for both, trend and also individual exposed groups versus controls.
For the female mice, histio sittic car commas, here in the controls, the asterisk indicates the occurrence in the other groups, 1, 1, and 4, statistically tistically significant trend.
It's not been ob served in the other Cornell study, good sample there, the rate historically is 2.5. So this is another rare tumor.
The conclusions then, we reached, for the results of the 2-year studies in the roots, evidence of carcinogenic activity in male rats based on increased incidences of the rarely occurring did I momma and mammary gland carcinoma. No evidence of carcinogenic activity in the female rats at any dose. And the lesions in the nose, male and female rats were increased.
In the mice, excuse me -- carcinogenic. Clear evidence of increased carcinogenic activity based on the -- or combined and -- carcinoma. Any equivocal evidence regs based on incidences of -- and again there were, well, there were more nonneo plastic lesions in the mice, they occurred in the nose, fore stomach, glan lar stomach and -- the mice.
Summary slide was promised. Here it is. We are looking at meth eugenol, Estragole, and Isoeugenol, here we have various endpoints of toxicity, the columns show, I think, that convincingly the toxicity of meth eugenol and Estragole are very similar, and different from the toxicity observed for Isoeugenol. So, this will be incorporated into our report, paper we publish, expand expanded on, drawing more from the liter lit ra. Approximately eight different chemicals in this class. Thank you.
Does the committee have questions at this time?
Dr. Crump: Thank you for the presentation. I have a general question. In the course of your presentation you indicated the NTD was not exceeded --
How does the NTD define the maximum tolerated dose, what basis for that comment?
What comes to mind for me in response to your question, back in the mid-80s there was a committee set up to address the design of these studies. It was called in the report in general the do-all committee report published in 84, 85. In there they talk about NTD survival, body weight effects. In general if there's no effect -- survival in the body decreases to do not exceed 15%. There's a published citation, authoritative on that point.
I just wonder where the 15% figure, good to know.
That part, and general experience, where I am coming from with that, statement. Seemed to be a concern with the est -- I added it on the fly.
Kenny Crump: As far as I could study, was well-conducted. I agree with the consolutions, think that's all the comments I have at this time.
Okay, Dr. Pino?
Dr. Pino: As far as any scientific criticisms, it was acknowledged in the discussion the lack of effect at high dose in rat study suggested high dose could be tolerated. That is acknowledged. I don't recall in the dose -- data, used as part of rationale, if that's not there maybe you could add that to part of the rationale.
Then again, I had some similar comments about -- clearly identifying those considered directly compound related, also secondary data, body weight changes. Then also, as far as identifying some of the, for the histo logic findings, lower doses not significant, clearly related to the compound. On page 78, discussing the finding in the spleen about cellular depletion, due to decreased -- cells. I want to ask the pathology is if that was really hem at owe pettic cells or limp owe cites, if it was lymphocytes, I would change it to reflect that maybe.
As far as the kidney findings, there was some neo plastic -- in the kidney, I think PapO and tubular nee cro sis, I would bring those, something you don't usually see as a background finding that often. I would bring that forward to the summary and mention non-neo plastic effects for the mice, kidney effects.
Then just a comment on the statistical methods section, calculation of incidents. This was also in other reports as well, but where it says when macroscopic examation required to detect certain -- denominator consisted z of certain number of an mals in which neo cropsy per formed -- the -- routine test necessary test necessary mammary gland or -- unless I am interpreting wrong, an example that should be included there, that's all.
Doctor
Dr. Bradfield field: Nothing to add.
Dr.
Yes. Again, thank you very much for the thorough review and comments. I would like to respond for Dr. Pino, some of the general comments you made about effects of toxicity, we will address all those as indicated earlier. Thank you for those. I think with regards to your question about the distinction between cellular depletion in spleen and liesm liesm owe lymphocytes --
The word was incorrect. Should have been lymphoid. We will change that. It was a mis-- the wrong word. We will indicate it was lymphoid depletion. While I have the mic, can I address -- the protocol required tissue, that statement is not entirely accurate. We will change it. Those organ systems are [audio choppy] thank you for finding that.
