UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C.  20460

TXR No. 0054204		OFFICE OF
		PREVENTION, PESTICIDES AND
		TOXIC SUBSTANCES

April 4, 2006

Memorandum

Subject:	Toxicology Disciplinary Chapter for the Re-Registration Eligibility Decision (RED) Risk Assessment  

		Active Ingredient: 	2-(thiocyanomethylthio)benzothiazole (TCMTB) 
		PC Code		035603	

DP Barcode:	D328184

From:		Deborah Smegal, Toxicologist/Risk Assessor
		Re-Registration Branch 1 (RRB1)
		Health Effects Division (7509C)

Through:	Whang Phang ,  Branch Senior Scientist 
		Re-Registration Branch 1 (RRB1)
		Health Effects Division (7509C)

To:		Kathryn Jakob, Chemical Review Manager
		Regulatory Management Branch II
		Antimicrobials Division (7510C)

1.0	HAZARD CHARACTERIZATION
2.0	REQUIREMENTS
3.0	DATA GAP(S)
4.0	HAZARD ASSESSMENT
4.1	Acute Toxicity
4.2	Subchronic Toxicity
4.3	Prenatal Developmental Toxicity
4.4	Reproductive Toxicity
4.5	Chronic Toxicity
4.6	Carcinogenicity
4.7	Mutagenicity
4.8	Neurotoxicity
4.9	Metabolism
5.0	HAZARD CHARACTERIZATION FOR 2-MERCAPTOBENZOTHIAZOLE  (2-MBT
6.0	TOXICITY ENDPOINT SELECTION
6.1	See Section 9.2 for Endpoint Selection Table.
6.2	Dermal Absorption
6.3	Classification of Carcinogenic Potential
7.0	FQPA CONSIDERATIONS
7.1	Special Sensitivity to Infants and Children
7.2	Recommendation for a Developmental Neurotoxicity Study
8.0	REFERENCES
9.0 APPENDICES
9.1	Toxicity Profile Summary Tables
9.2	Summary of Toxicological Dose and Endpoints  for 2-(Thiocyanomethylthio)benzothiazole (TCMTB) for Use in Human Risk Assessment1

1.0	HAZARD CHARACTERIZATION

The toxicology data base for TCMTB is considered complete for a food use pesticide.  No additional studies are required at this time.  TCMTB exhibits low acute oral and dermal toxicity (toxicity category III).  However, it is highly irritating to the eyes and skin (toxicity category I and II, respectively) and is also considered to be highly toxic via the inhalation route of exposure (toxicity category I).  TCMTB is a dermal sensitizer.  

Following subchronic oral exposures to rodents, TCMTB caused increased incidence of mild to severe stomach lesions characterized by inflammation, hyperplasia, necrosis, and ulceration.  Inflammatory cells infiltration and edema were also noted at a low incidence.  TCMTB resulted in decreased body weight gains (78 to 84% of control), food consumption (87-95% of control) and food efficiency.  In a 21-day rat dermal toxicity study, TCMTB produced dose-dependant dermal irritation in all dose groups beginning on treatment days 3-4, which progressed to eschar formation.  Rats in the mid and high dose group had ulcers, hemorrhages and chronic dermatitis.  Decreased body weight gain, food consumption, and hematological (changes in hemoglobin, hematocrit, and segmented neutrophils) and clinical chemistry changes (blood urea nitrogen, glucose, globulins, and elevated serum aspartate aminotransferase (AST) were also noted.  However, the clinical chemistry changes did not correspond to any treatment-related findings in the liver or kidney. 

Developmental toxicity studies were available in both the rat and rabbit for TCMTB.  Rabbits appear to be more sensitive to the toxicity of TCMTB than rats.  In rabbits, the maternal NOAEL and LOAEL were 16  and 32 mg ai/kg/day, respectively.   In rats, the maternal NOAEL and LOAEL were 21 and 64 mg ai/kg/day.  No developmental effects were noted in the rabbit at the highest dose tested (32 mg ai/kg/day).  However, in the rat adverse developmental effects including increased fetal and litter incidence of fused/wavy ribs, rudimentary cervical, thoracic and lumbar ribs and increased incidence of sternebrae and pelvic girdle anomalies were noted at doses of 105 mg ai/kg/day, which was maternally toxic.  

In a two-generation rat reproduction study, there were no treatment related effects noted at the highest dose tested for parental toxicity or on reproductive parameters examined in this study.  Slight, statistically significant effects were noted in mean body weight in the high dose offspring in the second mating (F2B) around lactation day 21.  This must be considered as systemic toxicity as the litters began with relatively similar mean body weights, and around lactation day 14 the pups began to consume diet while continuing to nurse.   Mean pup weight was not significantly reduced at days 7 or 14 in the F2B pups, and there were no consistent effects on pup weight in the F0 and F1a generation pups.  Thus, the Agency believes this finding in the F2B pups on day 21 is of questionable toxicological significance.     

Dogs appear to be the most sensitive species to TCMTB toxicity following chronic exposure. In the chronic dog toxicity study, adverse effects were noted at the lowest dose tested of 3.8 mg/kg/day.  Adverse effects included decreased body weight gain, hematological effects (changes in white blood cells, monocytes), alterations in clinical chemistry parameters (plasma ALT) and decreased uterine weight in females.     
 
In the chronic rat toxicity/carcinogenicity study, no adverse systemic toxicity effects were noted at the highest dose tested of 20 mg/kg/day.  However, there was a statistically significant increase in the incidence of testicular interstitial cell adenomas in males of mid (P=0.0155) and high-dose (P=0.0087) levels, that had a highly significant positive dose-related trend (P=0.0018).

Treatment was also associated with a possible increased incidence of thyroid C-cell adenomas in females of the mid- and high-dose levels, which had a highly significant (P=0.0067) positive dose-related trend, but did not attain a statistically significant level in the pairwise comparison with concurrent controls.  No historical data were available for review.  

In the chronic mouse toxicity/carcinogenicity study, decreased body weight gain was noted in both sexes, while there was a statistically increased incidence of focal and diffuse hyperplasia of duodenal mucosa in males (10/42 vs 1/41 in controls) at 150 mg TCMTB/kg/day (122 mg ai/kg/day).  There was no evidence of carcinogenicity.  

The available toxicity data do no indicate neurotoxicity in the experimental animals exposed to TCMTB by either oral or derma routes.   

TCMTB was negative for mutagenicity in the gene mutation assay with bacteria, and did not cause an increase in unscheduled DNA synthesis (UDS) in rat primary hepatocytes assay.  It was also negative for chromosomal aberrations in the in vivo micronucleus assay in mice. 

TCMTB has been classified  as Group C - possible human carcinogen - and the Agency recommended that for the purpose of risk characterization, the Reference Dose (RfD) approach be used for quantitation of cancer risk.  This was based on statistically significant increases in tumors in both sexes of the Sprague-Dawley rat: testicular interstitial cell adenomas in males and thyroid C-cell adenomas in females.  

2.0	REQUIREMENTS 

	The Toxicology Data Requirements (40CFR 158.340) for food uses of TCMTB are in Table 1. Use of the new guideline numbers does not imply that the new (1998) guideline protocols were used.

Test 
	
Technical	
	
Required	
Satisfied	

870.1100	Acute Oral Toxicity	
870.1200	Acute Dermal Toxicity	
870.1300	Acute Inhalation Toxicity	
870.2400	Primary Eye Irritation	
870.2500	Primary Dermal Irritation	
870.2600	Dermal Sensitization	
870.6100	Acute Delayed Neurotox. (Hen)	
870.6200a	Acute Neurotox. Screening Battery (Rat)		
Y 
Y 
Y 
Y 
Y 
Y 
N
N	
Y
Y
Y
Y
Y
Y

870.3100	Oral Subchronic (Rodent)	
870.3150	Oral Subchronic (Non-Rodent)	
870.3200	21-Day Dermal	
870.3250	90-Day Dermal	
870.3465	90-Day Inhalation		
Y
Y
N
N
Held in reserve	
Y (by chronic)
Y (by chronic)
Y

870.3700a	Developmental Toxicity (rodent)	
870.3700b	Developmental Toxicity( non-rodent)	
870.3800	Reproduction		
Y 
Y 
Y	
Y
Y
Y	

870.4100a	Chronic Toxicity (Rodent)	
870.4100b	Chronic Toxicity (Non-rodent)	
870.4200a	Oncogenicity (Rat)	
870.4200b	Oncogenicity (Mouse)	
870.4300	Chronic/Oncogenicity		
Y
Y
Y 
Y 
Y	
Y
Y
Y
Y
Y	

870.5100	MutagenicityGene Mutation - bacterial	
870.5300	MutagenicityGene Mutation - mammalian	
870.5395	MutagenicityMammalian erythrocyte micronucleus	
870.5xxx	MutagenicityOther Genotoxic Effects		
Y
Y
Y
N	
Y

Y
N	

870.6100	90-Day Neurotoxicity (hen)	
870.6200b	90 Day Neuro. Screening Battery (Rat)	
870.6300	Develop. Neuro		
N
N
N	
--
--
--	

870.7485	General Metabolism	
870.7600	Dermal Penetration		
Y
N	

--	

870.7200	Companion Animal Safety		
N	
--	

Special Studies for Ocular Effects	
Acute Oral (Rat)	
Subchronic Oral (Rat)	
Six-month Oral (Dog)		
N	
--	
Y - Yes;   N – no; -- not necessary

3.0	DATA GAP(S)

There are no toxicology data gaps for TCMBT at this time.   The Antimicrobials Division Toxicology Endpoint Selection Committee (ADTC) recommended that a repeated dose inhalation toxicity study for TCMTB be held in reserve, pending the outcome of inhalation risk assessments using the oral toxicity data. 

4.0	HAZARD ASSESSMENT

4.1	Acute Toxicity 

	Adequacy of data base for acute toxicity:  The data base for acute toxicity is considered complete.  No additional studies are required at this time.  TCMTB exhibits low acute oral and dermal toxicity (toxicity category III).  However, it is highly irritating to the eyes and skin (toxicity category I and II, respectively)) and is also considered to be highly toxic via the inhalation route of exposure (toxicity category I).  TCMTB is a dermal sensitizer.  The acute toxicity data on the TCMTB Technical (80% ai), or Busan 72 (containing 60% ai) is summarized below in Table 2.

Table 2. Acute Toxicity Data on  TCMTB Technical (80% ai)	
Guideline No./ Study Type	MRID No.	Results	Toxicity Category	
870.1100 Acute oral toxicity	41583801 	LD50 = 750 mg/kg (M+F); 80% ai	III	
870.1200 Acute dermal toxicity	41515401	LD50 > 2000 mg/kg (M+F); 80% ai	III	
870.1300 Acute inhalation toxicity	41640601	LC50=0.07 mg/L; 80% ai	I	
870.2400 Acute eye irritation	Acc No. 111991	Diluted Busan 72 (60 % ai):  primary irritation score (PIS)=2/110 (slight conjunctival redness, no corneal opacity); undiluted Busan 72 (60% ai) PIS=34/110 (blanched conjunctivae, chemosis, corneal opacity not reversible by day 7)	I	
870.2500 Acute dermal irritation	41583701	primary irritation index=7.42 with severe erythema and edema observed at 72 hours; 80% ai	II	
870.2600 Skin sensitization	MRID 42349201
Acc No. 259676	Busan 74 (80% ai) caused delayed contact hypersensitivity in guinea pigs when induced and challenged by a 40% w/v aqueous concentration of active ingredient.  Sensitizer.  	--	

4.2	Subchronic Toxicity 

	Adequacy of data base for subchronic toxicity:  The data base for subchronic toxicity is considered adequate for risk assessment purposes.  Although the subchronic rodent studies are unacceptable, there are chronic studies available to fulfill the guideline requirements

Following repeat oral exposures to rodents, TCMTB caused increased incidence of mild to severe stomach lesions characterized by inflammation, hyperplasia, necrosis, and ulceration.  Inflammatory cells infiltration and edema were also noted at a low incidence.  TCMTB resulted in decreased body weight gains (78 to 84% of control), food consumption (87-95% of control) and food efficiency.  In a 21-day rat dermal toxicity study, TCMTB produced dose-dependant dermal irritation in all dose groups beginning on treatment days 3-4, which progressed to eschar formation.  Rats in the mid and high dose group had ulcers, hemorrhages and chronic dermatitis.  Decreased body weight gain, food consumption, and hematological (changes in hemoglobin, hematocrit, and segmented neutrophils) and clinical chemistry changes (blood urea nitrogen, glucose, globulins, and elevated serum aspartate aminotransferase (AST) were also noted.  However, the clinical chemistry changes did not correspond to any treatment-related findings in the liver or kidney. 

870.3100	90-Day Oral Toxicity - Rat

In a 13 week range finding study (MRID 43112801), TCMTB (81.56% ai, Batch 1517 MRID 46698604, 92179001) was administered to Sprague-Dawley rats (n=10/sex/dose) in the diet at dose levels of 0, 10, 30, 70 or 100 mg/kg/day (actual doses: 10.2, 31, 70 and 100 mg TCMTB/kg/day for males and 10.3, 31, 72 and 99 mgTCMTB/kg/day for females using food consumption data).  These doses are equivalent to 8.3/8.4 (M/F), 24.5, 57/59 (M/F), 80.8/81.6 (M/F) mg ai/kg/day, respectively when considering the purity of the test substance as 81.56% ai.  This study examined limited parameters.

Systemic toxicity was noted at 30 mg/kg/day (24.5 mg ai/kg/day) and above in the form of increased incidence of squamous epithelial hyperplasia of the stomach.  There were also treatment related decreases in body weight gains (78 to 84% of control), food consumption (87-95% of control) and food efficiency (89-94% of control) in both sexes of to 70 and 100 mgTCMTB/kg/day (57/59 (M/F) and 80.8/82.6 (M/F) mg ai/kg/day).  The LOAEL for systemic toxicity is 30 mg TCMTB/kg/day (24.5 mg ai/kg/day) and the NOAEL for systemic toxicity is 10 mg TCMTB/kg/day (8.3.8.4 mg ai/kg/day) based on increased incidences of stomach lesions.

This 90-day oral toxicity study in the rat is UNACCEPTABLE-GUIDELINE and does not satisfy the guideline requirement for a subchronic (90-day) oral toxicity study OPPTS 870.3100 in the rat because is was only a range finding study, and based on major deficiencies (stability, diet concentrations and homogeneity were not measured, and hematology and clinical chemistry parameters were not measured).  This study can not be upgraded.  

 870.3100	90-Day Oral Toxicity - Rat

In a 90-day study (MRID 92179026), TCMTB (Lot 9-3573; approximately 80% as supplied by the registrant via email correspondence) was administered to Sprague-Dawley rats (n=30/sex/dose) in the diet at dose levels of 0, 333, 500 and 750 ppm for 90 days.  Dietary doses were approximately  0, 16.7, 25, 37.5 mgTCMTB/kg/day,  which is equivalent to 0, 13.4, 20 and 30 mg ai/kg/day, respectively based on a purity of approximately 80% ai. (as supplied by the registrant).  

There were no treatment-related effects on any of the parameters measured except histopathology.  Mild inflammatory changes were found in the gastric mucosa of males in the high dose group.  Mild to severe changes in the stomach, including inflammation, necrosis and ulceration, were found in the females in this group.  Females in the mid-dose group had a low incidence of inflammatory cells infiltration (5% vs. 0% in the control) and edema (10% vs. 0% in the control).  

The NOAEL is 500 ppm (25 mg TCMTB/kg/day or 20 mg ai/kg/day).  The LOAEL is 750 ppm (37.5 mg TCMTB/kg/day or 30 mg ai/kg/day).  The Maximum Tolerated Dose (MTD) in males is slightly higher than 750 ppm (based on histopathology findings); females is slightly lower than 750  ppm (based on incidence and severity of histopathology findings in the stomach).  

