Disulfiram

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 May 1989 - 21 Jun 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

Version / remarks:

adopted in 2018

Deviations:

yes

Remarks:

On several occasions there was only one observation per day for mortality and moribundity. Due to mistakes food consumption values for week 10 reflect 6-day consumption. Blood sampling prior to treatment (week -2) was done on non-fastened animals.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPP 83-1 (Chronic Toxicity)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Thiram

EC Number:

205-286-2

EC Name:

Thiram

Cas Number:

137-26-8

Molecular formula:

C6H12N2S4

IUPAC Name:

tetramethylthiuram disulfide

Test animals

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hazleton Research Products
- Age at study initiation: 4 -5 months
- Weight at study initiation: 4.0 - 6.8 kg
- Fasting period before study: not applicable
- Housing: Individually in stainless steel, screen-bottom cages. Animal housing and husbandry complied with standards outlined in the Guidance for the Care and Use of Laboratory Animals.
- Diet: Certified Canine Diet® #5007 (Purina Mills., Ltd.) ad libitum
- Water: Water ad libitum.
- Acclimation period: 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 23 May 1989 To: 25 May 1990

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet: weekly
- Mixing appropriate amounts with: A specified amount of basal diet was weighed into a labelled mixing bowl, from which about 200 g was removed and placed in a blender. The required amount of test material was weighed, added to the blender, overlaid with about 50 g of basal diet from the mixing bowl before blending. This premix was transferred to the mixing bowl. About 100 g of basal diet from the mixing bowl was then transferred to the blender, blended to recover residual test material and returned to the mixing bowl and thoroughly mixed. Samples for dose analyses were taken directly from the mixing bowl. Diets were stored frozen in covered containers until dispensed daily into food containers.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability was confirmed for all test material dietary concentrations used in the study. Samples of diet were analysed on the day of mixing except on holidays when they were analysed within one day of mixing. All samples were stored in the freezer until analysed. Mean values for stability assay results for samples on the diets on the day of preparation were 88%, 93.8% and 96% of the theoretical levels of 30, 90 and 250 ppm, respectively.

Homogeneity was determined at all dose levels, by analysing samples taken from the top, bottom and two opposing sides. The mean values for homogeneity assay results were 83%, 91.6% and 92% of the theoretical levels of 30, 90 and 250 ppm, respectively, indicating that the mixing procedure resulted in a homogeneous distribution in the diet.

Concentration at each dose level was analysed from samples taken directly from the mixing bowl. Test diet verification analysis demonstrated that the anticipated levels of the test substance were achieved in the test diets.

Duration of treatment / exposure:

52 weeks

Frequency of treatment:

continuously via the diet

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

30 ppm

Remarks:

corresponding to approx. 0.84 and 0.9 mg/kg bw/day for male and female dogs, respectively.

Doses / concentrations 2

Dose / conc.:

90 ppm

Remarks:

corresponding to an approx. 2.61 and 2.54 mg/kg bw/day for male and female dogs, respectively.

Doses / concentrations 3

Dose / conc.:

250 ppm

Remarks:

corresponding to approx. 7.35 and 7.23 mg/kg bw/day for male and female dogs, respectively.

No. of animals per sex per dose:

6

Control animals:

yes, plain diet

Details on study design:

- Fasting period before blood sampling for clinical biochemistry: yes, overnight (except for Week -2)

Positive control:

No

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed at least twice daily for mortality, morbidity and clinical signs.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded weekly before initiation of treatment, on the first day of treatment, weekly from Week 1 until Week 16 of treatment, and every 4 weeks thereafter. In addition, the body weight of each animal was recorded on the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No
Food consumption data were recorded daily and reported weekly beginning 1 week before initiation of treatment, for Weeks 1 through 16 and once every four weeks thereafter.

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before initiation of treatment and during Week 51 of treatment
- Dose groups that were examined: The pupils were dilated with 1% Mydriacyl, then the eyes were examined with an indirect ophthalmoscope.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week -2, 13, 26 and 52
- Anaesthetic used for blood collection: No
- Animals fasted: Yes, overnight (except for Week -2)
- How many animals: all animals of all dose groups including controls (for Week 26 and 52: all surviving animals)
- Parameters checked: erythrocyte count (RBC), haemoglobin (Hb), haematocrit (Hc), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), mean corpuscular volume (MCV), white blood cell count, differential white blood cell count (including nucleated red blood cell count, corrected white blood cell count, segmented neutrophil count, band neutrophil count, lymphocyte count, monocyte count, eosinophil count, basophil count), platelet count, prothrombin time (PT), reticulocyte count, cell morphology

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week -2, 13, 26 and 52
- Anaesthetic used for blood collection: No
- Animals fasted: Yes, overnight (except for Week -2)
- How many animals: all animals of all dose groups including controls (for Week 26 and 52: all surviving animals)
- Parameters checked: glucose, blood urea nitrogen, creatinine, total protein, albumin, globulin, total bilirubin, cholesterol, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, creatine kinase, calcium, inorganic phosphorus, sodium, potassium, and chloride.