Jon Mirsalis: An issue brought up yesterday, identifying positives in the micronucleus. This is a little more A egregious than yesterday. You are calling the female -- grace will smack me, but the POO 1, anybody looked at the numbers? The studies we have done in the last few days, the controls, space.five to 2.7, typical. This happened to be low, .5. The highest response was 1.6, we're classifying this size a clear positive based on a single group with 1.6 per thousand micronuclei, means you did eight mice, half had one, half had -- I accept there's an increase -- this is so -- even on the low end of normal control range, I cannot imagine how one would call this positive. I think we have to look at these things in the context of what wlab historical ranges are. You have an unusually low control, very low. That 1.6 on which we are calling this positive, the low-dose mail eugenol had 1.6 and the male is clearly negative.
I just -- I think you really have to -- significant, however well within the control range -- the same as the low dose on the male which we are calling negative, lower than the typical control. I think there's too much of a knee-jerk the P level says -- you have created a [indiscernible], in vivo vive tox Ico -- you have to put caveats on the report.
So we will look at this issue closely after hearing -- thank you.
Okay, anymore discussion? Dr. Howard? I think this is a very nice study. Enjoyed reading it. The one question, you make a good issue about the incidents of the thigh momma and mammary malig Nancy malignancy in the male rats --
I don't have an answer for that. Grace, do you --
Verification, are you asking separately or animals where you have seen both in the same --
Within the same dose.
The nature of rare occurrences makes answering that difficult, but we will search.
You do go into good length explaining how it's different than historical controls, but looking at induced might add weight to your argument.
Okay. We also had written comments on this report submitted by Dr. [indiscernible], New Zealand Limited, and Dr. Mark tp ton bee, MIT, and in Sweden. Are there any oral comments from the public? From these two individuals? Dr. Goodrich? Did you want to present --
You have -- minutes to present.
Goodrich: Thank you. I thank you for the opportunity to speak. I represent -- their U.S. agent responsible for providing regulatory consultation. A question ease New Zealand has -- they have been e been in the -- in their efforts to register the product internationally, and my charge for the United States, they have had to go through, obviously, quite a number of regulatory hurdles and in reading the report, we found something we felt was inconsistent with the types of things we are required to do in our studies. Basically, Isoeugenol, the methodology which Dr. Bristol indicated was one of the methods, the method we use, catalyzed, typically ice Jen Isoeugenol through oxidation goes through -- one of the things is -- can be effected by light, water, and by oxygen or exposure to air. It's this latter which led us to -- and the statement within the test article, used 99% pure, led us to the conclusion that's you may want to consider that particular statement.
This has to do with the methodology used, the gas cro mat graph methodology used to analyze the article. As I indicated, AQUI-S has a -- to meet regulatory requirements internationally. Although we acknowledge that the methodology the NTD used or -- used in the study is consistent with what's found in the literature, it's really inadequate to detect the polymerized compounds. These are the type of compounds we would see in a polymerized sample. In the bottom two, neither of those are detected in the methodology used in this study. Essentially what we are looking at is the NTD starts at roughly 80-degrees, ramps up over the first eight minutes, held for a total of 17. The methodology developed, modified to look for these polymerized compounds by AQUI-S New Zealand, starts at -- held for 13 minutes, over the next three ramped up, held for 14 additional minutes, total of 30-minute time continuum.
In looking at the -- fresh material by both methods, and polymerized. This is the typical for Isoeugenol in NTP method and the AQUI-S method. The scales are different because of the temperature and time differences. Even in fresh you are missing at least 9/tenths of a%. Doesn't show up because of the scale. 1% of the material that [lost audio] when we look at polymerize material with the NTP methodology you see the primary peaks of -- some mine oar weak peaks occur, but -- looking at the same material, polymerized color with the AQUI-S method, as we get out past 13 minutes, there's 6.5 of the material that's polymerized.
This is just an expanded scale with the NTP, and with the AQUI-S method showing this roughly 6 and a half percent polymerized. The conditions we normally look at is that for our product, we have about a two-year shelf life, pretty standard for a compound, the volatile nature of this particular compound. As we read the report, the understanding is a single or actually two lots were purchased back in 1997, these were stored, used throughout the course of the study, which, depending upon how much shelf life was left when first purchased, could be 7 to 9 years, exceeding normal shelf life. Light, water, the other one, keep forgetting -- air. Can cause breakdown or polymerization, when AQUI-S -- they purge with nitrogen to get rid of the air, I understand the conditions used in this one [indiscernible], trying to use a single-source for the entire study, but I would submit this is not in keeping with a typical gerks LT for a study we do for the GLP studies we conduct for regulatory studies. I appreciate your time ask my opportunity to present this.
