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EC number: 202-607-8
CAS number: 97-77-8
Source, RA-A, CAS 137-26-8, key, report number HLA 6111-112, chronic (52 weeks, dog):
NOAEL (oral) 30 ppm for males (corresponding to approx. 0.84 mg/kg bw/day), 90 ppm for females (corresponding to approx. 2.54 mg/kg bw/day)
After correction for molecular weight, the oral NOAEL for the target substance is ca. 1.04 mg/kg bw/day for males and ca. 3.13 mg/kg bw/day for females.
Source, RA-A, CAS 137-26-8, key, report number UCB 421/920767, subacute (21 days, rat):
NOAEL (local, dermal) <100 mg/kg bw/day (females, males)
NOAEL (general systemic toxicity, dermal) 300 mg/kg bw/day (females, males)
After correction for molecular weight, the NOAEL for the target substance is < 123.3 mg/kg bw/day for local effects and 369.9 mg/kg bw/day for systemic effects.
After correction for molecular weight, the oral NOAEL for disulfiram is ca. 1.04 mg/kg bw/day for males and ca. 3.13 mg/kg bw/day for females. In addition to the key study, several repeated dose studies in rats, mice and dogs were included in the technical dossier as supporting information.
After correction for molecular weight, the NOAEL for disulfiram is < 123 mg/kg bw/day for local effects and 369.9 mg/kg bw/day for systemic effects. The LOAEL per area was 3.3 mg/cm². It was calculated using the lowest body weight (2.6 kg), the lowest dose group (123 mg/kg bw/day) and the exposure area of 12 x 8 cm.
Please refer to the respective endpoint summary.
Repeated dose toxicity studies on disulfiram
Studies after oral repeated dose administration of the test material disulfiram in the rat and mouse are available (NTP, 1979). Range-finding oral feeding studies lasting 7 weeks were performed in both species to set dose levels for the 108-week chronic studies.
Five Fisher 344 rats per sex and dose were administered daily doses of 0, 1500, 2200, 3200, 4600 and 6800 ppm of the test material disulfiram in the diet for seven weeks. Five female and ten male B6C3F1 mice per sex and dose were administered daily doses of 2000, 3000, 4000, 4500, 5000, 6000 and 8000 ppm (males) and 250, 500, 1000, 1500, 2000, 2500, 5000 and 10000 ppm (females) of the test material disulfiram in the diet for seven weeks (NTP, 1979). Ten mice per sex per dose served as controls. Each animal was weighed twice per week and at the end of the administration period, all animals were sacrificed. Only survival, body weights and grosspathological changes were examined. Survival was adequate in all dose groups in both rats and mice. The body weights of both rats and mice were decreased compared to untreated controls at all dose levels. The LOAEL was therefore 44.6 mg/kg bw/d (corresponding to a daily dose of 1500 ppm) for male and female rats, 64 mg/kg bw/d (corresponding to a daily dose of 2000 ppm) for male mice and 16 mg/kg bw/d (corresponding to a daily dose of 500 ppm) for female mice.
In both rats and mice, the dose range for the chronic study was based on the LOAEL found in the 7-week studies, which was based on a reduction in body weight only.
The restricted study information severely affect the reliability of the studies which were conducted with the test material disulfiram.
Repeated dose toxicity studies on the source substance
Repeated-dose toxicity of disulfiram is therefore assessed using reliable read-across data from studies conducted with a surrogate substance, thiram (CAS No. 137 -26 -8).(For detailed information on the justification of read-across, please refer to the analogue justification document attached in IUCLID section 13).
After correction for molecular weight the NOAELs for thiram are used as NOAELs for disulfiram. The molecular weight of thiram is 240.44 g/mol, the molecular weight of disulfiram is 296.54 g/mol. Therefore, a correction factor of 296.54 g/mol / 240.44 g/mol = 1.233 was applied.
Repeated dose toxicity: oral administration
Data on repeated dose toxicity are available after exposure for 28 days (mouse, dog), 90 days, 52 weeks (dog) and 104 weeks (rat) to characterize the potency of the test substance to induce toxicity after short-term and long-term exposure. The 52-week oral toxicity dog study serves as a key study while the other studies serve as supporting studies.
A subacute (Report number: HLA-6111-127) supporting study was available that served as a dose range finding study for a carcinogenicity study (Report number: 798-223). Both studies were conducted similar to OECD guidelines and under GLP conditions.
