Guideline 3
III. Guideline for Threshold Assessment
A. INTRODUCTION
This guideline describes how FDA uses information to determine whether chronic bioassays are necessary to resolve questions concerning the potential carcinogenicity of the sponsored compound. The threshold assessment assumes that the potential of a sponsored compound to present a risk of cancer to people is based on the potential carcinogenicity of the compound and the exposure of people to its residues. The threshold assessment is not meant to substitute for a quantitative risk assessment or a comprehensive safety evaluation for the sponsored compound.
Each substance for use in food-producing animals will be subjected to a threshold assessment unless it is exempt. If not exempt, FDA recommends the particular procedure outlined in this guideline unless an acceptable alternative procedure is developed.
A substance is exempt from a threshold assessment if the purpose of a threshold assessment -- to determine whether chronic bioassays are needed -- does not apply. FDA has determined that it will not assess the potential for carcinogenicity for essential nutrients and endogenous substances. For demonstrated carcinogens, a threshold assessment is obviously not needed.
For certain other classes of substances, the purpose of a threshold assessment applies, but procedures and criteria other than those set forth in this guideline are more appropriate. Thus, alternative threshold assessments are appropriate. Such an alternative has been developed for biomass products (see guideline VII of this document), and will be developed for "other complex uncharacterized products" as needed. Other classes of substances may be exempted, as appropriate. Further, alternative assessments may be developed for classes not listed.
B. DEFINITIONS
1. Sponsored compound means any drug or additive proposed for use in or on food-producing animals.
2. Target animal means the production class of animals in which a sponsored compound is proposed or intended for use.
3. Essential nutrients mean compounds found in the tissues of untreated target animals and not produced by the animal in sufficient quantity to support the animal's growth, development, function, or reproduction. These compounds must be supplied from: external sources (e.g., vitamins, essential amino acids, essential fatty acids, and minerals).
4. Total residue means all compounds present in edible tissues of the target animal that result from the use of the sponsored compound, including the sponsored compound, its metabolites, and any other substances formed in or on food because of the sponsored compound's use.
5. Residue of carcinogenic concern means all compounds in the total residue minus any compounds shown not to be of carcinogenic concern.
C. POTENTIAL CARCINOGENICITY
FDA will consider the structure of the parent compound, data from short-term genetic toxicity tests predictive of carcinogenicity, data from subchronic or chronic toxicity studies, and any other relevant information to assess potential carcinogenicity. The sponsor should provide the results of a literature search on the biological activity of the compound, including relevant information on structurally related compounds. The sponsor should also provide all relevant information pertaining to pharmacological and physiological activity of the compound and its metabolism in target or experimental animals. This information can provide clues regarding the toxicity of the compound.
1. Structure
Because of uncertainties in identifying potential carcinogens based on molecular structure, FDA will use the structure guide (see Appendix 1) as a screening device. FDA scientists will consider all of the relevant structural and biological information on a specific compound before assigning that compound to a toxicity category based on its structure.
2. Genetic Toxicity Tests
Because the field of genetic toxicity testing is undergoing rapid development, sponsors are urged to seek guidance from FDA on the specific tests most acceptable for the specific compound of interest. The sponsor should test the compound in three test systems that have been demonstrated to have a high correlation between positive results in chronic bioassays for carcinogenicity and positive results in short-term genetic toxicity tests (see Appendix 2). The sponsor should include tests for gene mutations in two test systems and may use bacteria, Drosophila, or mammalian cells in culture. As the third test in the battery, the sponsor should include a test for DNA repair synthesis in mammalian cells. When a compound belongs to a structural class containing known carcinogens and for which these short-term genetic toxicity tests are known to be inadequate predictors of potential carcinogenicity, FDA will not use such tests to reduce the concern for carcinogenicity.
3. Feeding Studies
Data from subchronic feeding studies conducted on a sponsored compound may frequently raise the suspicion of carcinogenicity. Examples of such data include liver or kidney necrosis, biliary duct hyperplasia, endometrial changes indicative of a preneoplastic condition, or any other biological effects indicative of the development of cancer.
4. Other Information
Other information on the compound may also raise a suspicion of carcinogenicity. Examples of such information include evidence of covalent binding to cellular macromolecules (protein or nucleic acids) or intercalation into nucleic acids; evidence of non-endogenous gonadal steroid activity; evidence of persistent stimulation or inhibition of synthetic activity of a specific organ or cell type that may be a potential indicator of a preneoplastic condition; evidence of toxic effects on rapidly dividing cell populations.
5. Category Assignment
FDA will evaluate the information submitted by the sponsor and will assign the compound to a category (A, B, C, or D). FDA will assign compounds for which it has the lowest suspicion of carcinogenicity to category A, and those for which it has the highest suspicion to category D.
