Thiram

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 Oct 1990 - 29 Jan 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 484 (Genetic Toxicology: Mouse Spot Test)

Version / remarks:

adopted in 1986

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.24 (Mouse Spot Test)

Deviations:

yes

Remarks:

consecutively to negative results, statsitical analysis was not performed

GLP compliance:

yes

Type of assay:

mouse spot test

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:

thiram

Test animals

Species:

mouse

Strain:

other: NMRI (female), DBA/2 (male)

Details on species / strain selection:

The mouse is the most suitable species with strains expressing coat colour mutations which are well characterized with regard to the genetic background.
In addition, the mouse is an experimental animal in many physiological , pharmacological, and toxicological studies. Data from such experiments also may be useful for the design and the performance of the mouse spot test.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Laboratory for Mutagenicity Testing, Darmstadt, Germany (females), BRL- Tierfarm Füllinsdorf, Germany (males)
- Age at study initiation: minimum 10 weeks
- Weight at study initiation: approx. 30 g
- Housing: individually after treatment in Makrolon Type II cages with wire mesh top
- Diet: pelleted standard diet (ALTROMIN, Lage/Lippe, Germany)
- Water: tap water, ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24
- Humidity (%): 20 -80
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 4 Oct 1990 To: 29 Jan 1991

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose); 1%
- Justification for choice of solvent/vehicle: toxicity profile in animals

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The dose was adjusted to the most recent body weight.

Duration of treatment / exposure:

single oral dose at Day 9 of pregnancy

Frequency of treatment:

single oral dose

Post exposure period:

3 weeks. Offspring was examined between day 39-43 after birth. For a detailed study schedule, please refer to "Any other information on materials and methods incl. tables".

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

75 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

750 mg/kg bw/day

No. of animals per sex per dose:

Treated animals were:
Solvent control: 21 pregnant females
Positive control: 30 pregnant females
75 mg/kg bw: 29 pregnant females
750 mg/kg bw: 79 pregnant females

Control animals:

yes, concurrent vehicle

Positive control(s):

Ethylnitrosurea (ENU)
- Route of administration: orally, once
- Doses / concentrations: 20 mg/kg bw
- Volume: 10 mL/kg bw
- Solvent: physiological saline, pH 4.5
- The solution was prepared on the day of administration.

Examinations

Tissues and cell types examined:

The offspring were coded and scored for the occurrence of pigmented spots.
Three classes of spots were distinguished:

1. WMVS (white mid-ventral spots);
White spots within 5 mm of the mid-ventral line which are presumed to result from cell killing.

2. MDS (misdifferentiation spots):
Yellow, agouti-like spots associated with mammaet genital ia, throat, axillary and inguinal areas and mid-forehead, which are presumed to result from misdifferentiation.

3. RS (recessive spots) :
pigmented and white spots randomly distributed on the coat which are presumed to result from somatic mutation.

All three classes of spots were scored but only the last, RS, is of genetic relevance. Microscopic analysis of mounted coloured hairs was performed only if there were doubts in classification of a spot.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: A pre-experiment with 750 mg/kg bw was conducted.

Evaluation criteria:

The test article is classified as mutagenic if it induces a statistically significant dose-related increase in the frequency of genetically relevant spots or a statistically significant positive response for at least one of the test points compared with the negative control.

The test article is classified as non-mutagenic if it induces no statistically significant dose-related increase in the frequency of genetical ly relevant spots and no statistically significant positive response for at least one of the test points compared with the negative control.

Both biological and statistical significance should be considered together in the evaluation.

Statistics:

A statistical analysis of the results was not necessary to perform as after treatment with the test article the frequency of recessive spots which are presumed to result from somatic mutation were in the range of the negative control value.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

750 mg/kg bw = MTD

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 750 mg/kg bw
- Results: A pre-experiment was realised in order to evaluate the toxicity of the test susbtance. After a single oral dose of 750 mg/kg bw, the treated animals expressed toxic reactions (reduction of spontaneous activity, ptosis, apathy); this dose was estimated to be the MTD.

RESULTS OF DEFINITIVE STUDY
In the mutation assay the mean litter size was not reduced after treatment with the test article indicating no embryotoxic effects. Experimental conditions as well as criteria for the determination of test responses are well defined and appropriate. Controls gave the expected response. In the vehicle test group one recessive spot (RS) was observed. After treatment with 75 or 750 mg/kg bw thiram, the frequency of RS was 0.75 and 0.62%, respectively, which are in the range of the historical control data (0.00%- 1.00%). The positive control showed a distinct increase in induced RS frequency.

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

Applicant's summary and conclusion

Conclusions:

The study was conducted according to the OECD guideline 484 and under GLP conditions. During the mutagenicity assay described and under the experimental conditions reported, the test article did not induce mutations in somatic cells of the mouse.