4,6-bis(octylthiomethyl)-o-cresol

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

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

(only 1000 erythrocytes/animal were counted)

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

4,6-bis(octylthiomethyl)-o-cresol

EC Number:

402-860-6

EC Name:

4,6-bis(octylthiomethyl)-o-cresol

Cas Number:

110553-27-0

Molecular formula:

C25 H44 O S2

IUPAC Name:

2-methyl-4,6-bis[(octylsulfanyl)methyl]phenol

Details on test material:

- Storage: room temperature
- Physical state: liquid

Test animals

Species:

hamster, Chinese

Strain:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Ciba Geigy Tierfarm, Sisseln, Switzerland
- Age at study initiation: females 6- 10 weeks, males 4-9 weeks
- Weight at study initiation: females 22-32 g, males 22-34 g
- Diet: ad libitum; Standard diet: NAFAG No.924
- Water: ad libitum
- Acclimation period: 4-5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-21
- Humidity (%): 60-64
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: Arachis oil
- Source: Siegfried AG, Switzerland
- Justification for choice of solvent/vehicle: not specified
- Concentration of test material in vehicle:
- Amount of vehicle: 10 ml/kg bw
- Purity: no data

Details on exposure:

Application volume: 10 ml/kg bw

Duration of treatment / exposure:

single dose application

Frequency of treatment:

once

Post exposure period:

sacrifice was performed 16, 24 and 48 hours after gavage

Doses / concentrations

Dose / conc.:

5 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

8/sex/dose/sampling time

Control animals:

yes, concurrent vehicle

Positive control(s):

- Name: Cyclophosphamide (CPA)
- Source: ASTA-Werke, Germany
- Justification for choice of positive control(s): guideline recommended
- Number of animals: 8/sex/dose/sampling time
- Route of administration: gavage
- Vehicle: Arachis oil
- Application volume: 10 ml/kg bw
- Doses: 64 mg/kg bw
- Time for sacrifice: 24 hours after dosing

Examinations

Tissues and cell types examined:

Bone marrow; erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: the dose of 5000 mg/kg was determined as the highest applicable in the mutagenicity assay

DETAILS OF SLIDE PREPARATION: Bone marrow is harvested from the shafts of both femurs with fetal calf serum. After centrifugation small drops of the sediment mixture are transferred on the end of a slide, spread out with the aid of a polished cover glass and the preparations are air-dried. Within 24 hours, the slides are stained in undiluted May-Grünwald solution for 3 min then in May-Grünwald solution/water 1/1 for 2 min. After being rinsed in distilled water, the slides are left immersed in diluted Giemsa solution (16.6%), for 10 min. After rinsing with distilled water and air-drying, the slides are cleared in Xylene and mounted.

METHOD OF ANALYSIS: Prior to analysis the slides are coded. Thereafter the quality of staining is evaluated. The slides of five animals from each sex showing the best differentiation between mature and polychromatic erythrocytes are selected for later scoring. The slides of five female and five male animals each of the negative control group and of the dosage group sacrificed at 16, 24 and 48 hours post treatment are examined. From the animals of the positive control group which are sacrificed 24 hours after application, the slides of five female and five male animals are scored. 1000 polychromatic erythrocytes per animal each are scored for the incidence of micronuclei. The ratio of polychromatic to normochromatic erythrocytes is determined for each animal by counting a total of 1000 erythrocytes.

