3,3',5,5'-tetra-tert-butylbiphenyl-2,2'-diol

Genetic toxicity: in vivo

Administrative data

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (GLP)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

May 26, 1983

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

3,3',5,5'-tetra-tert-butylbiphenyl-2,2'-diol

EC Number:

407-920-5

EC Name:

3,3',5,5'-tetra-tert-butylbiphenyl-2,2'-diol

Cas Number:

6390-69-8

Molecular formula:

C28 H42 O2

IUPAC Name:

3,3',5,5'-tetra-tert-butyl-[1,1'-biphenyl]-2,2'-diol

Details on test material:

- Name of test material (as cited in study report): generic name CA 16-178
- Analytical purity: 97.7% (not specified in the study [Lab. sample], transfered from 28d study [BASF SE [Ciba-Geigy Ltd/884619]], with same Lot/batch)
- Lot/batch No.: A 88103 / Z 1572R
- Storage condition of test material: room temperature
- Stability under test conditions: ensured by sponsor
- Other: gloves and face masks as safety precautions

Test animals

Species:

hamster

Strain:

other:

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: chinese hamster (Cricetulus griseus) random outbred strain from Ciba-Geigy Tierfarm, Sisseln, Switzerland
- Weight at study initiation: females 25-32 g, males 28-35 g (tolerability test); females 24-35 g, males 25-35 g (mutagenicity test)
- Assigned to test groups randomly: yes
- Housing: individual caging
- Diet (e.g. ad libitum): standard diet: NAFAG No.924; ad libitum
- Water (e.g. ad libitum): tap water; ad libitum
- Acclimation period: at least 3 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-24°C (1°C above the range described in the protocol)
- Humidity (%): 44-49%
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: CMC (carboxymethyl cellulose); 0.5% aqueous solution

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The highest applicable dose was 5000 mg/kg bw (solubility limit)

Duration of treatment / exposure:

one single treatment

Frequency of treatment:

once

Post exposure period:

sampling was performed 16, 24 or 48 hours after treatment

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
5000 mg/kg bw
Basis:
actual ingested
in the mutagenicity main test

Doses / concentrations 2

Remarks:

Doses / Concentrations:
200, 1000 and 5000 mg/kg bw
Basis:
actual ingested
in the tolerability pretest

No. of animals per sex per dose:

2 (pretest) or 8 (per group and per sampling time; main test)

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: oral
- Doses / concentrations: 64 mg/kg in 20 ml/kg CMC 0.5%, sacrificed after 24 hours

Examinations

Tissues and cell types examined:

Bone marrow / bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
After pre-test treatment for determination of tolerability of the test substance, the treated animals were observed for a period corresponding to the interval between administration and sacrifice of the animals in the main test, plus one day. Depending on the outcome the highest dose causing no death is used as the highest in the main test, or if necessary the test is repeated with lower doses. In this experiment, the dose of 5000 mg/kg was determined as the highest applicable in the main assay.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
16, 24 or 48 hours after treatment, the animals were sacrificed by dislocation of the cervical vertebrae.

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-Gruenwald solution for 3 min then in May-Gruenwald 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:
The slides were coded prior to analysis and the quality of staining evaluated. 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 per dose group (negative control group, dosage group and positive control group) were examined at scheduled sampling time.

1000 polychromatic erythrocytes per animal each are scored for the incidence of micronuclei. To determine the mitotic activity of the red compartment, the ratio of polychromatic to normochromatic erythrocytes is calculated for each animal by counting a total of 1000 erythrocytes. A low proportion of polychromatic erythrocytes is indicative for a mitose inhibiting activity of the test substance.

Evaluation criteria:

The significance of difference is assessed by X2-test.

Statistics:

- A test substance is considered to be active in this test system if a statistically significant increase in the number of polychromatic erythrocytes with micronuclei in comparison with the negative control occurs at any sampling time.
- Assay acceptance criteria:
(1) the quality of the slides must allow a clear differentiation between polychromatic and normochromatic erythrocytes;
(2) the result obtained with the positive control has to fulfill the criteria given for a positive response;
(3) both the negative and the positive control are within range of the available historical negative and positive control data (mean control data from the studies performed within one year available).

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Remarks:

no significant increase in the number of micronucleated polychromatic erythrocytes compared to the vehicle treated animals

Toxicity:

no effects

Remarks:

but evaluated above the limit dose (at 5000 mg/kg bw)

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
In the tolerability test the highest applicable dose of 5000 mg/kg (as well as the mid and low doses) caused no death in a group of four animals. Therefore, this dose was taken as the highest in the mutagenicity test.

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 of test substance as compared with the negative control animals at all three sampling times. By contrast, the positive control yielded a marked increase of the percentage of micronucleated cells. Here the mean percentage of polychromatic erythrocytes with micronuclei was 1.82. In comparison with the negative control (0.03) this value was highly significant (p<0.05).
- Ratio of PCE/NCE (for Micronucleus assay): no significant changes compared to negative control values

Any other information on results incl. tables

Table 1: Summary of number of polychromatic erythrocytes with micronuclei and ratio of polychromatic to normochromatic (PCE/NCE) erythrocytes (arithmetic mean per sex and group).

Sampling time (hours)	Sex	Number of polychromatic erythrocytes	Number of Normochromatic erythrocytes	Ratio of PCE/NCE	Number of polychromatic erythrocytes with micronuclei	% of poly-chromatic erythrocytes with micronuclei
Negative control (0.5% CMC)
16	Males	560	440	1.3	1.2	0.12
	Females	493	507	1	0.2	0.02
24	Males	455	545	0.8	0.4	0.04
	Females	447	553	0.8	0.2	0.02
48	Males	450	550	0.8	0.6	0.06
	Females	476	524	0.9	0.2	0.02
Test substance (5000 mg test substance/kg bw)
16	Males	539	461	1.2	0.2	0.02
	Females	533	467	1.1	0.2	0.02
24	Males	494	506	1	0.4	0.04
	Females	465	535	0.9	0	0
48	Males	519	481	1.1	0	0
	Females	530	470	1.1	0	0
Positive control (64 mg cyclophosphamide/kg bw)
24	Males	415	585	0.7	24	2.4
	Females	422	578	0.7	12.4	1.24

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative