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Description of key information

Read-across from thiram: The dog appears to be the most sensitive species for thiram toxicity. NOEL for dogs after 52-week dietary administration of thiram is 0.84 mg/kg bw/d for males, and 2.54 mg/kg bw/d for females (Kehoe 1991, chronic, dog, RL2).

After correction for molecular weight, the oral NAEL for disulfiram is ca. 1.04 mg/kg bw/d for males and ca. 3.13 mg/kg bw/d for females.

After correction for molecular weight, using route to route extrapolation (oral to dermal) assuming a dermal absorption factor of 10%, the dermal NAEL for disulfiram is ca. 10.4 mg/kg bw/d for males and ca. 31.3 mg/kg bw/d for females.

After correction for molecular weight, using route to route extrapolation (oral to inhalation), the inhalative NAEC for disulfiram is ca. 1.82 mg/m3 for males and ca. 5.52 mg/m3 for females.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Dose descriptor:
NOAEL
1.04 mg/kg bw/day
Study duration:
chronic
Species:
dog

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Dose descriptor:
NOAEC
1.82 mg/m³
Study duration:
chronic
Species:
dog

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Dose descriptor:
NOAEL
10.4 mg/kg bw/day
Study duration:
chronic
Species:
dog

Additional information

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 and body weight 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 LOEL 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 8 mg/kg bw/d (corresponding to a daily dose of 250 ppm) for female mice.

In both rats and mice, the dose range for the chronic study was based on the LOEL found in the 7-week studies, which was based on a reduction in body weight only.

 

In subsequent chronic studies, 50 Fisher 344 rats per sex per dose were administered daily doses of 300 and 600 ppm of the test material disulfiram in the diet for 107 weeks. 20 untreated animals per sex per dose served as controls. Additionally 50 mice per sex per dose were administered daily doses of 500 and 2000 ppm (males) and 100 and 500 ppm (females) of the test material in the diet for 108 weeks (NTP, 1979). 20 untreated animals per sex per dose served as controls. All surviving animals were sacrificed at the end of the administration period of the test material. Mean body weights of dosed rats and mice of each sex were lower than those of the corresponding control groups and were dose-related. Mortality was not significantly affected by administration of the test material to either rats or mice, except for female rats, in which the mortality was higher in the control group than in the dosed groups; the survival at the end of the study period was >/= 65 % in all dosed or control groups in rats or mice of either sex, and sufficient numbers of animals were at risk in each group for the development of late-appearing tumours. No tumours occurred in rats or mice of either sex at incidences that were significantly higher in dosed groups than in corresponding control groups. Based on the fact that no treatment related adverse effects were observed in both species in either sex, the NOAEL was 12.5 mg/kg bw/d (corresponding to a daily dose of 600 ppm) for rats, 60 mg/kg bw/d (corresponding to a daily dose of 2000 ppm) for male mice and 14.7 mg/kg bw/d (corresponding to a daily dose of 500 ppm) for female mice.

 

The restricted study information and the lack of any effects at the highest dose level severely affect the reliability of the studies which were conducted with the test material disulfiram.

 

Repeated dose toxicity studies on thiram

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 NAELs 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.

 

Range-finding studies for dietary administration of thiram (CAS 137 -26 -8) were performed for short periods of exposure in mice and in dogs for 28 days. The studies were used to set dose levels for the subsequent 90-day studies.

 

In a sub-acute study, two beagle dogs per sex and dose were administered daily doses of 4, 12 and 27-21 mg/kg bw thiram for females and 4, 16 and 26-15 mg/kg bw thiram for males in the diet for a period of four weeks (Kehoe, 1988). Clinical signs and mortality were examined once daily. Body weight and food consumption were monitored once weekly.

Haematology parameters as well as clinical chemistry parameters were measured on study initiation and termination. At the end of the study all surviving animals were sacrificed and gross- and histopathology was conducted. 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/d as well as slightly lower platelet count in some dogs. One male given 27-21 mg/kg bw/d 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 dosed at the highest dose level had lower absolute organ weights. The liver of the surviving male dosed at 27-21 mg/kg bw/d 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/ dose group, the NOAEL was set at 4 mg/kg bw/d for both sexes.

After correction for molecular weight, the subacute NAEL for disulfiram is 4.94 mg/kg bw/d.

 

In a second sub-acute study, ten CD-1 mice per sex and dose were administered daily doses of 0, 300, 600, 1200 ppm thiram, corresponding to 0, 51-58, 101-115, 177-226 mg/kg bw for males and 0, 59-66, 111-127, 221-281 mg/kg bw for females in the diet for a period of four weeks (Kehoe, 1988).Clinical signs, mortality and food consumption were observed daily. Body weight was monitored immediately before start of treatment, on Day 0, weekly thereafter and at termination. Haematology and clinical chemistry parameters were measured after 4 weeks of treatment. At the end of the study all surviving animals were sacrificed and gross- and histopathology was conducted. Adverse clinical observations were confined to alopecia, hunched posture and squinting or lacrimation of the eyes. Some females showed also alterations on the tail. 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 Hc were slightly lower in all treated males. In females RBC count was decreased in the 600 ppm group while platelet count was increased at 600 and 1200 ppm. The only statistically significant alteration 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.The sub-acute NOAEL was set at 51-58 mg/kg bw/d for males and 59-66 mg/kg bw/d for females.