In the subacute oral toxicity study (Report number: HLA-6111-127), the test substance was administered at 300, 600 and 1200 ppm for 4 weeks to groups of 10 animals/sex and dose (corresponding to approx. 51-58, 101-115 and 177-226 mg/kg bw/day in males and to approx. 59-66, 111-127 and 221-281 mg/kg bw/day in females, respectively). There were no mortalities during the study. Body weights were significantly lower for males in the 300 and 1200 ppm groups during Week 3 and 4 and for males in the 600 ppm group for Week 4. Females showed no differences. Food consumptions were significantly lower for all treated animals throughout the study period. RBC count, Hb and Hct were slightly lower in all treated males (up to -8.6%). In females RBC count was decreased in the 600 ppm group (-6.2%) while platelet count was increased at 600 and 1200 ppm (up to +27.6%). The only statistically significant alteration in clinical chemistry was the decreased glucose value in females dosed at 1200 ppm. Absolute organ weights alterations were not dose-response-related and regarded as a result of the lower terminal body weight. There were no treatment-related macroscopic or microscopic observations noted.
No NOAEL was set for males, the LOAEL for males was set at 300 ppm, corresponding to 51 mg/kg bw/day. In females, 300 ppm, corresponding to 59 mg/kg bw/day was the NOAEL.
A subchronic (Report number: HLA 6111-110) and one combined chronic/carcinogenicity study (Report number: HLA 6111-113) were available, which are considered as supporting studies. All studies were conducted similar to OECD guidelines and under GLP conditions.
Description of the studies with rats
In a sub-chronic study (Report number: HLA 6111-110), the test substance was administered via diet to three groups, each consisting of ten male and ten female Sprague-Dawley rats. The test substance was administered for 13 weeks at dose levels 50, 500 and 1000 ppm, corresponding to 0, 3.5, 38 and 67 mg/kg bw/day for males and 0, 4, 38 and 80 mg/kg bw/day for females. The control group of ten males and ten females received plain diet. No treatment-related deaths or antemortem observations were noted. Body weights, cumulative body weight gains and food consumptions were significantly lower in animals dosed at 500 or 1000 ppm. Changes in clinical pathology occurred at 500, 1000 ppm or both and affected parameters typical for haemolysis, e.g. lower RBC count, Hb and Hc and higher MCV and MHC in both sexes. Besides that, higher leukocyte count, urea nitrogen and chloride and lower albumin in females and lower total protein and glucose in both sexes were noted. Corresponding to the lower terminal body weight animals dosed at 500 or 1000 ppm had lower absolute organ weights. Pathological examinations revealed the nonglandular stomach as target organ. However, since the observed mucosal hyperplasia may be caused by the irritating properties of the substance and the nonglandular stomach is not present in humans, the relevance for human hazard assessment may be questionable. Oral administration of the test substance to rats for a period of 13 weeks at a dose level up to 1000 ppm resulted in treatment-related adverse effects in animals treated with 500 and 1000 ppm. The NOAEL was set at 3.5-4 mg/kg bw/day.
A combined chronic and carcinogenicity study on rats was available (Report number: HLA 6111-113). The test substance was administered via dietary exposure to male and female Sprague-Dawley rats at dietary concentration of 30, 150 and 300 ppm (corresponding to approx. 1.46, 7.31 and 14.66 mg/kg bw/day in males and to approx. 1.80, 8.86 and 18.57 mg/kg bw/day in females, respectively). As also observed in the subchronic repeated dose studies, the test substance caused general systemic toxicity as evident by lower body weight gains and food intake as well as changes in blood parameters. Changes in blood parameters (haematological and clinical biochemistry) consisted of signs of haemolysis, evident by changes in the number of erythrocytes, haemoglobin and haematocrit concentration, as well as increase of mean corpuscular volume and mean corpuscular haemoglobin. Further, the relative and absolute kidney weights of rats treated with 300 ppm of the test substance (high dose) were decreased compared to the control group. Effects on gross pathology and histopathology were observed, the latter including extramedullary haematopoieses in the liver and spleen, suggesting a test material induced effect on haematopoiesis. Further organs showing abnormalities were the pancreas, the thyroid and the mesenteric lymph nodes. However, no clear dependency on the test material administration could be established for those. For the description of the carcinogenic effects, please refer to the respective Endpoint summary.
The NOAEL for systemic toxicity was set to 30 ppm, corresponding to 1.46 and 1.8 mg/kg bw/day for male and female rats, respectively.
