Iso(C10-C14)alkyl (3,5-di-tert-butyl-4-hydroxyphenyl)methylthioacetate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information

The test substance (as described in section 1.2) was not mutagenic in the Ames test (with and without metabolic activation) and in an HPRT test (with and without metabolic activation). The test article did not induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow cells of chinese hamster.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1989-07-04 to 1989-02-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline study, GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)

Qualifier:

according to guideline

Guideline:

other: EEC (19 September 1984), Mutagenicity (Micronucleus Test). Official Journal of the European Communities No L 251 137-139.

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

hamster

Strain:

other: Cricetulus griseus

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CIBA-GEIGY Tierfarm, Sisseln
- Weight at study initiation: females 31-34 g, males 30-35 g (tolerability test), females 21-31 g, males 22-34 g (mutagenicity test).
- Assigned to test groups randomly: yes
- Housing: Individually
- Diet: ad libitum (NAFAG No. 924)
- Water: tap water, ad libitum
- Acclimation period: At least 3 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15 - 23
- Humidity (%): 50 - 84
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: arachis oil

Details on exposure:

The preparation was administered orally to groups of 24 female and 24 male animals each in the negative and in the 5000 mg/kg bw dose group. The positive control group consisted of 8 female and 8 male animals. Treatment consisted of a single application. 16, 24 and 48 hours after application 8 female and 8 male animals per sampling time were sacrificed by dislocation of the cervical vertebrae.

Duration of treatment / exposure:

Single exposure

Frequency of treatment:

Once

Post exposure period:

16, 24, 48 hrs

Remarks:

Doses / Concentrations:
5000 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

In the tolerability test: 2 females and 2 males
In the mutagenicity test: 24 females and 24 males in the treatment groups and in the negative control groups. 8 females and 8 males in the positive control group

Control animals:

yes, concurrent vehicle

Positive control(s):

yes (CPA; Cyclophosphamide)

Tissues and cell types examined:

Bone marrow was harvested from the shafts of both femurs with fetal calf serum. After centrifugation small drops of the sediment mixture were transferred on the end of a slide, spread out with the aid of a polished cover glass and air-dried. Within 24 hours, the slides were 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 were left immersed in diluted Giemsa solution (16.6%), for 10 min. After rinsing with distilled water and air-drying, the slides were cleared in Xylene and mounted.

Details of tissue and slide preparation:

The slides of five animals from each sex showing the best differentiation between mature and polychromatic erythrocytes were 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 were examined. From the animals of the positive control group which were sacrificed 24 hours after application, the slides of five female and five male animals were scored. 1000 polychromatic erythrocytes per animal each were scored for the incidence of micronuclei. To determine the mitotic activity of the red compartment, the ratio of polychromatic to normochromatic erythrocytes was calculated for each animal by counting a total of 1000 erythrocytes. A low proportion of polychromatic erythrocytes is indicative for a mitosis inhibiting activity of the test substance.

Evaluation criteria:

- The quality of the slides must allow a clear differentiation between polychromatic and normochromatic erythrocytes.
- The result obtained with the positive control has to fulfill the criteria given for a positive response.

Statistics:

The significance of difference was assessed by X2-test.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
Three groups of four Chinese hamsters (two females and two males) are treated with three different doses, one receiving the maximum dose of 5000 mg/kg, or the highest applicable dose, and the other two doses of 1/5 and 1/25 of that amount respectively. The animals are treated with a single dose. The observation period corresponds to the interval between administration and sacrifice of the animals in the mutagenicity test, plus one day. In this experiment the dose of 5000 mg/kg was determined as the highest applicable in the mutagenicity assay

POSITIVE CONTROL
The positive control (cyclophosphamide, 64 mg/kg, sampling time 24 hours) yielded a marked increase of the percentage of micronucleated cells. Here the mean percentage of polychromatic erythrocytes with micronuclei was 1.00. In comparison with the negative control (0.08) this value is highly significant (p <0.05), confirming an appropriate sensitivity of the test system.

Experimental Result

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	510	490	1	0.6	0.06
		male	603	397	1.5	0	0
	5000 mg/kg	female	548	452	1.2	0.6	0.06
		male	632	368	1.7	0.8	0.08
24 h	Control	female	512	488	1	1	0.1
		male	573	427	1.3	0.6	0.06
	5000 mg/kg	female	544	456	1.2	0.4	0.04
		male	657	343	1.9	0.4	0.04
48 h	Control	female	581	419	1.4	0.6	0.06
		male	648	352	1.8	0.2	0.02
	5000 mg/kg	female	485	515	0.9	0.8	0.08
		male	605	395	1.5	0.4	0.04
Positive Control
48 h	Control	female	512	488	1	1	0.1
		male	573	427	1.3	0.6	0.06
	64 mg/kg	female	523	477	1.1	12.6	1.26
		male	581	419	1.4	7.4	0.74

Conclusions:

Interpretation of results (migrated information): negative

Executive summary:

The test item was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow cells of the Chinese hamster. The test item was administered orally to groups of 24 female and 24 male animals at 0 (vehicle control) or 5000 mg/kg bw. 16, 24 and 48 hours after application 8 females and 8 males per sampling time were sacrificed and bone-marrow smears were prepared. There was no significant increase in the number of micronucleated polychromatic erythrocytes in the animals treated with the dose of 5000 mg/kg bw of the test item as compared with the negative control animals at all three sampling times. The positive control group (cyclophosphamide) consisted of 8 female and 8 male animals and showed the appropriate response, thus confirming sensitivity of the test system. In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the Chinese hamster.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

In vitro

Ames test:

A Salmonella typhimurium/Escherichia coli reverse mutation assay was carried out according to OECD Guideline 471 and in compliance with GLP principles (CIBA-GEIGY, 1988). In the presence and absence of rat liver S-9-microsomal activation system and at concentrations of 5-5000 μg per plate, S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 as well as E. coli strain WP2uvrA were tested for the induction of base-pair substitutions or frameshift mutations. No evidence of a mutagenic potential was observed. No cytotoxicity was noted up to the highest dose tested. The positive controls induced the appropriate responses. Based on the results of these experiments and on standard evaluation criteria, it is concluded that the test article and its metabolites did not induce gene mutations in the strains of S. typhimurium and E. coli used.

HPRT Test:

In a GLP-compliant genotoxicity study according to OECD guideline 476 the test item was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster (Harlan, 2013). The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The concentration range of the main experiments was limited by phase separation of the test item. The test item was dissolved in DMSO. The tested concentrations in the main experiment ranged from 1.9 to 150 µg/ml. Phase separation occurred in both main experiments at 50.0 µg/mL and above with and without metabolic activation. No relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% in both parallel cultures occurred in experiment I and II up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment. No relevant and reproducible increase in mutant colony numbers/10⁶cells was observed in the main experiments up to the maximum concentration. The mutation frequency did not exceed the historical range of solvent controls. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells and is therefore considered to be non-mutagenic in this HPRT assay.

In vivo

Micronucleus test:

In an in vivo micronucleus test (Ciba-Geigy, 1989) performed according to OECD test guideline 474 and in compliance to GLP, the test article was investigated for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow cells of Chinese hamsters. The test item was administered orally to groups of 24 female and 24 male animals at 5000 mg/kg body weight. The positive control group (cyclophosphamide) consisted of 8 female and 8 male animals. 16, 24 and 48 hours after application 8 females and 8 males per sampling time were sacrificed and bone-marrow smears were prepared. No statistically significant increase in the number of micronucleated polychromatic erythrocytes compared to the negative control animals were observed at all three sampling times. The respective "positive control" experiment with cyclophosphamide (64 mg/kg) yielded an average of 1.00% polychromatic erythrocytes with micronuclei. This is significantly different from the controls (0.08%) 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 and the test substance is therefore considered to be non clastogenic.

Justification for selection of genetic toxicity endpoint
GLP compliant in vivo study following OECD guidelines

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available experimental test data is reliable and suitable for the purpose of classification under Directive 67/548/EEC. Based on the present data, classification for genotoxicity is not warranted.

 

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for the purpose of classification under Regulation (EC) No.1272/2008. Based on the present data, classification for genotoxicity is not warranted.