After correction for molecular weight, the sub-acute NAEL for disulfiram is ca. 62.93 mg/kg bw/d for males and ca. 72.81 mg/kg bw/d for females.

 

Rats and dogs received thiram in their diet either for a subchronic 90-day period or for a chronic period of 52 weeks.

In a sub-chronic study, thiram was administered via diet to three groups, each consisting of ten male and ten femaleSprague-Dawley rats (Kehoe, 1988). 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 for males and 0, 4, 38 and 80 mg/kg bw for females. The control group of ten males and ten females received plain diet.Clinical signs, body weight development and food consumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study as well as ophthalmoscopic examination was performed. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed. 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. Oral administration of thiram 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/d.

After correction for molecular weight, the NAEL for disulfiram is 4.32-4.94 mg/kg bw/d.

 

Thiram 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 0.00, 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 (Kehoe, 1990). Control dogs received plain diet. Clinical signs, food consumption, development of body weight, haematology, clinical chemistry, and ophthalmic examinations as well as macroscopic and microscopic pathological inspection were performed on all animals. No mortalities occurred. Clinical signs of toxicological consisted of food-like vomitus and 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 statistical 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/d.

After correction for molecular weight, the NAEL for disulfiram is 2.47 mg/kg bw/d.

 

Six dogs per sex and dose level received daily doses of 0, 30, 90 or 250 ppm thiram 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 in a chronic study for 52 consecutive weeks (Kehoe, 1991). All dogs were observed for mortality and clinical signs of toxicity twice daily. Body weights were determined weekly before initiation, on first day of treatment, weekly for weeks 1-16, then once every 4th week. Ophthalmoscopic, haematological, clinical chemistry examinations and urine analyses were performed prior initiation and in weeks 13, 26 and 52. Food consumption was recorded daily. A complete necropsy examination with detailed histopathology was conducted on all dogs surviving to scheduled termination and on dogs that died or were killed in moribund condition during the study period. 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, 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 thiram, these changes may be considered as an adaptation to treatment. There were no gross pathological and histopathological adverse findings at necropsy. 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 NOEL for dogs after 52-week dietary administration of thiram is 0.84 mg/kg bw/d for males, and 2.54 mg/kg bw/d for females.

After correction for molecular weight, the NAEL for disulfiram is ca. 1.04 mg/kg bw/d for males and ca. 3.13 mg/kg bw/d for females.

 

One study is available for sub-acute dermal repeated dose administration of the test material disulfiram in the rabbit (Edwards, 1982). Groups of five male and five female New Zealand White rabbits were administered 10, 300 and 1000 mg technical thiram/kg bw/day by dermal application over a 21 day (males) or 22 day (females) period. A concurrent control group was similarly treated with distilled water. 24 hours prior to the first application a dorsal region of 8 x 12 cm (approx. 10% of the total body surface) was clipped free of hair without abrading the skin. This clipping was repeated as necessary during the experimental period. The test substance was sufficiently moistened with water and applied evenly over the treatment area. The control animals received 0.6 mL of distilled water. The test substance remained on the skins for approximately 6 hours each day under an occlusive dressing. Afterwards the dressings were removed and the skin was washed with warm water. Clinical signs, local dermal irritation and body weight development as well as food consumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed. In all rabbits of the low-, mid- and high-dose group skin reactions consisting of slight or well-defined erythema with or without slight or well-defined oedema were noted. Well-defined responses were observed with greater frequency in mid- and high-dose rabbits. Reduced bodyweight gain and reduced food consumption was observed in females dosed at 1000 mg/kg bw/day.

Liver enzymes GOT, GPT, and cholesterol levels were increased in females dosed at 1000 mg/kg bw/day. Alkaline phosphatase activity was increased at 1000 mg/kg bw/day in both sexes. In the absence of histopathological changes to other tissues, the disturbance of body weight gain and biochemical parameters was considered to be of minor toxicological importance but sufficient to reject this dosage being considered a no-toxic effect level. The local NOEL for dermal irritation is 100 mg/kg bw/d, the NOEL for systemic effects is 300 mg/kg bw/d.

After correction for molecular weight, the NAEL for disulfiram is 123.3 mg/kg bw/d for local effects and 369.9 mg/kg bw/d for systemic effects.

 

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.

 

Justification for classification or non-classification

According to Annex I of DSD (67/548/EC) and Annex VI of CLP (1272/2008/EC), disulfiram has been legally classified as Xn, R48/22 and STOT RE 2, H373, respectively.