Statistics:

The significance of difference is assessed by X^2-test

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose: 200, 1000, 5000 mg/kg bw
- Number of animals/sex/dose: 2 per sex per dose
- Frequency of application: once
- Rationale for exposure: Determine the highest dosage of the test substance to be applied in the mutagenicity assay
- Harvest times: 16, 24, 48 and 72 hours after gavage

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): There was no significant increase in the number of micronucleated polychromatic erythrocytes in the animals treated with the dose of 5000 mg/kg as compared with the negative control animals.
By contrast, the positive control (cyclophosphamide, 64 mg/kg) yielded a marked increase of the percentage of micronucleated cells. Here the mean percentage of polychromatic erythrocytes with micronuclei was 1.73. In comparison with the vehicle control (0.16) this value is highly significant (p <0.05).
- Percent of polychromatic erythrocytes with micronuclei:
16 h: 0.20 for males (control: 0.20); 0.14 for females (control: 0.10)
24 h: 0.26 for males (control: 0.18); 0.14 for females (control: 0.14)
48 h: 0.30 for males (control: 0.18); 0.04 for females (control: 0.06)
pos. control, 24 h: 2.32 for males (control: 0.18); 1.14 for females (control: 0.14)
- Ratio of PCE/NCE (for Micronucleus assay):
16 h: 1.0 for males; 0.9 for females
24 h: 1.1 for males; 0.9 for females
48 h: 1.1 for males; 1.0 for females
pos. control, 24 h: 1.1 for males; 0.8 for females

Any other information on results incl. tables

SUMMARY OF EXPERIMENTAL RESULTS

Sacrifice	Treatment	Sex	polychromatic erythrocytes (average)	normochromatic erythrocytes (average)	ratio of p / n erythrocytes	number of polychromatic erythrocytes with micronuclei (average)	% of polychromatic erythrocytes with micronuclei (average)
16 h	Control	female	468	532	0.9	2.00	0.20
		male	453	547	0.8	1.00	0.10
	5000 mg/kg	female	492	508	1.0	2.00	0.20
		male	473	527	0.9	1.40	0.14
24 h	Control	female	484	516	0.9	1.80	0.18
		male	434	566	0.8	1.40	0.14
	5000 mg/kg	female	520	480	1.1	2.60	0.26
		male	460	540	0.9	1.40	0.14
48 h	Control	female	516	484	1.1	1.80	0.18
		male	493	507	1.0	0.60	0.06
	5000 mg/kg	female	526	474	1.1	3.00	0.30
		male	490	510	1.0	0.40	0.04
Positive Control
48 h	Control	female	484	516	0.9	1.80	0.18
		male	434	566	0.8	1.40	0.14
	64 mg/kg	female	514	486	1.1	23.20	2.32
		male	442	558	0.8	11.40	1.14

Applicant's summary and conclusion

Conclusions:

Under the conditions of this experiment, no evidence of mutagenic effects was obtained in Chinese hamsters treated with the test item.

Executive summary:

An in vivo micronucleus assay was performed to evaluate any mutagenic effect on polychromatic erythrocytes in bone marrow cells in vivo. Mutagenic effects present themselves in form of micronuclei in polychromatic erythrocytes in the bone marrow. These micronuclei are small particles consisting of acentric fragments of chromosomes or entire chromosomes which lag behind at anaphase stage during the mitotic process. After telophase, these fragments may not be included in the nuclei of daughter cells and form single or multiple micronuclei in the cytoplasm. The increase in micronucleated polychromatic erythrocytes shows a clear dose dependency, comparable to the occurrence of chromosome aberrations in metaphase preparations. In this experiment the animals were treated once with the highest applicable dose of 5000 mg/kg and sacrificed 16, 24 and 48 hours thereafter. The test article was administered by gavage. From the bone marrow smears were made.

The bone marrow smears from the animals treated with the dose of 5000 mg/kg of the test item showed no statistically significant increase (p >0.05) in the number of micronucleated polychromatic erythrocytes in comparison with the negative control animals at all three sampling times. The respective "positive control" experiments with cyclophosphamide (64 mg/kg) yielded an average of 1.73% polychromatic erythrocytes with micronuclei. This is significantly different from the controls (0.16%) treated with the vehicle (arachis oil) alone. It is concluded that under the conditions of this experiment, no evidence of mutagenic effects was obtained in Chinese hamsters treated with the test item.