This 90-day oral toxicity study in the rat is UNACCEPTABLE-GUIDELINE and does not satisfy the guideline requirement for a subchronic (90-day) oral toxicity study OPPTS 870.3100 in the rat because the percent recovery of TCMTB in the diets was low (81, 82 and 84% for the low, mid- and high dose groups, respectively), and ophthalmoscopic examinations were not done.  The approximate purity of 80% ai was submitted in late 2005 by the registrant.  

870.3200	21-Day Dermal Toxicity - Rat

Executive Summary:  In a dermal toxicity study (MRID 41655801), TCMTB (82.33% a.i. Lot OB-8483 via fax submission dated December 8, 2005) was applied to the skin of Sprague-Dawley rats (5/sex/group) at dose levels of 0, 25, 100, or 250 mg/kg/day for 21 days (6 hours/day).  These doses are equivalent to 0, 20.6, 82.3 and 206 mg ai/kg/day.  TCMTB produced slight to moderate dermal irritation in all dose groups beginning on treatment days 3-4. Dermal irritation was dose-dependent and progressed to eschar formation in 2 rats (1 male and 1 female) in the low dose group in all rats of the mid and high dose groups.  Blanching occurred in all rats of the high dose group and in all females and 3/5 males in the mid dose group. Decreased body weight gain was noted at the mid and high dose groups of male rats.  Statistically significant changes in hemoglobin, hematocrit, and segmented neutrophils were observed in male and female rats of the high dose level. Several clinical chemistry parameters were significantly increased in high dose males and females (blood urea nitrogen, glucose and globulins); while high dose females also had elevated serum aspartate aminotransferase (AST).  However, the clinical chemistry changes did not correspond to any treatment-related findings in the liver or kidney. 

The Systemic NOAEL = 20.6 mg ai/kg/day, and the Systemic LOAEL = 82.3 mg ai/kg/day, based on decreased body weight gain, food consumption, and hematological and clinical chemistry changes.  A Dermal  NOAEL was not identified.  The Dermal LOAEL is 20.6 mg ai/kg/day based on dose-dependent irritation which progressed to eschar formation in 2 rats of the low dose group and in all rats of the mid- and high-dose groups.  Mid- and high-dose groups exhibited ulcers, hemorrhages and chronic dermatitis.  

Based on the recent submission of purity information in 2005, this 21-day dermal toxicity study in the rat can be upgraded to ACCEPTABLE-GUIDELINE, and satisfies the guideline requirement for a subchronic dermal toxicity study OPPTS 870.3200 in the rat.  

4.3	Prenatal Developmental Toxicity 

Adequacy of data base for Prenatal Developmental Toxicity:  The data base for prenatal developmental toxicity is considered complete.  Developmental toxicity studies were available in both the rat and rabbit for TCMTB.  No additional studies are required at this time.   Rabbits appear to be more sensitive to the toxicity of TCMTB than rats.  In rabbits, the maternal NOAEL and LOAEL were 16  and 32 mg ai/kg/day, respectively.   In rats, the maternal NOAEL and LOAEL were 21 and 64 mg ai/kg/day.  No developmental effects were noted in the rabbit at the highest dose tested (32 mg ai/kg/day).  However, in the rat adverse developmental effects including increased fetal and litter incidence of fused/wavy ribs, rudimentary cervical, thoracic and lumbar ribs and increased incidence of sternebrae and pelvic girdle anomalies were noted at doses of 105 mg ai/kg/day, which was maternally toxic.  Thus, the Agency concludes that these studies show no evidence of primary developmental toxicity of TCMTB, which is consistent with the previous conclusion of the Health Effects Division’s RfD Peer Review committee (September 29, 1994; document # 011302).    

870.3700a Prenatal Developmental Toxicity Study - Rat

 In a prenatal developmental toxicity study (MRID 00154295, accession no. 260491); TCMTB (83.6% a.i. Lot 511230, MRID 46698601), was administered to 5 groups of  29 mated female Sprague-Dawley rats per group by oral gavage using technical TCMTB dissolved in corn oil/ 1% Tween 80 at doses of 0, 25.1, 76.5, and 125.5 mg/kg/day from gestation days 6 through 15.  These doses are equivalent to 0, 21, 64 and 105 mg ai/kg/day, respectively.  Maternal toxicity was evident at the 64 mg ai/kg/day (76.5 mg TCMTB/kg/day) dose level as ventral alopecia, rough coat, dyspnea/wheezing, oral discharge, nasal discharge, diarrhea/loose stool, urine staining, piloerection, and hunched gait.  Developmental toxicity was evident at the 105 mg ai/kg/day (125.5 mg TCMTB/kg/day) dose level in the form of increased fetal and litter incidence of fused/wavy ribs, rudimentary cervical, thoracic, and lumbar ribs, and increased number of sternebrae and pelvic girdle anomalies. 

The Maternal NOAEL = 21 mg ai/kg/day (25.1 mg TCMTB/kg/day), based on observations of clinical toxicity at 64 mg ai/kg/day (76.5 mgTCMTB/kg/day) (LOAEL). The Developmental NOAEL = 64 mg ai/kg/day (76.5 mg TCMTB/kg/day), based on increased fetal and litter incidence of skeletal anomalies. 

This developmental toxicity study in the rat is classified as acceptable/guideline and satisfies the requirement (OPPTS 870.3700) for a developmental toxicity study in the rodent. 

870.3700b Prenatal Developmental Toxicity Study - Rabbit

In a prenatal developmental toxicity (teratology) study (MRID 40075102), TCMTB (81.0 % a.i., Lot 5-13002, MRID 46698602) was orally dosed to presumed pregnant rabbits at dose levels of 0, 10, 20 and 40 mg/kg/day (20 rabbits/group) during gestation days 6-19 inclusive.    These doses are equivalent to 0, 8, 16, and 32 mg ai/kg/day, which were verified by the registrant. 

Maternal toxicity was evidenced by decreased body weight gain and food consumption in the 32 mg ai/kg/day dose group.  Statistically significant (p>0.01) body weight loss was noted in the high dose group on gestation days 6-12, 6-19 and 19-29.  Mean food consumption was significantly reduced for the high dose group during gestation days 6-12 and 6-19.  One high dose and two mid-dose group rabbits died during the study.  However, only the high dose death, which occurred on gestation day 18, was considered treatment related based on corroded gastric mucosa and reddened duodenal mucosa.  There were no clinical signs of toxicity in any dose level during the study.  Five dams aborted their litters prior to scheduled sacrifice (1 control, 2 in the low dose group and 2 in the mid-dose group).  No animals in the high dose group aborted, therefore, the abortions occurring in the lower dose groups were considered to be spontaneous, which is not uncommon in rabbits.  The Maternal Toxicity NOAEL is 16 mg ai/kg/day and the Maternal Toxicity LOAEL is 32 mg ai /kg/day based on decreased body weight gain and food consumption.

No statistically significant or treatment-related changes were noted in any treatment group for deaths/resorptions, altered growth, developmental anomalies or malformations.  The Developmental Toxicity LOAEL is greater than 32 mg ai/kg/day and the Developmental Toxicity NOAEL is greater than or equal to 32 mg ai/kg/day.

This study is classified as acceptable-guideline and satisfies the guideline requirement (§870.3700) for a developmental toxicity (teratology) study in rabbits.

4.4	Reproductive Toxicity 

	Adequacy of data base for Reproductive Toxicity:  The data base for reproductive toxicity is considered complete.  No additional studies are required at this time.  The available data showed that TCMTB did not cause reproductive effects in rats.

In a 2-generation reproduction toxicity study (MRID  41471401), TCMTB (81.6% ai, Batch 1517; MRID 92179011, 46698604) was administered to Sprague Dawley CD outbred albino rats from Charles River UK Ltd. in the diet at dose levels of 0, 25, 100, or 400 ppm (2.4, 9.6, and 38.4 mg/kg/day for males and 3.0, 11.7, and 45.5 mg/kg/day for females for the 25, 100, and 400 ppm groups, respectively) for 2 generations with 1 litter in the first generation and 2 litters in the second generation.  No treatment related effects were noted on reproductive parameters examined in this study.  Slight, statistically significant effects were noted in mean body weight in the high dose offspring in the second mating (F2B) around lactation day 21 (approximately 88% of control pups).  This must be considered as systemic toxicity as the litters began with relatively equal mean body weights and around lactation day 14, the pups began to consume diet while continuing to nurse, and thus mostly likely received more than 400 ppm TCMTB.   Mean pup weight was not significantly reduced at days 7 or 14 in the F2B pups, and there were no consistent effects on pup weight in the F0 and F1a generation pups.  Thus, the Agency believes this finding in the F2B pups  on day 21 is of questionable toxicological significance.    

The parental/systemic NOAEL = 400 ppm  (38.4/45.5 mg /kg/day  [males/females]) and the parental/systemic LOAEL > 400 ppm (> 38.4/45.5 mg /kg/day [males/females]).  The offspring NOAEL is 400 ppm (HDT) and the offspring LOAEL > 400 ppm. The Reproductive toxicity NOAEL > 400ppm and the Reproductive toxicity LOAEL > 400 ppm.  

This study is classified as acceptable-guideline and satisfies the guideline requirement for a 2-generation reproductive study (§870.3800; OECD 416) in rats

Consistent with both the RfD Peer Review’s conclusion and that of the California Environmental Protection Agency’s summary of TCMTB (February 22, 2001), there was no evidence of reproductive toxicity of TCMTB. 

4.5	Chronic Toxicity 

	Adequacy of data base for chronic toxicity:  The data base for chronic toxicity is considered complete, and no additional studies are required at this time.  

In a chronic toxicity study (MRID 41342201), technical TCMTB (81.6% ai, Batch 1517; MRID 92179008, 46698604) was administered to male and female beagle dogs at dose levels of 0, 100ppm (3.8 mg/kg/day in males; 4.0 mg/kg/day in females), 300ppm (11.7 mg/kg/day in males; 11.2 mg/kg/day in females), and 1000ppm (38.8 mg/kg/day in males; 43.2 mg/kg/day in females). Decreased body weight and body weight gain were observed in male dogs at the 100 ppm dose level for weeks 0-13 of the study, and overall body weight and weight gain were decreased greater than 10% in both male and female dogs at the 1000 ppm dose level.  Hematological effects include significant decreases in circulating white blood cells and monocytes  in male dogs at all dose levels tested, while effects on hematology in female dogs were limited.  Plasma ALT was significantly decreased in both sexes in a dose-related manner at all dose levels in both male and female dogs for the duration of the study. Decreased weight of the lung, thymus, and spleen were observed in male dogs at the 1000 ppm dose level, while decreased spleen, thymus, and uterine weight were observed in female dogs.  Involution of the thymus was the main histologic finding, and the severity of this lesion appeared to increase with increasing dose in both sexes.  The Systemic NOAEL < 100 ppm (males and females).  The Systemic LOAEL =  100 ppm [3.8 mg /kg/day for males; 4.0 mg /kg/day for females], based on  decreased body weight gain, white blood cells, monocytes and plasma ALT in males; decreased plasma ALT and uterine weight in females.     
 
This chronic study in the dog is classified as acceptable-non-guideline and does not satisfy the guideline requirement for a chronic oral study (OPPTS §870.4100, OECD 452) in dogs because a systemic NOAEL was not achieved.  However, this study is scientifically sound and adequate for use in risk assessment

4.6	Carcinogenicity 

	Adequacy of data base for Carcinogenicity: The data base for carcinogenicity is considered complete, and no additional studies are required at this time.  TCMTB produced an increase in testicular interstitial adenomas in male rats and thyroid C-cell adenomas in female rats.

1. Combined Chronic Toxicity/Carcinogenicity Study in Rats   (MRID No.:  42116301; 41529701; 41570301)

Executive Summary:   In a combined chronic/carcinogenicity study (MRID 42116301; 41529701; 41570301), TCMTB (81.6% ai purity, MRID 46698604) was administered to Sprague Dawley rats of both sexes (50/sex/dose, main group; and 20/sex/dose, interim kill group) for a period of up to 104 weeks in the diet at dose levels of 0, 2, 8, or 20 mg/kg/day.  Mortality was variable with survival rates of 80-100% in the interim kill group and 38-82% in the carcinogenicity group. No treatment-related clinical signs of toxicity were noted during the study.  No dose-related or significant effects were noted in the interim kill group.  The only significant reduction in body weight gains occurred in the 20 mg/kg/day males at week 80, and these do not appear to be treatment-related. No dose-related or significant effects were noted in the food consumption of either the interim kill group or the carcinogenicity group.
A dose-related decrease in platelets was noted in all treated females at week 104. All other changes in hematology parameters were not considered to be toxicologically significant. No changes in the clinical chemistry parameters were considered to be biologically significant due to the lack of a dose—response relationship. No treatment—related changes were noted in the organ weights of the interim kill group, but an increasing trend was noted in the female absolute liver weights in all treatment groups.  Since a dose response relationship was not present, it was not considered to be biologically significant.  Terminal organ weights were not reported.
Only one finding displayed a dose-response relationship in the microscopic pathology, interstitial cell tumors in the testes. The tumor incidences were control group 4/50, low dose 5/50, mid dose 13/50 and the high dose with 14/50. The mid and high doses were significantly different (p<0.05) from the controls. Additionally, the incidence of thyroid C-cell adenomas was increased in female rats of the high dose group, as compared to controls. The incidence of this lesion was 6/49, 4/49, 7/48 and 13/50 for the control, low, mid and high dose groups, respectively. Thus, TCMTB appears to be carcinogenic inducing the formation of interstitial cell tumors in the testes and thyroid C-cell adenomas in female rats.

Although the current study did not appear to test to high enough doses to produce systemic toxicity, on the basis of results from other toxicity studies (noted below) as cited in the Cancer Peer Review document, this study is classified as acceptable-guideline and satisfies the OPPTS 870.4300 test guideline.

Discussion of Tumor Data 

Two findings displayed dose-response relationships in the microscopic pathology, interstitial cell tumors in the testes and thyroid C-cell adenomas in female rats.  Male rats had a significant dose-related increasing trend, as well as significant differences in the pair-wise comparisons of the 8 and 20 mg/kg/day dose groups with the controls, for testicular interstitial cell tumors.

Female rats had significant dose-related increasing trends, and significant differences in the pair-wise comparisons of the 20 mg/kg/day dose group with the controls for thyroid C-cell adenomas and adenomas and/or carcinomas combined; however, only adenomas were present in treated groups (the control group had the single carcinoma noted).  Although there were no significant differences in the pair-wise comparisons of the dosed groups with the controls, female rats did have a significant increasing trend in thyroid C-cell hyperplasia at p < 0.01.

Adequacy of the Dose Levels Tested

The statistical evaluation of mortality indicated no significant incremental changes with increasing doses of TCMTB in male rats.  Female rats showed a significant decreasing trend in mortality with increasing doses of TCMTB.  

No treatment-related clinical signs of toxicity were noted during the study.  The only significant reduction in body weight gains occurred in the 20 mg/kg/day males at week 80, and this did not appear to be treatment-related.  No dose-related or significant effects were noted in the food consumption.

A dose-related decrease in platelets was noted in all treated females at week 104.  All other changes in hematology parameters were not considered to be toxicologically significant.  No changes in the clinical chemistry parameters were considered to be biologically significant.  An increasing trend was noted in the female absolute liver weights in all treatment groups; however, since a dose response relationship was not present it was not considered to be biologically significant. 

The systemic toxicity observed in this study in male and female rats was minimal and there were no major differences between the treated and control groups.  However, data obtained from two subchronic feeding studies in rats (the 13 week range finding study, MRID# 43112801 and the 90 day study in rats, MRID# 92179026) indicate that toxic effects (decreased body weight gain and increased incidence of squamous epithelial hyperplasia of the stomach) were observed at the dose level of 30 mg/kg/day (a dose slightly higher than the HDT in this study).  The Systemic LOEL is greater than 20 mg/kg/day and the NOEL is equal to or greater than 20 mg/kg/day.  The study is considered in combination with the 13 week range finding study (MRID# 43112801) and the 90 day study in rats (MRID# 92179026).  Based on this information, the dose levels used in the chronic/carcinogenicity study in rats (MRID#’s 42116301, 41529701, and 41570301) were adequate for testing for the carcinogenic potential of TCMTB.  

2. Carcinogenicity Study in Mice  (MRID No.:  42383001)

Executive Summary:  In a carcinogenicity study (MRID 42383001), TCMTB (81.6% ai, batch 1517/Lot number 6-14285, MRID 46698604) was administered to groups of CD-1 mice (50/sex) in the diet at  concentrations calculated to give dose levels of 0, 15, 50, or 150 mg/kg/day for 2 years. These doses are equivalent to 0, 4, 41 and 122 mg ai/kg/day, respectively.  No carcinogenic potential was seen.  Body weights were significantly reduced in high-dose males and high dose females compared to controls. Weight gains in high dose males and females were approximately 25% lower than controls at 78 weeks. Cumulative weight gain was reduced 36% and 28% for high dose males and females, respectively at week 104. There was no effect on food consumption in this study.  None of the gross findings were remarkable.  An increase in focal and diffuse hyperplasia of the duodenal mucosa was seen microscopically in males receiving 150 mg/kg/day: the incidence was 10/42 compared to 1/41 in controls (p<0.01).  A nonsignificant increase was seen for mucosal hyperplasia of the duodenum in high-dose females (8/45 compared to 4/46 in controls).  No other pathologic findings were considered treatment related. The Systemic NOAEL is determined to be 50 mg/kg/day (41 mg ai/kg/day), and the Systemic LOAEL is determined to be 150 mg/kg/day (122 mg ai/kg/day), based on histologic changes in the duodenum of high dose males and decreased weight gain in both sexes at the high dose.

This carcinogenicity study in the mouse is acceptable guideline and satisfies the guideline requirement for a carcinogenicity study (OPPTS 870.4200; OECD 451).  

Discussion of Tumor Data 

There were no compound-related tumors observed in male or female mice.  All tissues were examined.

Adequacy of the Dose Levels Tested

The statistical evaluation of mortality indicated no significant incremental changes with increasing doses of TCMTB in male mice.  Female mice showed a significant decreasing trend in mortality with increasing doses of TCMTB.  

Body weights were significantly reduced in high-dose males and high-dose females as compared to controls.  Weight gains in high-dose groups were about 88% of control in males and 97% of control in females at 13 weeks; however at 80 weeks the weight gains were 67% of control in the males and 78% of control in the females and cumulative mean gains for the entire treatment period (weeks 0-104) were 64% and 72% of the controls for the high-dose males and females, respectively.  No marked effects on food consumption were observed.  None of the gross findings were remarkable.  Based on the histologic changes in the duodenum in high-dose males and the decreased weight gain in both sexes at the high-dose, the systemic toxicity LOAEL is considered to be 150 mg/kg/day (122 mg ai/kg/day) and the systemic toxicity NOAEL is 50 mg/kg/day (41 mg ai/kg/day).

The high dose tested (150 mg/kg/day) is considered to be an adequate dose for assessing the carcinogenic potential of TCMTB in male and female CD-1 mice.

3. Classification of Carcinogenic Potential

The Health Effects Division Carcinogenicity Peer Review Committee (CPRC) met on June 28, 1995 to discuss and evaluate the weight-of-the-evidence on 2-(Thiocyanomethylthio) benzothiazole (TCMTB) with particular reference to its carcinogenic potential.  Based on statistically significant increases in tumors in both sexes of the Sprague-Dawley rat: testicular interstitial cell adenomas in males and thyroid c-cell adenomas in females, the CPRC concluded that TCMTB should be classified as Group C - possible human carcinogen - and recommended that for the purpose of risk characterization, the Reference Dose (RfD) approach should be used for quantitation of human cancer risk.  

4.7	Mutagenicity 

	Adequacy of data base for Mutagenicity:  The data base for Mutagenicity is considered adequate based on pre 1991 mutagenicity guidelines. TCMTB was negative for mutagenicity in the gene mutation assay with bacteria (MRID# 41386101), and did not cause an increase in unscheduled DNA synthesis (UDS) in rat primary hepatocytes assay (Accession No. 266150; MRID# 00165518).  It was also negative for chromosomal aberrations in the in vivo micronucleus assay in mice (MRID# 00165520). 

Study Type:  Mutagenicity Test on TCMTB in the Salmonella/Mammalian-Microsome Reverse Mutation (Ames Test) with Confirmatory Assay

In an Ames assay (MRID# 41386101) TCMTB was negative for inducing reverse gene mutations in Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98, or TA100 exposed in the presence or absence of S9 activation up to cytotoxic doses (33.3 g/plate -S9; 66.7 g/plate +S9).  The results were confirmed in an independently performed trial.

Study Type:  Evaluation for Structural Chromosomal Aberration of TCMTB (EPA Reg. No.: 1448-29): Clastogenic Evaluation in the In Vivo Mouse Micronucleus Assay

 In a structural chromosomal aberration assay (MRID# 00165520), the high dose of TCMTB (500 mg/kg) exhibited mortality and signs of clinical toxicity.  No evidence of an increase in micronucleated polychromatic erythrocytes was noted at the dose levels tested in this study (50, 167 and 500 mg/kg).  There was evidence of some PCE depression at the high dose in the 48 and 72 hour animals.

Study Type:  Evaluation of Other Genotoxic Effects of TCMTB (EPA Reg. No.: 1448-29).   In the Rat Primary Hepatocyte Unscheduled DNA Synthesis Assay  

In an UDS assay (Accession No. 266150; MRID# 00165518), TCMTB did not cause a significant increase in the unscheduled DNA synthesis (UDS) in rat primary hepatocytes as measured in this study.

4.8	Neurotoxicity 

	Adequacy of data base for Neurotoxicity:  These studies are not required at this time.  The available data do not indicate neurotoxicity in experimental animals exposed by oral or dermal administration.  

4.9	Metabolism 

2-Mercaptobenzothiazole (2-MBT) is the main mammalian urinary metabolite of TCMTB, and has been suggested as a biological marker of exposure to TCMTB (Manninen et al. 1996).  In this study, three doses of TCMTB in olive oil were given to male rats by gavage for 3 weeks.  Urine was collected daily and the metabolites were analyzed as thioethers by derivatization with pentafluorobenzylbromide by gas chromatography-mass spectrometry.  The parent chemical was not detected in urine samples, but two metabolites of TCMTB were identified.  

2-Mercaptobenzothiazole (2-MBT) was the main metabolite, and its excretion varied according to the dose.  The second metabolite was 2-(mercaptomethylthio)benzothiazole.  The amount of 2-MBT excreted in rat urine was 66  12% (SD),  51 20% and 44   9% for TCMTB doses of 15, 75, and 150 mg/kg, respectively.  Two doses, 75 and 150 mg/kg , caused diuresis in rats during the 1 week of dosing.  During the 3-week TCMTB treatment, rat liver microsomal CYP enzyme profile was not significantly changed.  Urine samples for sawmill workers exposed to TCMTB were collected after their work shifts for exposure assessment.  TCMTB could not be detected in the urine samples of exposed workers.  Most concentrations of 2-MBT were below the limit of detection, 0.12 mol/l, the concentrations were 0.12-0.15 mol/l only in few cases.  The authors conclude that the determination of 2-MBT in urine, when a sample is taken immediately after a work shift, is a suitable indicator of workers’ exposure to TCMTB.  

In a rat metabolism study (MRID 40884801) 14C-TCMTB as administered to Sprague Dawley rats (5/sex/dose) by oral gavage at single doses of 3 and 30 mg/kg, and at a repeated oral dose of 3 mg/kg.  Excretion of TCMTB derived radioactivity was monitored, as well as measurement of plasma levels and excretion of TCMTB derived radioactivity in bile.  Metabolites of TCMTB in urine, plasma, and bile were characterized using thin layer chromatography.  Absorption of TCMTB was rapid, with 80% excreted in urine by 24 hours post-dose at both the 3 and 30 mg/kg dose levels.  In bile, cannulated rats, there was a marked reduction in 0-24 hour urinary excretion (25-35%).