Thank you very much.
Dr. Tone bee?
I appreciate the opportunity to make comments. I am Mark Tom by. in the regulatory work we have been carrying out I have also laid out a number of studies for this product, and we have a recent study sponsored by [indiscernible], which could be of interest for the -- we have
I can add that -- full copy of that report, but for proprietary reasons it is kept at least confidential until now. the report concentrates on the -- study, and when it comes to the conclusion also presenting quite a lot -- some information on this -- which you have already seen.
The new study, which I will present some data on is also in vivo Jen owe toxicity study. The test made according to the -- 474 study, carried out by the laboratory in Denmark, now days called, LAD, carried out in MN MRI mice.
After preliminary study it was decided -- use corn oil as vehicle, and oral dosing by -- the design of this study, it's according to the -- guidelines, also design from the -- studies, the -- studies, 2-year studies. As you see, the vehicle and the doses, eugenol, and -- body weight -- they are not significantly different from the control, and pretty similar to each other. [indiscernible], a positive control at the lower dose, which is significantly different from the control. This is, once again, polly chromatic studies, carried out according to G EP. We have a second -- exposure of 48 hours, the original was 24 hours. Again, no significant difference. The conclusion of this report is that, Isoeugenol did not show any geno toxic activity in the test in the mouse. It is a citation from the actual report. Then I have also added the studies we have already seen today in the main presentation by Dr. Bristol, that are here are two entity studies, in vitro studies, both negative, and the in vivo study which is partly negative, in male mice, the report written as positive at highest dose, 600 million PKO. But when I looked on the actual data on page E nienl 9 of the report, I found the results were not easy, so to say, for the female to interpret as the clear positive result. The difference between control male and female mice is greater, actually greater than between the -- male and female mice treated. In order to understand the results here, I think that it's necessary to have some historical control data to compare with, and positive control data to compare with. I have not found such data and tried to search because it is quite new information.
on the other hand, we have from Dr. John -- your -- you have hit on this point in the report as well.
We have got at least verbally some historical control data on the control. We think that the weight of evidence clearly suggests that Isoeugenol can be regarded as known geno toxic. Based both on the NTP studies and we have this problem with evaluation of the female data. We have to mention. But still, and of course we base this -- on study. So, we have come to the conclusion that we would like to suggest that the report which has been so far -- toxicity, not only two-year, but all the other toxicity studies, but in the final conclusions, there is not written anything about the unitoxic aspects in the study, not even on the rodents. Therefore we would like to suggest that it is added, sentence here, where it is -- there is strong evidence to support geno toxic -- new strong evidence to support geno toxic -- statistically speaking -- in the evidence of -- mice. Thank you.
Thank you very much.
Is there discussion from the board?
Are there anymore comments from the public?
There being none, we will display the conclusions and entertain a motion on acceptance.
While he's getting that up. A point of clarification for Doug. I am a little troubled by the discussion about the polymerization in the exper pirration of the material. What may not be evident to the gentleman who made the comment is how long it takes to get from a study to this report stage. My guess is it hasn't been seven to 10 years, but could you comment, Doug, on when the in-life phase ended relative to the purchase of -- is it a couple years or was it in fact many years that the material was sitting on the shelf?
Dr. Smith went over reports, so on, that are pertinent to this, and put together the timeline that answers the question. I think the -- what we really need to refer to is the information included in the appendix of this report. Some of D r. Goodwin's information came from a different study of Isoeugenol, and the information from that report. But from this report here, this technical report and records in the NTP archive for the study, the [choppy audio] took three weeks to perform the identity, and -- study, up to the present it's been stored at low temperature, minus 20-degrees in sealed containers. Also, initial analysis for puter purity of the test article -- would have detected polymer if present, and saw nothing that would have exceeded 1%. Those same analysis were reported on stored samples of this lot, recently, in connection with another study, reproduced those analyses done more than two years ago, but approximately two plus years. So we feel confident about the test article and that it was handled properly. Looking at details of records, study, I think, clears up that question.
I have a question in had terms of what is the implication of having polymers there in terms of toxicity?
