N,N'-hexane-1,6-diylbis[3-(3,5-di-tert-butyl-4-hydroxyphenylpropionamide]

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

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:

1981

Report date:

1981

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OTS 798.5915 (In Vivo Sister Chromatid Exchange Assay)

Version / remarks:

(1998)

Deviations:

yes

Remarks:

(4 animals per sex and dose)

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OTS 798.5915 (In Vivo Sister Chromatid Exchange Assay)

Deviations:

yes

Remarks:

(4 animals per sex and dose)

Principles of method if other than guideline:

Procedure according to Perry et al. (1974), Goto et al. (1978), Allen et al. (1977), Perry et al. (1975), Marquardt et al. (1978)
PERRY, P. and S. WOLFF Nature 251 , 156-158 (1974).
GOTO, K., S. MAEDA, Y. KANO and T. SUGIYAMA Chromosoma (Berl.) 66, 351-359 (1978).
ALLEN, J.W., C F . SHULER, R.W. MENDES and S.A. LATT
Cytogenet. Cell Genet. 18, 231-237 (1977).
PERRY, P. and H.J. EVANS Nature 258, 121-125 (1975).
MARQUARDT, H. and U. BAYER Mutation Res. 56, 169-176 (1978).

GLP compliance:

no

Type of assay:

sister chromatid exchange assay

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-hexane-1,6-diylbis[3-(3,5-di-tert-butyl-4-hydroxyphenylpropionamide]

EC Number:

245-442-7

EC Name:

N,N'-hexane-1,6-diylbis[3-(3,5-di-tert-butyl-4-hydroxyphenylpropionamide]

Cas Number:

23128-74-7

Molecular formula:

C40H64N2O4

IUPAC Name:

N,N'-hexane-1,6-diylbis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propanamide]

Details on test material:

- Physical state: solid

Test animals

Species:

hamster, Chinese

Strain:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 20-28 g (females), 25-30 g (males)
- Diet (e.g. ad libitum): NAFAG No. 924
- Water (e.g. ad libitum): tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22- 24
- Humidity (%): 54-62
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 0.7% aqueous solution of CMC (carboxymethyl cellulose)
- Amount of vehicle (if gavage or dermal): 20 mL/kg bw

Details on exposure:

Two hrs before application of test or control substance each animal is applied with 5 -bromodeoxyuridine (BUdR) (45 mg tab as subcutaneous implantation in the neck (Fa. Heinrich Mack, D-Illertissen, Germany)). Twenty-four hours after administration of test substance and 2hrs after intraperitoneal injection with colcemide animals were sacrificed.

Duration of treatment / exposure:

24 hrs

Frequency of treatment:

single dose

Post exposure period:

24 hrs (test substance)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 500 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

3 000 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

6 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

4 (only chromatides of 2 animals per group and sex examined)

Control animals:

yes, concurrent vehicle

Positive control(s):

7,12-dimethylbenzanthracene
- Route of administration: oral, gavage
- Doses / concentrations: 100 mg/kg bw in 0.7% aqueous solution of sodium-carboxymethylcellulose (CMC)

Examinations

Tissues and cell types examined:

Bone marrow, 25 cells per animal in metaphase

Details of tissue and slide preparation:

Bone marrow from the shafts of both femurs was suspended in balanced salt solution and diluted to hypotonicity with distilled water, kept in a waterbath at 4 to 6°C for 23 min and then centrifuged for 10 min at 200 x g. The pellets were then fixed in methanol-acetic acid 3:1 for a period of 30 min, resuspended, centrifuged for 5 min at 150 x g, and stored in renewed fixative overnight at 4°C. Finally the pellets again were centrifuged for 5 min at 150 x g, and resuspended in some 0.5 mL fixative.
These specimens were pipetted onto wet slides and air-dried. The air-dried slides then were treated with a solution of bisbenzimide for 15 min, rinsed in McIlvaine-buffer pH 8.0 and irradiated in this buffer at 50°C with UV-light of 350 nm. Following the development of the fluorochrome-UV-light reaction in 60°C 2 x SSC (standard sodium citrate) for 90 min, the slides were stained in 40% Giemsa for 20-40 min, well rinsed, cleared in Xylol and mounted in Eukitt.
The slides of two female and two male animals each of the treatment groups and of the control groups were examined. Twenty-five differently stained metaphases of the second cell-cycle with 5-bromodeoxyuridine (BUdR)-substitution were analysed.

Evaluation criteria:

Analyze of Sister Chromatide Exchanges (SCE's) according to Marquardt et al. (1978)
MARQUARDT, H. and BAYER, U. Mutation Res. 56, 169-176 (1978).

Statistics:

Significance of differences between treatment and control group was assessed by the t-test (p < 0.01)

Results and discussion

Test results

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:

When comparing the number of SCE’s between negative control and the test groups (1500, 3000 and 6000 mg/kg bw test substance) no statistical difference between treated groups and control could be seen. In contrast, the positive control group showed a highly significant increase of SCE's per cell (12.8) in comparison with the negative control (5.28 SCE's/cell).

Any other information on results incl. tables

Table 1: The effect of test substance on bone marrow cells of chinese hamster

Group	Dose [mg/kg]	overall no of animals  (male+ female)	group mean no.  of SCE's	standard- deviation	t-value
Control (0.7% CMC in water)		2m +2f	5.28	2.95
Control (DMBA)	100	2m +2f	12.79	6.25	10.88*
Test substance	1500	2m +2f	6.02	3.16	1.72
Test substance	3000	2m +2f	4.81	2.37	1.24
Test substance	6000	2m +2f	4.88	3.91	0.82
* p≤ 0.01 DMBA= 7,12 Dimethylbenzanthracene