If the compound or its metabolites are structurally related to a demonstrated carcinogen, FDA will initially assign the compound to category D. If the sponsor submits results from a battery of short-term genetic toxicity tests and subchronic feeding studies that do not raise the suspicion of carcinogenicity, FDA will re-assign the compound to category B; if the results from the battery of short-term genetic toxicity tests, from subchronic feeding studies, or from other sources raise the suspicion of carcinogenicity, the compound will remain in category D.
If the compound or its metabolites are not structurally related to a demonstrated carcinogen, FDA will initially assign the compound to category C. If the sponsor submits results from a battery of short-term genetic toxicity tests and subchronic feeding studies that do not raise the suspicion of carcinogenicity, FDA will re-assign the compound to category A; if the results from the battery of short-term genetic toxicity tests, from subchronic feeding studies, or from other sources raise the suspicion of carcinogenicity, FDA will re-assign the compound to category D.
Instead of the above procedure, the sponsor may develop a defensible alternative procedure that is shown to be comparable or may elect to conduct chronic feeding studies. If adequate chronic bioassays in two test species demonstrate that the sponsored compound is not carcinogenic, FDA will re-assign the compound to category A. The sponsor should provide information from test and target species that demonstrates that the test animals will be exposed to a spectrum of metabolites similar to those produced by the target species. Discussion of factors to be considered in the conduct of adequate bioassays for carcinogenicity may be found in the current literature (see for example, "Long-term and short-term screening assays for carcinogens: A critical appraisal," IARC Monograph Supplement 2, pp. 5-83, Lyon 1980; "Identification of potential carcinogens and risk estimation," Report of the Interagency Regulatory Liaison Group, J.N.C.I., 63, pp. 240-268, #2. July 1979).
There may be cases where chronic testing of a compound has been performed, but the data do not meet FDA standards for demonstrating that it is not carcinogenic. These data, depending on their quality, may be of equal or greater value than the evidence in the other areas considered in the threshold assessment. In such a case, FDA will assign the compound to an appropriate category after consideration of all the evidence.
D. EXPOSURE
If a compound is assigned to category B, then FDA will consider the potential exposure of people to its residues. When considering potential exposure, FDA evaluates the proposed conditions of use of the compound in target animals and the residue concentration under those conditions of use.
1. Proposed Use
The frequency of human exposure to residues of the compound is related to the extent of its use in food-producing animals. For example, if a large number of food-producing animals are treated with the compound, then people would frequently ingest its residues; conversely, if a small number of animals are treated with the compound, then people would intermittently ingest its residues. FDA will determine the use classification for each individual sponsor's product from the labeling claim that has the greater potential for human exposure to residues of the product. FDA will assign a sponsored compound to category H or category L by the criteria described below.
L - Administration on a selective basis for specific disease treatment or prevention, for specific reproductive uses, or administration for minor uses only.
H - Administration on a non-selective basis to a herd or flock for production improvement or prevention of common diseases.
Category H includes products that claim growth promotion, improved feed efficiency, or a increased rate of weight gain. Category H also includes products used for prevention of common diseases as a standard practice without waiting for the appearance of signs of disease in members of the group. This situation may occur when disease is known to be prevalent in the particular species, and there is a high likelihood of its occurrence. The potential for frequent human exposure to residues of these compounds is high.
Category L includes products used for treatment or prevention of specific diseases where signs of a disease have occurred and a diagnosis has been made. Category L will also include compounds intended for minor uses only, as defined in 21 CFR 514.1(d)(1). The potential for frequent human exposure to residues of these compounds is low.
The following discussion provides examples of the application of the above use categories. Coccidiosis in fowl and atrophic rhinitis in swine are common conditions. These species often receive treatment to prevent these conditions in the absence of signs of disease. Accordingly, FDA would assign to category H sponsor compounds used to prevent these conditions. FDA would also assign to category H a sponsored compound used for the prevention of round worms in swine because infection with round worms is a common condition in swine. However, FDA would assign to category L an anthelmintic used for a specific worm condition after diagnosis. Likewise, FDA would assign to category L a sponsored compound used to treat a condition diagnosed as fowl cholera. FDA would assign to category L a sponsored compound used to treat an individual animal diagnosed as having signs of shipping fever or used to treat the remainder of the herd to prevent shipping fever. Category H would not be appropriate for such a compound because shipping fever is not a common condition that receives routine treatment in the absence of signs of disease. FDA would assign to category L a sponsored compound used to treat individual dairy cows showing signs of mastitis.
2. Total Residue Factor
The amount of total residue ingested in a single exposure is equal to the amount of tissue consumed times the measured concentration of residue in the tissue. FDA will estimate the amount of tissue consumed and calculate the exposure as described in GUIDELINE FOR ESTABLISHING A TOLERANCE. The concentration of residue in the tissue will usually be determined from a total residue depletion study in target animals.