Conclusion for all studies in rats
In all of the repeated dose toxicity studies with rats, the read across source substance caused decreased body weight gain and food intake. Further, changes in blood parameters suggesting haemolysis were observed. These were consistent decreased mean red blood cells (RBC), haemoglobin (Hb) and haematocrit (Hct) concentrations, increased mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC). The haematological changes occured at an actual ingested dose of 38 mg/kg bw/day (males and females) in the 13-week repeated dose oral toxicity study and at an actual ingested dose of 7.3 mg/kg bw/day (males) and 8.9 mg/kg bw/day (females) in the combined chronic/carcinogenicity study.
Based on the subchronic and combined chronic/carcinogenicity rat studies, the test substance affected several target organs with the following treatment-related tissue changes: erosive lesions of the non-glandular epithelium in the stomach, changes in mesenteric lymph nodes, extramedullary haematopoiesis (EMH) in the spleen and liver. These histopathological findings were accompanied by gross pathological findings (stomach, mesenteric lymph nodes, liver, kidneys). In the 104-weeks dietary combined chronic/carcinogenicity study, increased incidences of steatosis/fatty infiltration were found in the pancreas of mid- and high-dosed animals compared to the control group. This finding cannot be specifically attributed to treatment but could also be age-related or a secondary effect to the presence of acinar atrophy (Covance 6111 -113).
The principal effect caused by the test substance is haemolysis indicated by a decrease in red blood cells and haemoglobin and by an increase in the mean corpuscular volume and mean corpuscular haemoglobin concentration is accompanied by subsequent effects on spleen and liver, seen as extramedullary haematopoiesis.
The carcinogenicity of the test item is discussed in the corresponding section. The ED properties of the substance are discussed in the respective robust study summaries.
In total, the lowest oral NOAEL in rats was 30 ppm (corresponding to 1.46 mg/kg bw/day for males and 1.8 mg/kg bw/day for females) from the 2 years combined chronic/carcinogenicity study.
Three oral repeated dose toxicity studies in dogs are available, covering different study durations including a 28-days supporting study (Report number: HLA 6111-109), a 90-days supporting study (Report number: HLA 6111-121) and a 52-weeks (Report number: HLA 6111-112) study. All three studies were conducted similar to the respective OECD guidelines and under GLP conditions.
Description of studies with dogs
In a sub-acute supporting study (Report number: HLA 6111-109), two beagle dogs per sex and dose were administered daily doses of 125, 500 and 2000/1500 ppm (corresponding to approx.4, 12 and 27-21 mg/kg bw test substance for females and 4, 16 and 26-15 mg/kg bw/day) test substance for males in the diet for a period of four weeks. Due to mortality, the dose level of the high dosed group was reduced from 2000 ppm to 1500 ppm during week 3 and 4.
Clinical observations noted included ptyalism and convulsion. One female of the highest dose group died during week 2 and one male of the same group was moribund and sacrificed during week 4. Body weights in both sexes were decreased at all doses. During week 4, food consumptions in males/females at all doses were lower than those of controls. Treatment-related changes in blood parameters appeared to be lower erythrocyte count, haemoglobin and haematocrit in males given 12 and 27-21 mg/kg bw/day as well as slightly lower platelet count in some dogs. One male given 27-21 mg/kg bw/day had higher ALT, AST and AP. Slightly lower absolute lymphocyte and slightly higher total bilirubin and urea nitrogen occurred in some dogs. Corresponding to the lower body weights, surviving animals given the highest dose level had lower absolute organ weights. The liver of the surviving male dosed at 27-21 mg/kg bw/day had hepatocellular degeneration with sinusoidal cell proliferation and pigmentation. Based on reduced body weight and food consumption and alteration in clinical blood parameters in the 16/12 mg/kg bw/day dose group, the NOAEL was set at 4 mg/kg bw/day for both sexes.
In the sub-chronic study, the test substance was administered via diet to groups of 4 male and 4 female Beagle dogs for a period of 13 weeks at dietary concentrations of 75, 250 and 500 ppm, corresponding to 1.94 -2.58, 6.17-7.85, 10.55-14.69 mg/kg bw/day for males and 0.00, 2.14-2.55, 6.67-8.01, 11.75-13.42 mg/kg bw/day for females (Report number: HLA 6111-121). Control dogs received plain diet. No mortalities occurred. Clinical signs of toxicological relevance consisted of food-like vomitus, reduced food consumption, and reduced body weights (up to 18% and 21% lower as compared to control animals for males and females of the 500 ppm group in week 13).