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: Yes
- Time schedule for collection of urine: Week -2, 13, 26 and 52 before blood sampling
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes (food, water was provided ad libitum)
- Parameters checked: Appearance, volume, specific gravity, pH, protein, glucose, ketones, bilirubin, urobilinogen, and occult blood were determined on urine samples. The sediment was examined using a microscope.

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All animals, including two animals killed in extremis during the treatment period and those surviving after 52 weeks of treatment were subjected to a complete necropsy. Animals were sacrificed, after an overnight fasting period, by exsanguination under pentobarbital sodium anaesthesia and subjected to macroscopic examination. Macroscopic examination included: examination of the external surface of the body, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the nasal cavity and paranasal sinuses, and the thoracic, abdominal, and pelvic cavities and viscera.

Organs weighed (paired organs were weighed separately): brain, kidneys, liver, ovaries, testes, and thyroids with parathyroids. Organ-to-body weight percentages and organ-to-brain weight ratios were calculated.

HISTOPATHOLOGY: Yes
- Tissues collected: adrenals, aorta, brain, caecum, cervix, colon, duodenum, epididymides, oesophagus, eyes, femur and bone marrow (articular surface of the distal end), gallbladder, heart, ileum, jejunum, kidneys, all gross lesions, liver, lungs, lymph node (mandibular and mesenteric), mammary gland (females only), muscle (thigh), ovaries, pancreas, pituitary, prostate, rectum, salivary gland (submandibular), sciatic nerve, skin, spinal cord (cervical, mid-thoracic and lumber), spleen, sternum and bone marrow, stomach, testes, thymus, thyroid with parathyroid, trachea, urinary bladder, uterus, and vagina.
- Fixative: 10% phosphate-buffered formalin (except eyes: Zenker’s solution)
- Embedding media: Paraffin
- Staining: haematoxylin and eosin
- Animals examined: All animals of all dose groups, including controls

Optional endpoint(s):

Bone marrow smears from the sternum were prepared and retained for possible examination.

Statistics:

Levene’s test was performed to test for variance homogeneity. In the case of heterogeneity of variance at p < 0.05, transformations were used to stabilise the variance.
Analysis of variance (ANOVA) was performed on the homogeneous or transformed data. If significant; Dunnett’s t-test, Student’s t-test, or Games and Howell Modified Tukey-Kramer test was used for pairwise comparisons between groups.
One-way ANOVA was used to analyse initial body weights, food consumption, clinical chemistry and haematology values (except red blood cell morphology), urine (pH, volume, and specific gravity), organ weight, organ-to-body weight percentages, and organ-to-brain weight ratios.
One-way analysis of covariance (ANCOVA) was used to analyse body weight, with initial body weight as a covariate. If the ANCOVA was significant, Dunnett’s t-test, Student’s t-test, or Games and Howell Modified Tukey-Kramer test was used for pairwise comparisons between groups.
Group comparisons were evaluated at the 5% two-tailed probability level.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

There were no test material related observations that were considered abnormal. No effect on the neurological system was observed.

Summarized results can be found in Attachment 1 in the attached background material.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Two animals were sacrificed moribund during the course of the study, but their conditions did not appear to be test material related. One female treated with 250 ppm was observed in clonic seizures with opisthotonos before sacrifice during week 26. This death was attributed pathologically to hydrocephalus. One female treated with 90 ppm was observed in clonic convulsions with pulmonary congestion before sacrifice during week 25. This death was attributed pathologically to meningoencephalitis.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No statistically significant differences in body weights were observed in males and females at any of the applied doses. At Week 52, mean body weights of the male animals given 30, 90 and 250 ppm were 93%, 105% and 87%, respectively, of the control group mean. Mean body weights of the female animals given 30, 90 and 250 ppm were 108%, 94% and 98%, respectively, of the control group mean.

Summarized results can be found in Attachment 1 in the attached background material.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No statistically significant effects on male food consumption were observed. Food consumption for females given 250 ppm diet was significantly lower than those of the controls during Week 20, but as this was a single occurrence, it was not considered adverse or treatment-related. There were no consistent effects for males or females throughout the study.

Summarized results can be found in Attachment 1 in the attached background material.

Food efficiency:

not examined

Description (incidence and severity):

not applicable

Water consumption and compound intake (if drinking water study):

not examined

Description (incidence and severity):

not applicable

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No ophthalmic lesions were observed for any animal.

Summarized results can be found in Attachment 1 in the attached background material.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

- 30 ppm: No adverse effect was observed. MCV was slightly increased in females, but was not considered adverse.
- 90 ppm: increased MCV (males, females), increased MCH (males, females). All changes noted were statistically significantly different compared to the control group.
- 250 ppm: decreased RBC (males, Week 13, 26 and 52, up to approx. -11.4%, females, Week 13, 26 and 52, up to approx. -9.7%), increased MCV (males, up to approx. +9.2%, females, up to approx. +14.5%), increased MCH (males, up to approx. +10.1%, females up to approx. 14.9%). All changes noted were statistically significantly different compared to the control group. However, the data on increased MCV and MCH were considered inconclusive by the study director because of similar differences present in the study animals before initiation of treatment.

Other statistically significant differences were considered to represent normal biological variation and were not test material related.

Summarized results can be found in Attachment 1 in the attached background material.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- 30 ppm: No treatment-related differences were observed between the control and treatment group.
- 90 ppm: decreased total protein (males), increased cholesterol (males)
- 250 ppm: decreased total protein (males, up to approx. -11.9%), increased cholesterol (males, up to approx. +55.9%, females, up to approx. +95.5%), decreased albumin (males, up to approx. -15%, females up to approx. -14.3%). These changes were considered to be treatment-related.

Summarized results can be found in Attachment 1 in the attached background material.

Endocrine findings:

not specified

Description (incidence and severity):

The following ED-related parameters were investigated in the study: histopathology of cervix, epididymis, mammary gland, ovary, prostate, testis, thyroid, uterus, vagina and adrenals, organ weights were recorded for liver, prostate, testis and brain. For details, please refer to the respective result fields and the endpoint summary.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Urinalysis revealed no significant differences after treatment with the test substance.

Summarized results can be found in Attachment 1 in the attached background material.

Behaviour (functional findings):

not examined

Description (incidence and severity):

not applicable

Immunological findings:

not examined

Description (incidence and severity):

not applicable

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Administration of the test substance was associated with few changes in organ weight. Statistically
significant increases were:
- 30 ppm: statistically significantly increased relative liver weight (males, +17.5%) compared to the control group
- 90 ppm: statistically significantly increased absolute (+26.6%) and relative (+19.9%) liver weight (males) compared to the control group
- 250 ppm: statistically significantly increased absolute (males +29.4%) and relative (males +44.5%, females +28.1%) liver weight compared to the control group

The increase in of ≥20% liver weight (at 90 and 250 ppm) is considered adverse, irrespective of histopathological findings which were also found in control animals and indicate no adverse effect.The significant increase in liver-to-body weight in male animals given 30 ppm was not considered test material related. Additionally, a statistically significant increase in male liver-to-brain weight was observed after treatment with 250 ppm.

Summarized results can be found in Attachment 1 in the attached background material.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related macroscopic changes found at necropsy.

Summarized results can be found in Attachment 1 in the attached background material.

Neuropathological findings:

not examined

Description (incidence and severity):

not applicable

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There was a variable incidence across all treatment groups (including controls) of extramedullary haematopoiesis (EMH) in the liver, characterised by multifocal accumulations of mixed haematopoietic cells with the sinusoids or around vascular channels. Some higher severity scores for EMH were observed for a few animals. The severity scores for hepatic EMH were minimal in the control animals and most of the animals that received the test material. 1/6 males given 30 ppm and 1/6 males given 250 ppm received a moderate EMH score. 1/6 males given 90 ppm and 2/6 females given 250 ppm received a slight EMH score.
Scattered microgranulomas were observed in liver, but these were similar to the observations made in control animals. 1/6 females given 90 ppm showed some focal hepatic necrosis with minimal severity and the incidence was too low to be considered conclusively treatment related.
All other microscopic findings were considered incidental and unrelated to test material administration.
The conditions of the two animals sacrificed moribund did not appear to be related to the test material.

Summarized results can be found in Attachment 1 in the attached background material.

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

not applicable

Other effects:

not examined

Description (incidence and severity):

not applicable

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

30 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No adverse effects were observed at this dose level.

Remarks on result:

other: corresponding to an actual ingested dose of 0.84 mg/kg bw/day

2

Key result

Dose descriptor:

NOAEL

Effect level:

90 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No adverse effects were observed at this dose level.

Remarks on result:

other: corresponding to approx. 2.54 mg/kg bw/day.

3

Key result

Dose descriptor:

LOAEL

Effect level:

90 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical biochemistry

haematology

organ weights and organ / body weight ratios

Remarks on result:

other: corresponding to approx. 2.61 mg/kg bw/day. The effects on haematology and clinical chemistry were not clearly attributable to treatment, but are included here as a worst-case scenario. At higher doses, histopathological changes were observed.

4

Key result

Dose descriptor:

LOAEL

Effect level:

250 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical biochemistry

haematology

organ weights and organ / body weight ratios

Remarks on result:

other: corresponding to an actual ingested dose of 7.23 mg/kg bw/day.

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

90 ppm

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

The present study was conducted to assess the toxicity of test substance on dogs when given for a time frame of approximately 52 weeks. The study was similar to the OECD guideline 452 and was performed under GLP conditions. The test substance was administered via dietary exposure to groups of 6 male and 6 female dogs at dose levels of 30, 90 and 250 ppm corresponding to
approximately 0.84, 2.61 and 7.35 mg/kg bw/day for males and 0.9, 2.54 and 7.23 mg/kg bw/day for
females. Under the conditions of the test, the test substance caused changes in haematology, clinical biochemistry parameters, organs weights and histopathology at 90 and/or 250 ppm in males and at 250 ppm in females. Therefore, the NOAEL was set at 30 ppm for males (0.84 mg/kg bw/day) and at 90 ppm for females (2.54 mg/kg bw/day).