Ordinarily, the sponsor performs the depletion study by administering a radiolabeled compound to a sufficient number of previously unmedicated animals to permit serial sacrifice of groups of animals at intervals after the last treatment. The sponsor should use a dosing schedule consistent with the intended use of the product. FDA will calculate the total residue factor using the concentration of total residue in the tissue at the time the animals are expected to be marketed as food as indicated by the label recommended withdrawal period proposed by the sponsor, provided that the proposed withdrawal period is consistent with normal husbandry practices and reasonably certain to be followed in practice. If the sponsor changes the conditions of intended use or the proposed withdrawal period, FDA will re-evaluate this factor.
In the absence of information about the composition of the total residue in the edible tissue, FDA will assume that the entire residue is of carcinogenic concern. However, if the sponsor submits information on the composition of the residue, FDA will reduce the total residue factor by whatever amount has been identified and shown not to be of carcinogenic concern. For example, a portion of the radioactive residue may be a compound for which adequate studies have already been conducted to show that its presence as a residue is not of carcinogenic concern.
If the total residue factor is used to decide that chronic bioassays for carcinogenicity are not needed, the total residue factor will also establish the maximum tolerance that can be assigned to that compound under the general food safety requirements.
E. DECISION ELEMENTS
1. After all relevant information is evaluated, FDA will ask for chronic bioassays for carcinogenicity for a compound assigned to category D or C. However for a specific compound, if the half-life of its residues in edible tissue is short and it is administered a long time before slaughter (for example, several months), then FDA may not ask for chronic bioassays. Under these circumstances, FDA can conclude that any potential risk to people will be too low to Justify chronic testing. In reaching this conclusion, FDA will also consider whether the proposed conditions of use are reasonably certain to be followed in practice.
2. FDA will not ask for chronic bioassays for carcinogenicity for a compound assigned to category A.
3. FDA will ask for chronic bioassays for carcinogenicity for a compound assigned to category B if it is assigned to the high use category and the total residue factor exceeds 0.25 micrograms/ kilogram body weight/day (equivalent to a concentration of 10 parts per billion in the total diet of people, assuming a 60 kilogram body weight and a total solid diet of 1500 grams).
4. FDA will ask for chronic bioassays for carcinogenicity for a compound assigned to category B if it is assigned to the low use category and the total residue factor exceeds 6.25 micrograms/ kilogram body weight/day (equivalent to a concentration of 250 parts per billion in the total diet of people).
5. FDA will continue to evaluate each compound for other toxicological endpoints under the general food safety provisions of the act. If, during that testing, data are obtained that raise the suspicion of carcinogenicity, FDA will apply the threshold assessment to determine if chronic bioassays for carcinogenicity are needed.
CARCINOGEN STRUCTURE GUIDE
Over the past fifty years of research in chemical carcinogenesis, it has become apparent that the ability to induce cancer in laboratory animals and humans is associated with the presence of certain structural features or functional groups in molecules, and not with others. This was recognized empirically, and for many years textbooks and monographs on the subject of carcinogenesis have been organized according to structural classes of chemical carcinogens (1, 2, 3, 4, 5, 25). These empirical classifications helped stimulate the research over these years which has demonstrated that reactive properties of specific functional groups in chemical carcinogens toward critical biological targets, either before or after host metabolism, are responsible for the initiation of the carcinogenicity induced by these compounds. So, there has been established a firm scientific basis that structure, and, in particular, the presence or absence of certain functional groups, plays a dominant mechanistic role in carcinogenesis by chemical compounds.
The presence of a structural feature that is responsible for carcinogenicity in other compounds greatly increases the probability that another compound with the same features will also be carcinogenic. Unfortunately, there are many little-understood subtleties in these structure-activity relationships and, in spite of valiant attempts, structure-activity relationships or theoretical models based on them never have been able to reliably replace experiment in determining carcinogenicity. Nevertheless, it is sound to use the cumulative knowledge about classes of chemical carcinogens to aid in setting priorities for biological testing.
CLASSES OF CHEMICAL CARCINOGENS
1. Aromatic Amino-, Amido-, Hydroxylamino-, Nitroso-, Azo-, Azoxy-, Nitro-
(1, 2, 3, 8, 9, 14, 16, 17, 22, 24)
Typical Examples:
2-Naphthylamine
4-Hydroxylaminoquinoline-l-oxide
2-Acetylaminofluorene
4-Amino-2-nitrophenol
4,4-Dimethylaminoazobenzene
Mechanism:
Cellular metabolism is necessary for activity. The best evidence indicates that the hydroxylamino compounds are proximate carcinogenic forms. All of the above functional groups are related by oxidation state and can be converted to hydroxylamines by hydolases, oxidases or reductases endogenous to most tissues.
Comments:
Aromatic nucleus, Ar-, can potentially be any type, including heterocyclic aromatics like furan, quinoline, etc. Carcinogenic potency depends dramatically upon the number of fused or conjugated aromatics rings. Monocyclic derivatives usually have lower activity. Recent testing indicates that ring substitution by alkyl, hydroxyl-, alkoxyl-, or amino- groups enhances the potential carcinogenic activity of monocyclic aromatic nitrogen compounds. But, even aniline has now been demonstrated to be carcinogenic (26).