There's some reports in the literature of biological activity of the DI-Isoeugenol, one more step, dehydro Isoeugenol. As model compounds for studying lignin chemistry, kind of interesting. And thal in of -- nothing is very noticeable, but they have some biological, some commercial interests are exploring.
Thank you. Anymore discussion?
Paul?
If a statement should be added to a materials, section -- page 31, 32 to reflect an analysis of polymer content was conducted and what was found, give that information to the public at least.
Is there any discussion -- for the conclusions?
There's actually nothing in the conclusions, typically you don't put -- I have no other comments.
I entertain a motion to motion to accept the conclusions?
One comment. I still suggest adding the kidney as non-neo plastic finding for the mouse.
Dr. Crump made the motion to accept the conclusion, is there a second?
I will second, Keith Soper.
All in favor, raise your hand.
opposed?
Dr. Pin Dr. Pino?
I would like to add --
Carries, with the assumption that will be added.
All right. Then I guess we are ready to move on to 5 high to hide hydroxymethyl. Furfural.
Conditions present during the cooking, baking of most food stuffs. Examples of commonly occurring food stuffs containing five hide drox meth -- breakfast cereal, coffee, pastries, dried fruits, beer. Many people don't consider that a food item, but I thought worth mentioning. It's ubiquitous in the human diet.
The major metabolites in it humans are normal constit wepts of human your in. Some industrial uses in the synthesis of several types of pollins and resins, undocumented, and major exposure is through the diet.
NTP first became interested in the compound, based on a series of papers published in the early 1990s suggesting that compounds form during cooking could possibly be involved in the promotion of colon tumors. In the first paper here, by, what was done is to initiate a group of rats with oxy meth -- feed different diets, one with -- one with [indiscernible], in ole, and one control, that study showed that the rats receiving either -- increased numbers in -- the gastrointestinal tract, the colon. They also showed 5 hide drox meth alone had the -- the activity much weaker than oxy methane. In the second paper the authors hypothesized possible intermediate, they ingt sized the sulfate ester, showed it was mute owe Jenic, and also singt sighed the clor owe metic, but since the pathway of that particular metabolite was not clear, whereas the cul fa transfer as and involvement of -- is well-known, they hypothesize the the cul feat ester might be ght be might be a -- so this suggested -- for mages of sulfate ester, of course, the active compound, DNA mutations, and lead to cancer.
So it was against this backdrop that this compound was nominated by NIEHS, and based on the papers I just summarized. The fact there was essentially no data on the cars carcinogenic -- widespread, probably everyone on the planet. And additionally would provide information about the role of naturally occurring dietary chemistries that would be potentially cars car carcinogen. So it was administered by gavage and water. We did absorption distribution, metabolism, discretion studies. I summarized the studies in the report, the details were actually published in a separate publication. We did three-week and three month toxicology studies. During the 3-month study we collected urine three different --
The range findings indicated that in rats there was really no treatment related pathology, a slight decrease in the mean body weights of -- males, but very little else happened in that study. So we used the same doses from 94 to 1500 -- in mice no treatment related pathology, however we saw reduced survival at 1500-milligram in male and females, therefore we backed off for the three-month study to use 750-milligram per kilogram as the high dose.
During the 3-month study there was in rats, reduced body weights at the top two dose levels, reduced survival at 1500-milligrams per kilograms, but no biologically significant reduce in weightings or -- females, reduced survival, again no clinical signs, biologicalally significant body weight changes, or histopathic lesions. We found at 375-milligrams per kilogram and above the ease trough cycle was prolonged in females. Because of the reduced survive will in both sexes we selected 750 as the top dose in the two-year study.
In mice, males, we saw reduced body weights at 750 in the 3-month studies. Didn't see treatment related changes in organ weights or -- there was histopath logical region, at 180 and above. There was no significant treatment related pathology in females, a slight body weight reduction. We selected 750-milligrams per kilogram as the high dose for mice.
The results of the A DME studies confirmed -- is met metabolized. The urine analysis conducted at the thee different time points indicated no evidence of glia keen depletion.
