Disulfiram

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 Jul - 9 Nov 1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted in 2014

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

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:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratory, Kingston, NY, USA
- Age at study initiation: 6 - 8 weeks
- Weight at study initiation: 27.1 - 35.9 g (males), 23.8 - 30.7 g (females)
- Assigned to test groups randomly: yes
- Housing: up to 5 per cage in plastic autoclavable cages with wire lids
- Diet:certified laboratory rodent chow, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 26.67
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 20 Jul 1987 1987 To: 15 Oct 1987

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

CMC (carboxymethyl cellulose), 1%
- Justification for choice of solvent/vehicle: toxicity profile in animals
- Concentration of test material in vehicle: 3.8, 18.9 and 37.7 for the low, mid and high dose, respectively
- Volume administered: 10 mL/kg bw
- Lot/batch no.: 7-2-87

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The dosing solutions were prepared freshly and based on the most recently measured body weight (directly before injection).

Duration of treatment / exposure:

single dose

Frequency of treatment:

single dose

Post exposure period:

24, 48 and 72 h

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

38 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

189 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

377 mg/kg bw/day

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

triethylenemelamine
- Route of administration: IP injection
- Doses / concentrations: 0.25 mg/kg bw
- Vehicle: water

Examinations

Tissues and cell types examined:

Bone marrow from femur was prepared.

Details of tissue and slide preparation:

Toxicits test: A toxicity test was conducted with 6 groups of five male and five female rats each. Animals were observed after dose administration and daily thereafter for 7 days for clinical signs of chemical effect. Body weights were recorded prior to dose administration and 1 and 3 days after dose administration

TREATMENT AND SAMPLING TIMES: Animals were observed after dosing for clinical signs. At the scheduled time of sacrifice, 5 mice/sex were sacrificed by CO2 asphyxiation. Immediately following sacrifice, the femurs were exposed, cut just about the knee and the bone marrow was aspired into a syringe containing fetal bovine serum. The bone marrow cells were transferred to a capped centrifuge tube containing approximately 1 mL FBS. The bone marrow cells were pelleted by centrifugation.

DETAILS OF SLIDE PREPARATION: After centrifugation, the pellet was were resuspended by aspiration with a capillary pipet and a small drop of bone marrow suspension was spread onto a clean glass slide. Two to four slides were prepared from each animal. The slides were fixed in methanol stained with May-Gruenwald-Giemsa and permanently mounted.

METHOD OF ANALYSIS: Using medium magnification, an area of acceptable quality was selected such that the cells were well spread and stained. Using oil immersion, 1000 polychromatic erythrocytes were scored for the presence of micronuclei. The proportion of polychromatic erythrocytes to total erythrocytes and the number of micronucleated normocytes in the field of 1000 polychromatic erythrocytes were also enumerated.

Evaluation criteria:

The test article is considered to induce a positive response if a treatment—related increase in micronucleated polychromatic erythrocytes is observed relative to the vehicle control (P<0.05, Kastenbaum—Bowman Tables) . The positive response must be dose—dependent or must be observed at a single dose level at adjacent sacrifice times. If a single treatment group is significantly elevated at one sacrifice time, the assay is considered a suspect or unconfirmed positive and a repeat assay will be recommended.

The mean incidence of micronucleated polychromatic erythrocytes must not exceed 5/1000 polychromatic erythrocytes in the negative (vehicle) control. The incidence of micronucleated polychromatic erythrocytes in the positive control group must be statistically significantly increased relative to the negative control (P<0.05, Kastenbaum—Bowman Tables) .

Statistics:

The incidence of micronucleated polychromatic erythrocytes per 1000 polychromatic erythrocytes was presented for each animal and treatment group.

Statistical significance was determined using the Kastenbaum—Bowman tables which were based on the binomial distribution.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF TOXICITY STUDY
- Dose range: 200 - 1000 mg/kg bw
- Results: Deaths were observed in all test article—treated groups, with 1, 6, 9, 7 and 9 deaths occurring at dose groups of 200, 400, 600, 800 and 1000 mg/kg bw, respectively. Clinical observations for the above groups included lethargy, paralysis, and piloerection. The LD50 was calculated by probit analysis to be approximately 471 mg/kg bw. 377 mg/kg bw was estimated to be 80% of the LD50 and was selected as the high dose for the micronucleus test.

RESULTS OF DEFINITIVE STUDY
At the highest dose level, mortality (15/38 animals), clinical signs and bone marrow toxicity (reduced ratio PCE/Total E) were observed. These effects were also apparent in some animals in the mid-dose group. No increase in the number of micronucleated polychromatic erythrocytes was detected at any of the sampling times. Experimental conditions as well as the criteria for the determination of test responses are well defined and appropriate. Positive controls gave the expected response.

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

Applicant's summary and conclusion

Conclusions:

The study was conducted similar to OECD guideline 474 and under GLP conditions.

Under the conditions of the test, the test substance did not increase the incidence of micronucleated polychromatic erythrocytes in bone marrow and was concluded to be negative in the micronucleus test using male and female CD-1 mice.