2. N-Nitroso- (2, 3, 10, 16, 17, 18, 22, 25)
Examples:
N-nitrosodimethylamine
(DMN)
N-Methyl-N'-nitro-N-nitrosoguanidine
(MNNG)
Mechanism:
For N-nitrosylated secondary amines, (a), metabolic activation is necessary. The best evidence indicates that alpha-carbon atom hydroxylation of an alkyl group is a prerequisite to the expression of carcinogenic potential.
For N-nitroso compounds derived from ureas, urethanes, carbamates, carboxylic amides and guanidines, (b), direct action without prior metabolism is the most likely mode of action.
Comments:
There is a relatively high correlation between the presence of a N-nitroso moiety and the capability of a compound to induce cancer. There may be exceptions due to structural prevention of metabolic activation. But these are likely to be few in number. All N-nitroso compounds should be viewed with extreme suspicion.
3. 0rganohalogens
a. Mustards (6, 15, 22)
b. Di-to poly-halogenated alkanes and cycloalkanes (6, 12, 20, 22, 25)
c. Alpha-halogenated ethers (6, 16, 22)
d. Halogenated alkenes (6, 13, 20, 22, 25)
e. Polyhalogenated aromatics (19, 22)
Typical Examples:
a. 
Di(2-chloroethyl) methlamine
b. 
Carbon Tetrachloride
Mirex
1,2-Dibromoethane
c. 
Bis(2-chloromethyl) ether
d. 
Vinyl chloride
e. Polychlorinated Biphenyls
Mechanism:
Mustards, haloethers and some haloalkanes are alkylating agents. They are thought to be direct acting carcinogens and not need any metabolic alteration for activity. The mechanisms of action for other subclasses of organohalogens have not been well studied. Enzymic epoxidation has been suggested as required for chlorinated alkenes and free radical pathways have been postulated for 1,1,1 trihaloalkanes
Comments:
This is a very diverse group of compounds. Prediction of carcinogenic activity is made difficult by the lack of knowledge of metabolism and mechanism of action of some of these compounds.
4. Hydrazo-(Hydrazines), Azoxy-, and Azo- (2, 10, 16, 22)
Typical Examples:
1-methyl-2-benzylhydrazine
Azoethane
Elaiomycin
Mechanisms:
Metabolic activation is considered to be necessary. The best evidence indicates that N-hydroxylation and further metabolic oxidations are prerequisite for activity.
Comments:
This class of compounds, particularly the hydrazines, has many members which are carcinogenic. The presence of these substructural moieties gives a compound a high possibility of carcinogenic activity.
5. Alkyl (Aryl) Sulfates, Sulfonates, and Sultones (1, 2, 6, 13, 16, 22)
Specific Examples:
Diethylsulfate
1,3 propanesultone
Mechanism:
Classic alkylating agents. These are direct acting carcinogens requiring no prior metabolism.
Comments:
Carcinogenic activity is greater for gamma-sultones than delta-sultones, presumably due to more strain, and hence, higher reactivity of the 5-membered ring compounds.
6. Strained Ring Heterocycles
a. 
Epoxides (1, 2, 6, 21, 22, 25)
b. 
Aziridines (1, 2, 6, 15, 22)
c. 
Beta-Lactones (1, 2, 6, 16, 21, 22)
Typical Examples:
Glycidaldehye
Aziridine Ethanol
beta-Butyrolactone
7. Fused Polynuclear Aromatic (1, 2, 4, 7, 11, 22, 25)
Examples:
7,12 Dimethylbenz (a) anthracene
4, 11-Diazadibenzo(b, d, e, f) chrysene
7H-dibenzo (c, g) carbazole
Mechanism:
The best evidence indicates that these aromatic compounds require metabolism for activity. Epoxidation of aromatic ring double bonds is the best candidate for the metabolic pathway leading to ultimate carcinogenic forms.
Comments:
Structure-activity is very subtle in this group of compounds. Even slight changes in structure can dramatically change carcinogenic potency. In spite of many years of study no simple reliable models of prediction are available. Any compound that contains three or more fused aromatic rings must be considered suspect, including those with nitrogen and even sulfur containing heterocyclic aromatic residues. Compounds based on the phenanthrene substructure are more often carcinogenic than those containing the linear anthracene moiety. The fact that quinoline has been reported to be carcinogenic, and several derivatives mutagenic, suggest that two fused rings are enough to produce an active compound if N-heterocyclic aromatic rings are involved. Recent epidemiological data indicate that benzene is carcinogenic to humans.
8. Aryldialkyltriazenes (10, 22, 27)
Example:
1- ( 3-pyridyl ) -3,3-dimethyltriazine
Mechanism:
Both non-metabolic and metabolic pathways have been proposed for the critical reactions responsible for inducing cancer.
