2,4-di-tert-butylphenyl 3,5-di-tert-butyl-4-hydroxybenzoate

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:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1978

Report date:

1978

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Deviations:

no

GLP compliance:

no

Type of assay:

other: micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

2,4-di-tert-butylphenyl 3,5-di-tert-butyl-4-hydroxybenzoate

EC Number:

224-166-0

EC Name:

2,4-di-tert-butylphenyl 3,5-di-tert-butyl-4-hydroxybenzoate

Cas Number:

4221-80-1

Molecular formula:

C29H42O3

IUPAC Name:

2,4-di-tert-butylphenyl 3,5-di-tert-butyl-4-hydroxybenzoate

Test animals

Species:

hamster, Chinese

Strain:

other: Cricetulus griseus

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Housing: Kept in an air-conditioned room
- Diet (e.g. ad libitum): NAFAG No. 196
- Water: Tap water ad libitum.
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature: 23 °C +/- 1 °C
- Humidity: 55 % +/- 5 %
- Photoperiod: The room was illuminated for 12 hours

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: Sesame oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The preparation was administered orally to groups of 6 female and 6 male animals each.
- Test substance: 500, 1000 and 2000 mg/kg in 20 mL/kg sesame oil.
- Cyclophosphamide (ENDOXAN ): 128 mg/kg in 20 mL/kg sesame oil (positive control).
- 20 mL sesame oil/kg (negative control).

DIET PREPARATION
- Rate of preparation of diet (frequency): daily one application on 2 consecutive days

Duration of treatment / exposure:

48 hours

Frequency of treatment:

daily one application on 2 consecutive days

Post exposure period:

none

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

3

Control animals:

yes

Positive control(s):

- Cyclophosphomamide
- Route of administration: orally
- Doses / concentrations: 128 mg/kg

Examinations

Details of tissue and slide preparation:

TREATMENT AND SAMPLING TIMES:
Treatment consisted of daily one application on 2 consecutive days. 24 h after the second application the animals were sacrificed.

DETAILS OF SLIDE PREPARATION:
Bone marrow was harvested from the shafts of both femurs. In a siliconized pipette filled with 0.5 yl rat serum the bone marrow was drawn up. In order to receive a homogeneous suspension the content of pipette was aspirated gently about three times. Small drops of the mixture were transferred on the end of a slide, spread out by pulling it behind a polished cover glass and the preparations were air-dried. At the next day the slides were stained in undiluted May-Grunwald solution for 2 min then in May-Griinwald solution/ water 1/1 for 2 min and then in Giemsa's, 40% for 20 min. After being rinsed in methanol 55% for 5-8 sec. and washed off twice in water, they are left immersed in water for approx. 2 min. After rinsing with distilled water and airdrying the slides were cleared in Xylol and mounted in Eukitt.

METHOD OF ANALYSIS:
The slides of three female and three male animals per group were examined. 1000 bone marrow cells each were scored per animal and the following anomalies were registered: Single Jolly bodies, fragments of nuclei in erythrocytes, micronuclei in erythroblasts, micronuclei in leucopoietic cells, polyploid cells.

Statistics:

The significance of difference was assessed by X^2 test.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

In all dosage groups the percentage of cells displaying anomalies of nuclei did not differ significantly from the negative control. By contrast, the positive control (cyclophosphamide, 128 mg/kg) yielded a marked increase of -the percentage of cells with anomalies. Here the mean percentage of anomalies was 5.17, whereas the negative control yielded a percentage of 0.083. The difference is highly significant (p<0.05).

Any other information on results incl. tables

	Number of animals	Sex of animals	Single Jolly bodies	Fragments of nuclei in erythrocytes	Micronuclei in erythroblasts	Micronuclei in leucopoietic cells	Polyploid cells	Total
Control	1	f						0.0
(Sesame oil)	2	f	0.2					0.2
	3	f						0.0
	4	m	0.1					0.1
	5	m					0.1	0.1
	6	m					0.1	0.1
Cyclophosphamide	1	f	4.8	1.1	0.1	0.1	0.3	6.4
(128 mg/kg)	2	f	3.7	0.8	0.2		0.3	5.0
	3	f	2.9	0.4	0.3	0.1	0.2	3.9
	4	m	3.3	0.6	0.2	0.1	0.4	4.6
	5	m	1.8	0.1	0.1		1.7	3.7
	6	m	4.3	2.2	0.3	0.2	0.4	7.4
Test item	1	f	0.2					0.2
(500 mg/kg)	2	f					0.1	0.1
	3	f					0.1	0.1
	4	m						0.0
	5	m	0.1					0.1
Test item	6	m						0.0
(1000 mg/kg)	1	f					0.2	0.2
	2	f				0.1	0.1	0.2
	3	f	0.3				0.1	0.4
	4	m					0.1	0.1
	5	m						0.0
	6	m						0.0
Test item	1	f	0.3					0.3
(2000 mg/kg)	2	f	0.1					0.1
	3	f	0.1				0.1	0.2
	4	m					0.2	0.2
	5	m	0.1					0.1
	6	m						0.0

Applicant's summary and conclusion

Conclusions:

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