Distribution data indicated that red blood cells and kidney were the only sites of significant retention of residual radioactivity.  However, these levels were low (0.02-2.41 g equiv./g tissue).  Measurement of elimination of radioactivity from blood showed that the half-life in red cells might be in excess of 7 days.  

Excretion of TCMTB derived radioactivity was mainly in urine, with between 80-85% of the total dose excreted in non-cannulated rats.  Fecal excretion comprised about 5% of an administered dose, and was not altered in cannulated rats, suggesting reabsorption of radioactivity from the gastrointestinal tract and excretion in urine.  Pharmacokinetic data indicated that clearance was approximately equal at both the low and high dose, and the AUC was proportional to the dose, suggesting first order elimination at both the low and high dose. 

Metabolite characterization using radio-TLC showed the presence of one and possibly 2 metabolites of TCMTB in urine and bile.  However, definitive identification was not made, other than to suggest that at least one metabolite could be a glucuronide conjugate.  

This metabolism study in the rat is classified as unacceptable guideline and does not satisfy the guideline requirement for a metabolism study [OPTTS 870.7485, OECD 417] in rats because there are incomplete data for the repeated low oral dose group, incomplete identification of metabolites and the purity of  TCMTB was not reported.  

5.0	HAZARD CHARACTERIZATION FOR 2-MERCAPTOBENZOTHIAZOLE 	(2-MBT)

2-Mercaptobenzothiazole (2-MBT) is the main mammalian metabolite of TCMTB (Manninen et al., Arch. Toxicol. 70: 579-584, 1996).  Although the current uses of 2-MBT have been cancelled, it is instructive to summarize the hazard of 2-MBT relative to that of TCMTB, as this will be the main metabolite that mammalian organisms are exposed to when exposed to TCMTB.

2-MBT shows low acute toxicity by the oral route (Toxicity Category IV, MRID 41571901) and dermal route (LC50 > 2000 mg/kg, Toxicity Category III, MRID 41571902). 2-MBT was minimally irritating to the eyes (Toxicity Category III, MRID 41571903) and slightly irritating to the skin (Toxicity Category IV, MRID 41571904).  A dermal sensitization study submitted for the zinc salt of 2-MBT showed no evidence of hypersensitivity when tested using the modified Buehler test method (MRID 41571905).

A 90-day oral toxicity study conduced with 2-MBT (96.3% a.i.)  by the National Toxicology Program at doses of 187.5, 375, 750, and 1500 mg/kg/day (technical report no. 332) in rats showed  decreased body weight gain at 1500 mg/kg/day in both sexes compared to control, and increased liver weight at 1500 mg/kg/day.  A 90-day toxicity study by the NTP with 2-MBT in mice at doses of 0, 94, 188, 375, 750, and 1500 mg/kg/day showed mortality at the high dose (5/10 males and 7/10 females), clonic seizures, lacrimation, and salivation at 750 and 1500 mg/kg/day, and lethargy and rough coat at 375 and 750 mg/kg/day. There were no gross or microscopic pathological effects noted in mice.  

In a 13-week dermal toxicity study with 2-MBT (98.22%), administration of 2-MBT at doses of  0, 200, 1000, and 2000 mg/kg/day resulted in increased relative liver weight at 1000 and 2000 mg/kg/day, the only effect noted in this study (MRID 42146301). 

In a developmental toxicity study in rats (MRID 41422202), 2-MBT (98.5% a.i.) was administered to mated female SD Crl:COBS CD BR VAF rats (26/dose) at doses of 0, 300, 1200, and 1800 mg/kg/day on gestation days 6 through 15 inclusive.  At 1200 mg/kg/day, maternal toxicity was observed in the form of salivation and urine staining.  There was no evidence of developmental toxicity related to administration of 2-MBT in this study.  The Maternal NOAEL = 300 mg/kg/day and the Maternal LOAEL = 1200 mg/kg/day, based on increased salivation and urine staining.  The Developmental NOAEL > 1800 mg/kg/day and the Developmental LOAEL > 1800 mg/kg/day.  

In a 2-generation reproduction toxicity study (MRID 41912501), 2-MBT(98.0-98.5% a.i.) was administered in the diet to groups of COBS BR rats at doses of 0, 194, 695, and 1195 mg/kg/day [males] and 0, 218, 783, and 1327 mg/kg/day [females]).  At the high dose, decreased body weight and weight gain were observed as well as increased incidence of microscopic changes in the kidney. Fertility, length of gestation, and pup viability were unaffected by 2-MBT treatment. Pup growth (in the form of decreased body weight) was decreased at the 695/783 and 1195/1327 mg/kg/day dose levels by 9% and 15% respectively on day 14 and by 13% and  21% on day 21 in the first generation, and was decreased significantly (9-13%) at all dose levels in the second generation on days 14 and 21.  The systemic/reproductive toxicity NOAEL = 194 mg/kg/day and the systemic/reproductive toxicity LOAEL = 695 mg/kg/day based on decreases in body weight in parental animals and in offspring at this dose. 

In carcinogenicity studies conducted by the NTP, there was some evidence of carcinogenicity for 
2-MBT in male and female rats.  The evidence included increased incidences of pituitary 
adenomas/adeno-carcinomas and adrenal gland pheochromocytomas in both female dosed  groups and increased incidences of adrenal gland pheochromocytomas/malignant pheochromocytomas and  preputial gland adenomas/carcinomas in both male dosed groups along with an increased incidence of mononuclear cell leukemia and pancreatic acinar cell adenomas in the low dose males.  For both male dosed groups, however, the treatment levels appeared to be excessive.

Based on the available data from the NTP report, 2-MBT appears to be carcinogenic in female mice based on increased incidences of  hepatocellular adenomas or carcinomas (combined) in the low dose females.  The high dose tested in this study was excessive based on the increased mortality noted in high dose females. 

2-MBT was negative in a salmonella (Ames) assay, a rat dominant lethal test, a CHO/HGPRT test [gene mutation], and a mouse micronucleus test [chromosomal aberration].  2-MBT was positive both with and without metabolic activation in a mouse lymphoma test [gene mutation].  MBT-sodium salt was negative in a rat hepatocyte unscheduled DNA synthesis assay.

The HED RfD/Peer Review committee met in April of 1994 to establish a Reference Dose for 2-MBT.  The committee recommended that the RfD be established on the basis of the 2-generation reproduction toxicity study using the NOAEL value of 194 mg/kg/day.  Using a total uncertainty factor of 300 (10x interspecies extrapolation, 10x intraspecies variation, 3x for lack of a chronic toxicity study in non-rodents [per the RfD peer review committee memo]), the RfD committee calculated a Reference Dose for 2-MBT of  0.6 mg/kg/day. Note that this chronic RfD value is considerably higher than that for the parent TCMTB, consistent with the toxicity database which shows lower toxicity for 2-MBT than TCMTB. 

The HED Carcinogenicity Peer Review Committee decided that 2-MBT should be classified as Group-C possible human carcinogen and recommended that for the purpose of risk characterization the Reference Dose (RfD) approach should be used for quantification of human risk.  This decision to use the RfD approach was based on some evidence of adrenal gland tumors in male and female rats and some evidence of preputial gland tumors in male rats.  There is equivocal evidence for pituitary gland tumors in male rats.  The evidence for hepatocellular tumors in mice was also equivocal.  2-MBT was also considered to have genotoxic activity.  The q1* approach to risk assessment was not used since the tumors were found in only 1 species and there was no clear evidence of carcinogenicity from any of the tumor types.

6.0	TOXICITY ENDPOINT SELECTION

6.1	See Section 9.2 for Endpoint Selection Table.

6.2	Dermal Absorption  

For TCMTB, a dermal absorption value is currently not needed based on selection of dermal endpoints from a route-specific study (21-day dermal toxicity study in rats, MRID 41655801).

A dermal absorption study was submitted by the registrant as an unaudited draft report (MRID NR42516601).  This study was reviewed by the Office of Pesticide Programs. The report was considered unacceptable for review based on the unaudited nature of the submission and other errors noted in the review. However, the executive summary of the unaudited submission is of interest in noting that the dermal absorption of TCMTB appears to increase with time of contact as well as dose level.  This study will require formal review of a final report to verify these conclusions. 

6.3	Classification of Carcinogenic Potential 

See Section 4.6.   

7.0	FQPA CONSIDERATIONS

7.1	Special Sensitivity to Infants and Children 

On the basis of the results of developmental and reproductive toxicity testing as well as consideration of the conclusions of the RfD Peer Review committee, the ADTC recommended that the hazard-based FQPA safety factor be reduced to 1x.  

7.2	Recommendation for a Developmental Neurotoxicity Study 

A developmental neurotoxicity study for TCMTB is not required at this time. 

8.0	REFERENCES 

MRID 00154295, 92179009.  Goad P. T., et al. 1985.  Teratogenicity study of TCMTB in rats.   HED Doc no. 005769.

MRID No. 40075101, 40075102, 92179011.  Adam, C. P. 1986.  TCMTB—A teratology study in rabbits.  

MRID 40884801.  Cameron, B.D.  Scott, G.  1987.  The Metabolism of 2-(Thiocyanomethylthio)benzothiazole  (TCMTB) in the rat.  Buckman Laboratories, Inc., Memphis TN.  Inveresk Research International Ltd., Scotland.   

MRID No. 41342201, 92179008.  Goburdhun, R., and Greenough, R.J.  1989. 2-(Thiocyanomethylthio)benzothiazole (TCMTB):  52-Week dietary toxicity study in dogs.  

MRID No. 41471401, 92179011.  Hazelden, K., Pl. and Wilson, J.A.  1988.  TCMTB-Two generation reproduction study in rats.  HED Doc 010607

MRID No. 41529701, 41570301, 421165301.  Everett, D. J. et al. 1989.   104-Week dietary toxicity/carcinogenicity study n rats with 52-week interim kill.  

MRID No. 42383301.  Everett, D.J., et al. 1990.  2-(Thiocyanomethylthio)benzothiazole- 104-Week dietary carcinogenicity study in mice.  HED Doc. 010332.  

MRID No. 43112801.  Atkinson, C., et al. 1987.   2-(Thiocyanomethylthio)benzothiazole (TCMTB).  13-Week dietary dose range finding study in rats.  

MRID No. 92179026.  Rao, G., No. 1980.  90-Day subchronic oral toxicity study of TCMTB in rats.  

Manninen, A., Auriola S, Vartiainen M., Leisivuori J., Turunen, T., Pasanen M.  1996.  Determination of urinary 2-mercaptobenzothiazole (2-MBT), the main metabolite of 2-(thiocyanomethylthio)benzothiazole (TCMTB) in humans and rats.  

9.0 APPENDICES
Tables for Use in Risk Assessment

9.1	Toxicity Profile Summary Tables 

9.1.1	Acute Toxicity Table  - See Section 4.1

9.1.2	Subchronic, Chronic and Other Toxicity Tables 

Guideline No./ Study Type	MRID No. (year)/ Classification /Doses	Results	
870.3100
13-week oral toxicity rodents (Range finding)
(Diet)

Batch 1517
	43112801 (1987)
unacceptable/
guideline  (range finding study)
0, 10, 30, 70 and 100 mgTCMTB/kg/day in diet (0, 8.2, 24.5, 57 and 81.6 mg ai/kg/day)

M: 0, 10.2, 31,70 and 100 mg/kg/day 
F: 0, 10.3, 31, 72, 99 mg /kg/day

81.56% ai purity (46698604, 92179001)	NOAEL = 10.2 mg/kg/day(M); 10.3 mg/kg/day (F) (8.3 mg ai/kg/day)
LOAEL = 31 mg/kg/day (M+F) (25 mg ai/kg/day) based on increased incidence of squamous epithelial hyperplasia of the stomach and decreased in body weight gains, food consumption and food efficiency in both sexes of the 70 and 100 mg/kg/day dose groups. 	
870.3100
90-Day oral toxicity rodents (diet)

Lot 9-3573
	92179026 (1980)
unacceptable/
guideline  

0, 333, 500, 750 ppm in diet
0, 16.7, 25, 37.5 mgTCMTB/kg/day which is equivalent to
13.4, 20 and 30 mg ai/kg/day
Purity approximately 80% ai.  (as supplied by the registrant via email)	NOAEL = 25 mg TCMTB/kg/day (20 mg ai/kg/day)
LOAEL = 37.5 mg TCMTB/kg/day(30 mg ai/kg/day)  based on increased incidence of histopathology of the stomach, including inflammation, necrosis and ulceration.

870.3700a
Prenatal developmental in rodents

Lot 511230	00154295, 92179009 (1985)
0, 25.1, 76.5, 125.5 mg/kg via gavage 
acceptable guideline
83.6% ai purity (46698601)

Equivalent to 21, 64 and 105 mg ai/kg/day 	Maternal NOAEL = 21 mg ai/kg/day (25.1 mg TCMTB/kg/day)
LOAEL = 64 mg ai/kg/day (76.5 mg TCMTB/kg/day) based on clinical signs of ventral alopecia, rough coat, dyspnea/wheezing, oral discharge, diarrhea/loose stool, urine staining, piloerection and hunched gait along with reduced body weight gain.

Developmental NOAEL =64 mg ai/kg/day (76.5 mg TCMTB/kg/day)
LOAEL = 105 mg ai/kg/day (125.5 mg TCMTB/kg/day) based on skeletal anomaly observations consisting of fused/wavy ribs, rudimentary cervical thoracic and lumbar ribs, increased number of sternebrae and pelvic girdle anomalies.  	
870.3700b
Prenatal developmental in nonrodents

Lot 5-13002	40075101, 40075102, 92179011 (1986)
acceptable guideline 
0, 10, 20, 40 mgTCMTB/kg/day via gavage of 81% ai purity  (MRID 46698602)

Equivalent to 0, 8, 16 and 32 mg ai/kg/day 	Maternal NOAEL = 16 mg ai/kg/day (20 mg TCMTB/kg/day )
LOAEL = 32 mg ai/kg/day (40 mg TCMTB/kg/day) based on decreased body weight gain and food consumption.

Developmental NOAEL≥  32 mg ai/kg/day (40 mg TCMTB /kg/day)
LOAEL=.32 mg ai/kg/day (40 mg TCMTB /kg/day).
	
Two generation reproduction study in rats

Batch 1517	41471401, 92179011 (1988)
acceptable guideline 
0, 25, 100 and 400 ppm; 
M: 0, 2.4, 9.6, 38.4 mg/kg/day; 
F: 0, 3, 11.7, 45.5 mg/kg/day
81.6% ai purity (MRID 46698604)	Offspring NOAEL (HDT) =38.4 mg/kg/day (M); 45.5 mg/kg/day (F) mg/kg/day (400 ppm)  
Offspring  LOAEL = Not identified 

Parental/systemic  NOAEL =38.4 mg/kg/day (M); 45.5 mg/kg/day (F) (HDT) (400 ppm)
Parental/systemic LOAEL = Not identified.