Comments:
The Ar- can be either substituted phenyl or pyridine rings. Other aromatics may also be active.
9. Purine and Pyrimidine Analogues (5, 8)
Purine N-Oxides
or N-Hydroxides
Substituted Pyrimidines
Examples:
Xanthine-3-oxide
1-Beta-D-arabinofuranosylcytosine
Mechanism:
The purine-N-oxides are tautomeric forms of N-hydroxypurines. These hydroxylamines are thought to act similarly to other aromatic hydroxylamines. The mechanism of action of carcinogenic pyrimidine analogs is unknown. Most speculation about their activity centers around their ability to inhibit pyrimidine metabolism or perturb template function.
Comments:
The highest potency analogs are the purine-3-oxides. Some purine-l-oxides are also active, but to lesser degrees.
10. Thioamides (1, 2, 13, 22)
Examples:
Thioacetamide
Thiouren
REFERENCES
1. Clayson, D. B., Chemical Carcinogenesis, Little, Brown and Company, Boston, 1962.
2. Hueper, W. C. and Conray, W. D., Chemical Carcinogenesis and Cancers, C.C. Thomas, Springfield, Ill., 1964.
3. Arcos, J. C., Argus, M.F. and Wolfe, G., Chemical Induction of Cancer, Volume 1, Academic Press, New York, 1968.
4. Arcos, J. C. and Argus, M.F., Chemical Induction of Cancer, Volume IIA, Academic Press, New York, 1974.
5. Arcos, J. C. and Argus, M.F., Chemical Induction of Cancer, Volume IIB, Academic Press, New York, 1974.
6. Lawley, P. D., "Carcinogenesis by Alkylating Agents," in: Chemical Carcinogens, C. E. Searle, ed., American Chemical Society, 1976, p. 83.
7. Dipple, A., "Polynuclear Aromatic Carcinogens," in: Chemical Carcinogens, C. E. Searle, ed., American Chemical Society, 1976, p. 245
8. Clayson, D. B. and Garner, R. C., "Carcinogenic Aromatic Amines and Related Compounds," in: Chemical Carcinogens, C. E. Searle, ed., American Chemical Society, 1976, p. 366.
9. Parkes, H. G., "The Epidemiology of Aromatic Amine Cancers," in: Chemical Carcinogens, C. E. Searle, ed., American Chemical Society, 1976, p. 462.
10. Magee, P. N., Montesano, R. and Preussmann, R., "N-Nitroso Compounds and Related Carcinogens." in: Chemical Carcinogens, C. E. Searle, ed., American Chemical Society, 1976, p. 491.
11. World Health Organization, IARC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Man, "Certain Polycyclic Aromatic Hydrocarbons and Heterocylic Compounds," Volume 3, International Agency for Research on Cancer, Lyon, 1973.
12. World Health Organization, IARC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Man, "Some Organochlorine Pesticides," volume b, International Agency for Research on Cancer, Lyon. 1974.
13. World Health Organization, IARC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Man, "Some Anti-Thyroid and Related Substances, Nitrofurans and Industrial Chemicals," Volume 7, International Agency for Research on Cancer, Lyon, 1974.
14. World Health Organization, IARC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Man, "Some Aromatic Azo Compounds," Volume 8, International Agency for Research on Cancer, Lyon, 1975.
15. World Health Organization, IARC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Man ," Some Aziridines, N-, S- and O- Mustards and Selenium," Volume 9, International Agency for Research on Cancer, Lyon, 1975.
16. World Health Organization, IARC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Man, "Some Aromatic Amines, Hydrazine and related substances N-Nitroso Compounds and Miscellaneous Alkylating Agents, Volume 4, International Agency for Research on Cancer, Lyon, 1974.
17. World Health Organization, IARC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Man, "Some Aromatic Amines and Related Nitro Compounds," Volume 16, International Agency for Research on Cancer, Lyon, 1978
18. World Wealth Organization, IARC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Man, "Some N-Nitroso Compounds," Volume 17, International Agency for Research on Cancer, Lyon, 1978.
19. World Health Organization, IARC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Man, "Polychlorinated Biphenyls and -Polybromated Biphenyls," Volume 18, International Agency for Research on Cancer, Lyon, 1978.
20. World Health Organization, IARC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Man, "Some Halogenated Hydrocarbons," Volume 20, International Agency for Research on Cancer, Lyon, 1979.
21. World Health Organization, IARC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Man, "Cadmium, Nickel, Some Epoxides, Miscellaneous Industrial Chemicals," Volume II, International Agency for Research on Cancer, Lyon, 1976.
22. Fishbein, L., Potential Industrial Carcinogens and Mutagens, Environmental Protection Agency, Washington, D.C., 1977.
23. World Health Organization, IARC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Man, "Some Carbamates, Thiocarbamates and Carbazides," Volume 12, International Agency for Research on Cancer, Lyon, 1976.