During the two year stud nee rats there was no significant treatment related to survival or mean body weight, no treatment reported neo blast lesions -- of the nose, degeneration and -- both male and females. Scwameous and hype -- in male and [indiscernible]. Disbliefer during the two-year study in mice, the survival and body weights of the 750 oar 750 -- therefore these groups could not be used -- seizure activity -- occasion, other neurological signs, initial pathology review indicated no neo plasms in the prain but currently involved in a much more in-depth review of the brain, central nervous system, and we will include the results of that review in this report which it's complete
In male and females we saw significant increase in nonneo plastic lesions of the nose in -- and in females there was a significant increase in the incidences of and add noma and carcinoma combined ombined in the -- I note this trend, the control and the 188 and 375 group, nothing happened as far as carcinomas were concerned.
So based on those results, we conclude there's no evidence of carcinogenic activity in rats, no evidence of cars carcinogenic activity in male mice. Some evidence of carcinogenic activity in mice based on incidences of -- in the -- groups, and administration of 5 hydroxy -- the ole factory olfactory and -- the end.
Thank you very much. Are there comments from the boards, to begin with? We will go straight to review. Dr. Mirsalis? I have no major criticisms. This is a nice study, nice report. A couple things. You saw a lot of what I would call clinical neuro behavioral effects, unconsciousness, salvation, cat tone yea, some death, you are studying that, that's excellent and you should include that in the report. In light of the human exposure, I would like to see a little discussion on the average dietary intake in humans, what it might be in milligram per kilogram. Can would be useful, pull a calculator, determine average dietary intake the average person consumes from this to this, that would help in the discussion.
A trivial point, something I brought up yesterday, more to add. I would like to see you mention in the body of the report what the feed was used. You have to dig in the appendix to find that. Under the, now the way FDA is enforcing analysis, we subsequently determined after talking -- the -- are not doing what FDA recommended, secondary independent analysis of feed constituents. Something for your entire program to talk to -- go back, talk to contractors, something FDA is issuing a tions on now.
Didn't see lesions in the colon, some discussion, calling that out, one of the first things I looked for and you don't really address it other than to say there was carcinogenic -- and another thing -- put nip% 9%, very nice report. Otherwise I agree with the conclusions.
Dr. Soper: I thought this was a nice report. I thought it was well-done. I had one comment on the statistical analysis, typically the trend test. Since these 750-milligram per kilogram dose was deemed above the maximum dose in mice, not included in the evaluation of carcinogenic potential. It would also be appropriate for them not to be included in the polly 3 trend test. You will come up with the correct conclusions with the pair-wise comparisons, but to show the difference it can make, for the hep at owe cellular, carcinoma and -- you got a value of -- and I think I saw in the report you gave without the top dose the P value is .001, big change. Thanks.
Thank you. Dr. Pino?
Dr. Pino: My comments mainly concern the rat study. In the report, the results, there was talk about some -- adenomas not addressed anyplace else. I would like a better idea of bhoots thought to be going on there. Also, there were some clear cell -- in the liver, I think mentioned in the discussion buts not mentioned in the summary, in another report they were. So for consistency maybe they should be -- not in conclusions, at least the summary.
Then there's a statement that says two hep atee sight -- in 375, but not, no indication of if those are considered compound related or not. I think they are not, but that should clearly be stated and why.
Thank you. Would you like to respond? D r. Irwin?
Let me start with Dr. Pino. The add adenomas in male rats, a single occurrence of two animals, no dose response. We will definitely explain that, why we considered it not treatment related in the report. As far as -- cell adenomas, again, there's really -- they were increased only not in highest dose group but in the 375 group, and there's no dose response there. The control rate in the study, 1 out of 50 is very low, the historical control is 14%, about seven out of 50. There were no effect s in females, the reason we considered the adenomas not to be treatment related. I will delineate in the report. We agree they increase with dose, not preneo plastic, but we will -- [indiscernible].
Dr. Soper, I put that in the slide for your comment. Actually in table 13 we show the P value, but will make sure we get that in the body of the report, mention it so we use the correct P value just using the control and the 188 and 375 group and delete the high dose.
I have tried, Dr. Mirsalis, specifically looked for quantification of the two metabolites, in -- attempt to calculate what might be considered an average exposure. I will continue to look at that. Depending on what dietary substances you want to add together, but you can put together what you might consider an average human diet, figure something from that. I will certainly pursue that.
I think it's really -- I agree, worth mentioning the fact we found no lesions in the colon. Actually, was, when I first started going through this, discovered that HMF wasn't all that stable in feed it made me wonder about the first study when they administered in feed, I thought we might find something different here. I would be glad to answer any other questions.
Anymore discussion? Dr. Howard? Dr.Howard: I agree with Dr. Mirsalis' suggestion, putting the daily average intake. The discussion, as far as drawing comparisons with the doses using the study, but based on comment you just made I would also suggest if it's not already published, you put a statement in the -- no reports of average daily intake could be found. I do not recommend you try to calculate it.
I really looked, almost all publications are concerned with determining the levels, various types of food, but almost none deal -- except one particular one in which it was actually shown that these were components of the human urine, but not quantitated. It's a good suggestion.
Not try to move beyond what's published as far as average daily intake. That could be fraught with a lot of problems.
Comment from the public?
Ron Lawrence from FDA. Is since this material is a product of thermal degradation of food, it's going to occur in all cooked food, processed food, they turn out to be quite good once you get there, but you have do a lot of Monte Carlo A flail sis, analysis to get a good figure. You start taking the figure, level, 1000 foods, add them up, you don't get what you want. You get -- there's -- gluttony has it's limits, you know. I suggest that unless it's been done, I doubt it has, that you don't do this. I will just point out that once you start going down that road, folks, you are doing risk assessment. But because what you want to do is compare this to -- what humans are getting to what rats are getting, that's risk assessment to me, and whatever --
Thank you for those comments.
Anymore discussion? Dr. Crump?
I wanted to make another comment on the C cell. Adenoma, carcinoma, and the tie thyroid. That's very striking response, in the controls, one to 14 in the mid-dose. I am not sure that's dose related, but I don't think we can attribute that to chance either. It either says it's dose related or some of the assumptions about our study are not being met. If you start distributing animals at random, dose has no effect, not one time in a 1000 will you get that large a difference. To me, just -- if it's not dose related calls into question some of the assumptions or some of the ways we're conducting our responses or something. I think it's a very striking response. I will also comment on the liver tumors, female mice. Going back to the thyroid, no effect in the male rat -- female rats. But in this case, if the liver adenoma, mice, significant decrease in males, significant increase in all struck me as quite strange.
Well, the control here is really far below what the historical control is. That would probably not be a significant increase if that control were nearer what the historical control is. Now, that combined with the fact that in my mind if this were really treatment related certainly we should see some type of dose response. It's hard to imagine why it would be so high just in that one mid-dose, not increase also at a higher dose. I realize it seems to be sort of a freak response, but occasionally we see these types of things. So also the fact that actually the carcinomas did not increase at all, they were 0122. No indz station Indication this is -- led us to conclude it was not treatment related.
Could you repeat the historical background --
14.16% -- 750 --
We're basically almost twice that in the --
Correct.
Dr. Howard: I can agree with your argument in relation to historical controls, but I I don't using the one middose, you use the same logic to drop the high dose in the mice from your trend test, because of a significant decrease, looking at historical controls is probably the best way to look at --
Did that make sense?
Well, when you say high dose in the mice --
Table 13 you are dropping the 750-milligram per kilogram, using two doses. You use that same logic to -- to say I can drop the high dose and do polly K test --
In the case of rats there were no mitigating circumstances in the high dose, like in the case of the high-dose mice. Many of them died. There was obvious, significant compound related toxicity. Nothing to indicate there was a problem in the high-dose rats at all.
Good response, thank you.
Thank you.
Sometimes a little disconnect comes across, different end points get --
I will try to explain that better.
In the context of in the historical control data or not --
Thank you. Anymore comments before we display the conclusions?
Public comments?
Okay.
Any discussion?
Just asking the question. Adenoma incidences sufficient for some level of carcinogenic activity ? yes, okay.
There being no discussion, I will entertain a motion to accept the conclusions?
Jon Mirsalis salis, I move we accept.
Second?
Somebody brave?
Keith Soper, I second.
All in favor?
carries unanimously. Brings us to end of reports. Dr. did you have comments?
I can't express enough our appreciation of the staff, the, you put into the review, I thank the staff. The presentations this time were really excellent and I hope enjoyed your stay with us this time and you have a nice trip home. Thank you very much for your efforts.
Thank you. I would echo the presentations were outstanding, very clear, very thorough. Dr. Shane, did you have parting comments?
Thank you very much for coming, and the bus will be ready in about five minutes of noon. To take you to the airport.
Thank you.
Thank you, and the meeting is adjourned.
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