There were several statistically significant differences for clinical pathology variables between control and treated dogs. In addition, there were some consistent differences that were not statistically significant. The differences considered to represent treatment-related effects were lower RBC count and higher MCV and MCH in males and females given 75, 250, or 500 ppm; lower haemoglobin and haematocrit in females given 500 ppm, higher platelet count in males given 250 or 500 ppm; lower total protein and albumin in males and females given 75, 250, or 500 ppm; and higher cholesterol in males and females given 250 or 500 ppm. Other differences were considered normal biological variation, not related to the test material, and not toxicologically meaningful. Organ weight analyses, ophthalmological examinations and gross pathology revealed no treatment-related effects. Microscopic changes (chronic inflammatory cells, and hepatocellular degeneration with granulomatous inflammation) were found in the livers of several animals. These changes were thought to be variations of a process caused by low-grade bacterial infection. The treatment appeared to facilitate development and progression of the changes, but the changes were not considered to be toxicological effects. The NOAEL was set at 2 mg/kg bw/day.
In the 52-week study considered as the key study, six dogs per sex and dose level received daily doses of 0, 30, 90 or 250 ppm test substance in diet, corresponding to 0, 0.84, 2.61, 7.35 mg/kg bw/day for males and 0, 0.90, 2.54, 7.23 mg/kg bw/day for females for 52 consecutive weeks (Report number: HLA 6111-112).
One female at 90 ppm was sacrificed during week 25, due to clonic convulsions with pulmonary congestion. Pathology examinations revealed meningoencephalitis. Another female of the high-dose group was sacrificed during week 26 due to clonic seizures with opisthotonus. Pathology examinations revealed hydrocephalus. However, both deaths were not considered treatment-related. No treatment-related effects were noted in any of the other dogs surviving until termination. There were also no observations that indicated an effect on the neurological system. There were no statistically significant differences in body weights of males and females at any dose level. Food consumption of females at 250 ppm was significantly lower than controls during week 20. However, there were no consistent effects on food consumptions in both sexes throughout the study. Ophthalmoscopic examinations revealed no lesions in any animal. RBC values were reduced in high dose males. Higher MCV and MCH values were also observed in treated animals. However, since there were similar differences noted before initiation of treatment, no conclusion could be drawn from these findings. Clinical pathology examination revealed lower protein and higher cholesterol levels in males at 90 or 250 ppm, and in females at 250 ppm. Albumin values were lower in both sexes at 250 ppm. These effects were considered not treatment-related. Absolute and relative liver weights were significantly increased in males at 90 and 250 ppm. Relative liver weights were also significantly increased in high-dose females, as were liver-to-brain ratios in high-dose males. The changes in liver weights, in conjunction with the altered protein, albumin, and cholesterol levels suggest a test material-related effect on liver function and size. As the liver has an important role in the metabolism of the test substance, these changes may be considered as an adaptation to treatment. There were no gross pathological and histopathological adverse findings at necropsy. However, based on the observed magnitude of ≥ 20% liver weight increase compared to control, this effect may be considered adverse (ECETOC Technical Report No. 85, 2002). Based on the increased liver weights in mid- and high-dose males, and on changes in clinical chemistry parameters in the same groups, as well as for high-dose females, the NOAEL for dogs after 52-week dietary administration of the test substance is 0.84 mg/kg bw/day for males, and 2.54 mg/kg bw/day for females.
Conclusion for all studies in dogs
Dogs were noted with reduced body weight and food consumption in the subchronic key study. Decreased RBC, increased MCV and MCH were noted in all key studies. In the subchronic study, also decreased Hct, Hb and decreased MCHC were noted. In the subchronic and chronic study, the following clinical chemistry parameters were altered: total protein and albumin were decreased while cholesterol was increased. Hepatocellular degeneration was seen in the subacute and subchronic study, in the 52-week study, the absolute liver weight was increased but no histopathological changes were observed. The ED properties of the substance are discussed in the respective robust study summaries.
In total, the lowest most relevant oral NOAEL was 30 ppm for males (corresponding to 0.84 mg/kg bw/day) in the 52-week dog study. For females, the lowest NOAEL was 75 ppm (corresponding to approx. 2 mg/kg bw/day) in the subchronic study.