24. Carcinogenesis, A Comprehensive Survey, Volume 4: "Nitrofurans," G. T. Bryan, ed., Raven Press, New York, 1978.
25. Structural Correlates of Carcinogenesis and Mutagenesis, I. M. Asher and C. Zervos, eds., Department of Health, Education and Welfare, Washington, D.C., 1977.
26. National Cancer Institute, Bioassay of Aniline Hydrochloride for Possible Carcinogenicity, Technical Report Series No. 130, Department of Health, Education and Welfare, 1978.
27. Preussmann, R., Ivanovic, S., Landschutz, C., "Carcinogene Wirkung Von 13 Aryldialkyltriazenen an BD-Ratten," Z. Krebsforsch., 81, 285, 1974.
APPENDIX 2
A high positive correlation has been observed between the results of point mutational and DNA repair tests with bioassays for carcinogenicity. Unfortunately, negative test data in the mutagenicity assays do not correlate as well. However, positive data from the less time consuming and less expensive short-term tests are considered useful for determining the use of resources for bioassays for carcinogenicity. The threshold assessment will use short-term test data for this purpose. The following provides guidance to the types of tests and acceptable protocols to be found in the literature as well as considerations to be used by FDA in evaluating submitted test data.
Many of the short-term tests that may be considered as screens for carcinogenicity are, in fact, tests for mutagenicity. While the hypothesis that the initiation of tumor growth results from mutations in somatic cells is a reasonable one, the validity of using mutagenic tests as screens for carcinogenicity does not depend upon the validity of this hypothesis. The fact that most known carcinogens are mutagenic in at least some test systems and that most mutagens that have been adequately tested are carcinogenic provides the primary rationale for the inclusion of mutagenicity tests in the battery of screening tests for carcinogenicity.
The full mutagenic potential of a chemical is unlikely to be assessed in any single test for either technical or design reasons. Therefore, the information from a battery of short-term tests is required to evaluate as fully as possible the mutagenic activity of a chemical. An ideal battery would contain tests that would provide not only corroboration but also complementation so that all classes of carcinogens would be detected. However, as yet there are insufficient data both from the short-term tests and the rodent bioassay tests to select such a battery with a high degree of confidence.
FDA has considered a number of types of short-term tests that have been developed and proposed for use as predictors of carcinogenicity. The criteria used for recommending a particular assay include: (1) correlations of results with known carcinogens and non-carcinogens; (2) reproducibility of results, both within a laboratory and between laboratories; (3) relative ease of scoring and interpreting test responses; and (4) availability or potential availability of laboratory capability for performing the test.
The battery of short-term tests generally recommended includes: (1) a bacterial gene mutation assay, the Ames Salmonella assay is suggested; (2) a mammalian cell gene mutation assay, the most widely employed are the L5178Y thymidine kinase system and the Chinese hamster ovary (CHO) HGPRT assay, and, to a lesser extent, the Chinese hamster lung cell (V79) HGPRT assay; and (3) a generalized assay for DNA repair (unscheduled DNA synthesis, UDS) in mammalian cells, the UDS rat hepatocyte system is suggested. There is evidence that some carcinogens do not yield a positive response in a particular short-term test. Therefore, when a compound is of a structural class for which there is reason to believe that a particular short-term test is inadequate as a screen for carcinogenicity, its use will not be accepted to reduce the concern for carcinogenicity.
The sex-linked recessive lethal test in Drosophila is considered a suitable alternative gene mutation test when circumstances preclude the use of either of the above mentioned gene mutation assays. An example of such a situation is when an antimicrobial or antifungal animal drug possesses such extreme toxicity in the Ames test as to preclude the use of sufficiently high doses for negative results in bacterial tests to be considered significant. Other tests or batteries of tests, may be acceptable as supporting evidence to reduce the potential carcinogenicity factor of the threshold assessment. Such substitutions will be evaluated according to the criteria listed earlier in this guideline. Sponsors are advised to consult with FDA prior to performing the studies.
In general, reproducible data indicating that a sponsored compound is positive in one of the tests in the battery will result in a requirement for carcinogenicity testing. Factors such as dosage, metabolism, test sensitivity and reproducibility will be considered in FDA's evaluation of data submitted. It is recommended that original data be included in all submissions.
Bacterial mutagenicity tests are suggested because there is an extensive data base on the correlation between results in such tests and carcinogenicity as determined by long-term animal studies (McCann et at., 1975; McCann and Ames, 1976; Sugimura et al., 1976). These data indicate that mutagenicity in bacteria is a generally reliable indication that a chemical is likely to be carcinogenic. It appears, however, that there are chemical classes of carcinogens that fail to be detected as mutagens in bacterial and other mutational assays. In some instances the problems may be overcome by a change in protocol (Prival et al., 1979).