Reproductive 
NOAEL =38.4 mg/kg/day (M); 45.5 mg/kg/day (F) (HDT) (400 ppm)
LOAEL = Not identified.	
83-1a
Chronic toxicity in rats

Batch 1517	41529701, 41570301, 42116301 (1989)
acceptable guideline
0, 2, 8 and 20 mg/kg/day

81.6% ai purity(MRID 46698604)
	NOAEL=20 mg/kg/day
LOAEL= not identified.
Statistically significant increase in the incidence of testicular interstitial cell adenomas in males of mid (P=0.0155) and high-dose (P=0.0087) levels, that had a highly significant positive dose-related trend (P=0.0018).  Treatment was also associated with a possible increased incidence of thyroid C-cell adenomas in females of the mid- and high-dose levels, which had a highly significant (P=0.0067) positive dose-related trend, but did not attain a statistically significant level in the pairwise comparison with concurrent controls.  No historical data were available for review.  	
83-2
Chronic toxicity/
Carcinogenicity in mice

Batch 1517	42383001 (1990)
acceptable guideline
0, 5, 50, 150 mg/kg/day  which is  
equivalent to 4, 41 and 122 mg ai/kg./day  based on 
81.6% ai purity(MRID 46698604)	NOAEL=50 mg TCMTB/kg/day (41 mg ai/kg/day)
LOAEL= 150 mg TCMTB/kg/day (122 mg ai/kg/day) based on decreased body weight gain in both sexes, statistically increased incidence of focal and diffuse hyperplasia of duodenal mucosa in males (10/42 vs 1/41 in controls).  No evidence of carcinogenicity.  	
One-year dog

Batch 1517	41342201, 92179008 (1989)
unacceptable guideline (No NOAEL)
0, 100, 300 and 1000 ppm; 
M: 0, 3.8, 11.7 and 38.8 mg/kg/day
F:0, 4, 11.2, 43.2 mg/kg/day
81.6% ai purity(MRID 46698604)	NOAEL= not identified (<100 ppm or 3.8 mg/kg/day)
LOAEL= 3.8 mg/kg/day (M) and 4 mg/kg/day (F) based on decreased body weight gain, white cells, monocytes and plasma ALT in males and decreased plasma ALT and uterine weight in females.  

	
21-Day dermal toxicity study 

Lot OB-8483	41655801 (1990)

0, 25, 100 and 250 mg/kg/day which is equivalent to
0, 20.6, 82.3 and 206  mg ai/kg/day based on 
82.33% ai  purity
	Systemic NOAEL=20.6 mg ai/kg/day (25 mgTCMTB/kg/day)
Systemic LOAEL=82.3 mg ai/kg/day (100 mg TCMTB/kg/day) based on decreased body weight gain and food consumption in males, in addition to significant hematalogical (increased neutrophils, decreased hemoglobin, hematocrit and erythrocytes) and clinical chemistry changes (increased urea nitrogen, glucose and globulins) 

Dermal NOAEL= not identified
Dermal LOAEL= 20.6 mg ai/kg/day (25 mg T CMTB/kg/day) for dermal effects based on dose-dependent irritation which progressed to eschar formation in 2 rats of the low dose group, and in all rats of mid- and high-dose group.  Mid and high dose group exhibited ulcers, hemorrhages and chronic dermatitis.  	
870.5100/84-2
Mutagenicity study: Gene mutation (S. Typhimurium)	MRID 41386101
acceptable	TCMTB was negative for inducing reverse gene mutations in Salmonella typhimurium strains TA1535, TA1537, TA1538, TA 98 or TA 100 exposed in the presence of absence of S9 activation up to cytotoxic concentrations (33.3 ug/plate -S9; 66.7 ug/plate +S9).  The results were confirmed in an independently performed trial.	
870.5395/84-2
Structural Chromosomal Aberrations: Clastogenic Evaluation in in vivo mouse micronucleus assay	MRID 00165520 
Acceptable; EPA Accession No. 266152
TRID 470330-022
Purity not provided
	In a structural chromosomal aberration assay, the high dose of TCMTB (500 mg/kg) exhibited mortality and signs of clinical toxicity.  No evidence of an increase in micronucleated polychromatic erythrocytes (PCE) was noted at the dose levels tested in this study (50, 167, and 500 mg/kg).  There was evidence of some PCE depression at the high dose in the 48 and 72 hour animals.  	
870.5550/84-4 
Rat Primary Hepatocyte Unscheduled DNA Synthesis Assay	MRID 00165518 
Acceptable;
TRID 470330-020
EPA Accession No. 266150
Purity not provided; Lot No. 5-13002	TCMTB did not cause a significant increase in the unscheduled DNA synthesis (UDS) in rat primary hepatocytes as measured in this study.  Concentrations used were 0.05-25 ug/ml in DMSO. 	

9.2	Summary of Toxicological Dose and Endpoints  for 2-(Thiocyanomethylthio)benzothiazole (TCMTB) for Use in Human Risk Assessment1

Exposure
Scenario	
Dose Used in Risk Assessment
(mg/kg/day)
 	
Target MOEs/UFs FQPA safety factor for Risk Assessment	
Study  and Toxicological Effects	

Acute Dietary
(general population including infants and children) 	
NOAEL=  25.1  mg/kg/day

83.55% ai purity
	
 UF = 100  (10x inter-species extrapolation, 10x intra-species variation)

FQPA SF = 1x

Acute PAD = 0.25
mg TCMTB/kg/day	
Developmental toxicity study in rats (MRID 00154295, 92179009 (1985))

Maternal LOAEL = 76.5 mg TCMTB/kg/day, based on clinical signs of  toxicity (ventral alopecia, rough coat, dyspnea/wheezing, oral discharge, diarrhea/loose stool, urine staining, piloerection, and hunched gait). 

Acute Dietary 
(females 13-49) 	 

  An endpoint for females 13-49 was not identified in the available database for TCMTB. This risk assessment is not required. 

 	

Chronic Dietary 
(all populations) 	
LOAEL =  3.8 mg/kg/day
 
81.6% ai purity
	
 UF =  300  (10x inter-species extrapolation, 10x intra-species variation, 3x for use of a LOAEL)

FQPA SF = 1x

Chronic PAD = 0.01 
mg ai/kg/day	
 Chronic toxicity study in dogs (MRID 41342201, 92179008 (1989))

 LOAEL = 3.8 mg/kg/day (males), based on decreased body weight gain, decreased white cells, monocytes, and plasma ALT in males;  decreased uterine weight in females.	

Short- and Intermediate-Term 
Incidental Oral 
(1-30 days; 30 days-6 months)	
NOAEL=16.2 mg ai/kg/day 
 
81% ai purity
(20 mg TCMTB/kg/day) 
	
 MOE = 100	
Developmental toxicity study in rabbits (MRID 40075101, 40075102, 92179011 (1986))
 
LOAEL = 32 mg ai/kg/day (40 mg TCMTB/kg/day), based on decreased body weight gain and food consumption in maternal animals. 	

   Dermal (all durations)
(1-30 days; 30 days-6 months; > 6 months) 	
NOAEL= 25 mg/kg/day

82.33% ai purity
(20.6 mg ai//kg/day) 
 	
 MOE = 100 (ST and IT)

MOE = 300 (LT)	
21-Day dermal toxicity study in rats (MRID 41655801)

LOAEL =  100 mg TCMTB/kg/day (82.3 mg ai/kg/day), based on decreased body weight gain, food consumption, and hematological and clinical chemistry changes	

Inhalation
(short- and intermediate-term)	
NOAEL=16.2 mg ai/kg/day 
 
81% ai purity
(20 mg TCMTB/kg/day) 

Absorption=100% (inhalation and oral equivalent)	
  
MOE = 300 (ST and IT)
 	
 Developmental toxicity study in rabbits (MRID 40075102)

LOAEL = = 32 mg ai/kg/day (40 mg TCMTB/kg/day), based on decreased body weight gain and food consumption in maternal animals

	

Inhalation (long-term)	
LOAEL =  3.8 mg/kg/day
 
81.6% ai purity

Absorption=100% (inhalation and oral equivalent)
	
MOE = 1000	
Chronic toxicity study in dogs (MRID 41342201, 92179008 (1989))

 LOAEL = 3.8 mg/kg/day (males), based on decreased body weight gain, decreased white cells, monocytes, and plasma ALT in males;  decreased uterine weight in females.	

Carcinogenicity	
 The CPRC concluded that TCMTB should be classified as Group C - possible human carcinogen - and recommended that for the purpose of risk characterization, the Reference Dose (RfD) approach should be used for quantitation of human risk.  This was based on statistically significant increases in tumors in both sexes of the Sprague-Dawley rat: testicular interstitial cell adenomas in males and thyroid C-cell adenomas in females.