Repeated dose toxicity: dermal administration
A 21-day percutaneous study on rabbits (Report number: UCB 421/920767) was conducted according to OECD 410 and under GLP conditions. The test substance caused dermal irritation at all dose levels when applied dermally for three weeks to the intact skin of male and female rabbits (100, 300 and 1000 mg/kg bw/day). Treatment-related disturbances in body weight, food consumption and various biochemical parameters were observed for rabbits receiving the test substance at 1000 mg/kg bw/day. No treatment-related changes were observed between the control and treatment groups regarding haematological changes. The only statistically significant differences were slightly increased RBC and MCHC values in females of the highest dose group. As these were minor changes and not dose related, they were not considered treatment related or adverse. Thus, no NOAEL was set for local skin effects and the NOAEL for systemic effects was 300 mg/kg bw/day for males and females.
However, the local effects observed for the source substance Thiram after repeated exposure via the dermal route are not applicable to the target substance disulfiram. The target substance was tested for skin irritation in two valid and acceptable studies similar to OECD TG 404 and the substance was found to be not irritating to the skin of rabbits. Therefore, the repeated dose toxicity study in rabbits via the dermal route conducted with the source substance is included in the technical dossier of the target substance for completeness, but the observed local effects are neither used for the human hazard assessment nor for classification and labelling according to Regulation (EC) 1272/2008.
Overall, the lowest most relevant oral NOAEL was 30 ppm (corresponding to 0.84 mg/kg bw/day for males and 0.9 mg/kg bw/day for females) from the 52-weeks dog study. The lowest dermal NOAEL was 300 mg/kg bw/day for systemic effect in rabbits.
Further, it was concluded that the test substance affected the target organs liver (all animals), non-glandular stomach (rat and mice) and spleen (rat and mouse). The dog is considered to be the most sensitive species. An assessment on endocrine disruption was performed during the CoRAP substance evaluation of Thiram. The outcome of this evaluation in 2018 was that Thiram has no ED concern.
Assessment of Specific target organ toxicity after repeated exposure
Effects on target organs or haematology parameters were observed at dose levels below 10 mg/kg bw/day in the combined carcinogenicity/chronic toxicity study in the rat as well as in the subchronic and chronic dog studies. However, according to the Regulation (EC) No 1272/2008, the reference value of 10 mg/kg bw/day for classification as Specific Target Organ Toxicity after Repeated Exposure (STOT-RE), Category 1 is referring to a subchronic study in rats. The corresponding guidance values for a study period of 52 weeks are <= 2.5 mg/kg bw/day for STOT RE, Category 1, and > 2.5 - <= 25 mg/kg bw/day for STOT RE, Category 2. Moreover, it is stated that the effects observed should be “consistent and significant adverse change[s] in clinical biochemistry, haematology, or urinalysis parameters” (p. 469). Even though the changes are consistent over study periods and species, the effect at doses below 10 mg/kg bw/day cannot be seen as significantly adverse. The guideline states as an example of a significant adverse effect a reduction of haemoglobin of at least 10% compared to the control group (p. 470). A reduction of this extent has not been observed in any of the studies with the test substance. Therefore, significant adverse effects are only seen above doses of 10 mg/kg bw/day in the sub-chronic studies and above doses of 2.5 mg/kg bw/day in chronic studies over 52 weeks. Therefore, classification as Specific Target Organ Toxicity after Repeated Exposure (STOT RE), Category 1 is not warranted and the classification criteria for Specific Target Organ Toxicity after Repeated Exposure (STOT RE), Category 2, H373 (Regulation (EC) No 1272/2008) are met which is in line with the harmonised classification according to Annex VI of CLP Regulation (Index No.: 006-005-00-4).
After correction for molecular weight, the NOAEL for disulfiram via the oral route is ca. 1.04 mg/kg bw/day for males and ca. 3.13 mg/kg bw/day for females derived from the 52-week study in dogs. After correction for molecular weight, the dermal NOAEL for disulfiram is < 369.9 mg/kg bw/d for systemic effects derived from the dermal repeated dose toxicity study.
The dog appears to be the most sensitive species for long term systemic toxic effects caused by thiram. Thus, the systemic long term DNELs are based on studies which were conducted in dogs.
Covance 6111 -113, 104-week dietary combined chronic toxicity and Carcinogenicity study with thiram in rats, Supplementary Information on Pancreas Steatosis/Fatty Infiltration, 2016
Based on the observations of systemic toxic effects (significant changes in the spleen, stomach and liver) in the repeated dose toxicity studies conducted with the structurally related source substance at relevant dose levels, classification criteria for Specific Target Organ Toxicity after Repeated Exposure (STOT RE), Category 2, H373 (Regulation (EC) No 1272/2008) are met. This is in line with the harmonised classification of the target substance disulfiram (Annex VI of Regulation (EC) 1272/2008, index number 006-079-00-8).
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