Although the published data for tests in mammalian cells and Drosophila are not as extensive as those for bacterial mutagenicity tests, current indications are that these tests are useful as carcinogenicity screens. The most widely used test for gene mutations in cultured mammalian cells is probably the L5178Y mouse lymphoma assay which measures mutations at the thymidine kinase (TK) locus (Clive and Spector, 1975; Clive et el., 1979; Amacher et al., 1979, 1980). Two others assays for which the data bases are rapidly growing are Chinese hamster CHO (O'Neill et al., 1979) and V79 (Chu and Mulling, 1968; Huberman and Sachs, 1976). The marker commonly used in these assays is resistance to 6-thioguanine which arises from a loss of the hypoxanthine-guanine ribosyl transferase (HGPRT) activity.
Drosophila is one of the most extensively studied species from a genetic viewpoint and the sex-linked recessive lethal test is clearly the oldest assay for detecting mutations. Correlative studies for oncogenic potential of sixty chemicals have demonstrated that this assay has a high degree of detection capability both for direct acting carcinogens and procarcinogens (Vogel, 1975, 1977; Sobels, 1974; Wurgler et al., 1977).
A variety of techniques have been employed to measure DNA repair or unscheduled DNA synthesis either by radioautography (Painter and Cleaver, 1969) or by liquid scintillation counting (Stich and San, 1970). Only with radioautography is it possible to unequivocally distinguish repair synthesis from replicative synthesis and is, therefore, the preferred technique. Many types of cell lines can be employed. However, their use requires that replicative DNA synthesis be inhibited by hydroxyurea treatment or arginine deprivation. Since the metabolic capabilities of these cell lines are limited, the addition of an exogenous system for metabolic activation is required for the assay to be valid. Both of these problems are overcome in the UDS assay using primary rat hepatocytes (Williams, 1976, 1977). The freshly isolated nondividing liver cells that are used have the capacity to activate procarcinogens. The data base for this assay is growing rapidly and its sensitivity and reliability are becoming well documented (Williams, 1980; Probst et al., 1981).
All tests used in this battery should be conducted with and without a demonstrated metabolic activation system. While the cell systems used in short-term tests do not possess the full metabolic capabilities of whole animals, the incorporation of liver enzyme systems is considered the most reliable means for overcoming this limitation for routine evaluation of chemicals. Some test systems (Drosophila, rat hepatocyte) have these enzyme systems; for others, the addition of an exogenous enzyme system is necessary. The most commonly used exogenous enzyme system is that obtained using a 9,000 g supernatant (S9) of a rat liver homogenate. Other approaches such as the incorporation of cell feeder layers into the assay system have also been successfully used.
Due to continuing advances being made in the development of reliable short-term tests, it is difficult to select precise protocols that would be most highly recommended for each type of test. Among the references cited, the following contain useful considerations:
Ames test: Ames et al., 1975
de Serres and Shelby, 1978
Yahagi et al., 1975
Drosophila:Abrahamson and Lewis, 1971
L5178Y: Irr and Schnee, 1982
CH0: Hsie et al., 1981
V79: Bradley et al., 1981
The last two references are review papers that were part of the EPA Gene-Tox program. Additional papers on other assay systems will be published in Mutation Research. These reports are useful not only for their content but also for their extensive lists of references.
References
Abrahamson, S. and E. B. Lewis, 1971, In Chemical Mutagens: Principles and Methods for Their Detection, Ed. A. Hollaender, Vol. 2, pp. 461-487.
Amacher, D. E., S. Paillet and V. A. Ray, 1979, Point Mutations at The Thymidine Kinase Locus in L5178Y Mouse Lymphoma Cells, I. Application to Genetic Toxicological Testing, Mut. Res. 64:391-406.
Amacher, D. E., S.C. Paillet, G. N. Turner, V. A. Ray and D.E. Salsburg, 1980, Point Mutations at the Thymidine Kinase Locus in L5178Y Mouse Lympnoma Cells. II. Test Validation and Interpretation, Mut. Res. 72: 447-474.
Ames, B. N., J. McCann and E. Yamasaki, 1975, Methods for Detecting Carcinogens and Mutagens with the Salmonella/mammalian Microsome Mutagenicity Test, Mut. Res. 31:347-364.
Bradley, M. O., B. Bhuyan, M. C. Francis, R. Langenbach, A. Peterson, and E. Huberman, 1981, Mutagenesis by Chemical Agents in V79 Chinese Hamster Cells: A Review and Analysis of the Literature, Mut. Res. 87:81-142.
Chu, E. H. Y., and H. V. Malling, 1968, Mammalian Cell Genetics. II. Chemical Induction of Specific Locus Mutations in Chinese Hamster Cells in vitro, Proc. Natl. Acad. Sci. USA 61:1306-1312.
Clive, D., and J. F. S. Spector, 1975, Laboratory Procedures for Assessing Specific Locus Mutations at the TK Locus in Cultured L5178Y Mouse Lymphoma Cells, Mut. Res. 31:17-29.
Clive, D., K. O. Johnson, J. F. S. Spector, A. G. Batson and M. M. Brown, 1979, Validation and Characterization of the L5178Y/TK/- Mouse Lymphoma Mutagen Assay System, Mut. Res. 59:61-108.
DeSerres, F. S. and M.D. Shelby, 1979, Recommendations on Data Production and Analysis Using the Salmonella/Microsome Mutagenic Assay, Mut. Res. 64:159-165.
Hsie, A. W., D. A. Casciano, D. B. Couch, D. F. Krahn, J.P. O'Neill, and B. L. Whitfield, 1981, The Use of Chinese Hamster Ovary Cells to Quantify Specific Locus Mutation and to Determine Mutagenicity of Chemicals, Mut. Res. 86:193-214.
Huberman, E., and L. Sachs, 1976, Mutability of Different Genetic Loci and Mammalian Cells by Metabolically Activated Carcinogenic Polycyclic Hydrocarbons, Proc. Nat. Acad. Sci. USA 73:188-192.
Irr, J. D. and R. D. Schnee, 1982, A Statistical Method for Analysis of Mouse Lymphoma L5178Y Cell TK Locus Forward Mutation Assay, Mut. Res. 97:371-392.
McCann, J., E. Choi, E. Yamasaki, and B. N. Ames, 1975, Detection of Carcinogens as Mutagens in the Salmonella/Microsome Test: Assay of 300 Chemicals, Proc. Nat. Acad. Sci. USA 72:5135-5139.
McCann, J. and B. N. Ames, 1976, Detection of Carcinogens as Mutagens in the Salmonella/Microsome Test: Assay of 300 Chemicals: Discussion, Proc. Nat. Acad. Sci. USA 73:950-963.
O'Neill, J.P., et al., 1977, A Quantitative Assay of Mutation Induction at the Hypoxanthine-Guanine Phosphoribosyl Transferase Locus in Chinese Hamster Ovary Cells (CHO/HGPRT System): Development and Definition of the System, Mut. Res. 45:91-101.
Painter, R. B. and J. E. Cleaver, 1969, Repair Replication, Unscheduled DNA Synthesis and the Repair of Mammalian DNA, Radiat. Res. 37:4151-4166.
Prival, M. et al., 1979, Modifications of the Ames' Test to Detect Nitrosamines, Env. Mutagenesis Vol. 1:2, 1-15.
Probst, G. S., R. E. McMahon, L. E. Hill, C. Z. Thompson, J. K. Epp and S. B. Neal, 1981, Chemically-Induced Unscheduled DNA Synthesis in Primary Rat Hepatocyte Cultures: A Comparison with Bacterial Mutagenicity Using 218 Compounds, Environ. Mutagen. 3:11-32.
Sobels, F. R., 1974, The Advantage of Drosophila for Mutation Studies, Mut. Res. 26:277-284.
Stich, H. F. and R. H. C. San, 1970, DNA Repair and Chromosome Anomalies in Mammalian Cells Exposed to 4-Nitroquinoline 1-Oxide, Mut. Res. 10:389-404.
Sugimura, T., S. Sato, M. Nagao, T. Yahagi, T. Matsushima, Y. Seingo, M. Takeuchi, and T. Kawachi, 1976, Overlapping of Carcinogens and Mutagens, in Fundamentals in Cancer Prevention, ed. P.N. Magee et al., University Park Press, Baltimore, pp. 191-215.
Vogel, E., 1975, Some Aspects of the Detection of Potential Mutagenic Agents in Drosophila, Mut. Res. 29:241-250.
Vogel, E., 1977, In The Origins of Human Cancer, Cold Spring Harbor Laboratory, pp. 1483-1497.
Williams, G. M., 1976, Carcinogen-Induced DNA Repair in Primary Rat Liver Cell Cultures: A Possible Screen for Chemical Carcinogens, Cancer Lett. 1: 231-236.
Williams, G. M., 1977, The Detection of Chemical Carcinogens by Unscheduled DNA Synthesis in Rat Liver Primary Cell Cultures, Cancer Res. 37:1845-1851.
Williams, G. M., 1980, The Predictive Value of DNA Damage and Repair Assays for Carcinogenicity, In Applied Methods in Oncology Vol.3, Eds. G. M. Williams et al., pp. 213-230, Elsevier North-Holland Biomedical Press, Amsterdam.
Wurgler, F. E., Sobels, F. H., and Vogel, E., Drosophila as an Assay System for Detecting Genetic Changes, Handbook of Mutagenicity Test Procedures, Ed. B. Kilbey, 1977.
Yahagi, T., M. Degawa, Y. Seino, T. Matsushima, M. Nagao, T. Sugimura and Hasimoto, 1975, Mutagenicity of Carcinogenic Azo Dyes and Their Derivatives, Cancer Letters 1:91-96
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