A mixture of branched and linear C7-C9 alkyl 3-[3-(2H-benzotriazol-2-yl)-5-(1,1-dimethylethyl)-4-hydroxyphenyl]propionates

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

Oct. 7, 1987 to Mar. 3, 1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP and OECD guideline compliant study. A default reliability of 2 is assigned because of use as read-across according to ECHA guidance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

Version / remarks:

(1987)

Deviations:

no

GLP compliance:

yes

Type of assay:

DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro

Test material

Test material information

Constituent 1

Reference substance name:

A mixture of: α-3-(3-(2H-benzotriazol-2-yl)-5-tert-butyl-4-hydroxyphenyl)propionyl-ω-hydroxypoly(oxyethylene); α-3-(3-(2H-benzotriazol-2-yl)-5-tert-butyl-4-hydroxyphenyl)propionyl-ω-3-(3-(2H-benzotriazol-2-yl)-5-tert-butyl-4-hydroxyphenyl)propionyloxypoly(oxyethylene)

EC Number:

400-830-7

EC Name:

A mixture of: α-3-(3-(2H-benzotriazol-2-yl)-5-tert-butyl-4-hydroxyphenyl)propionyl-ω-hydroxypoly(oxyethylene); α-3-(3-(2H-benzotriazol-2-yl)-5-tert-butyl-4-hydroxyphenyl)propionyl-ω-3-(3-(2H-benzotriazol-2-yl)-5-tert-butyl-4-hydroxyphenyl)propionyloxypoly(oxyethylene)

Molecular formula:

The substance is a mixture of a number of chemical entities, of which there are two principal components : (C2H4O)6-7C19H21N3O3 (major product) (C2H4O)6-7C38H40N6O5 (minor product)

IUPAC Name:

400-830-7

Details on test material:

- Lot/batch No.: EN 20043.42
- Stability under test conditions: Ensured by sponsor
- Composition of test article: 100% UVCB (On file with the sponsor.)
Substance of the same batch number was used in the 90 d repeated dose toxicity study (CIT 874232) where analytical analysis was given in the study report.
- Structural formula attached as image file (if other than submission substance): see Fig.

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

hepatocytes: rat

Details on mammalian cell type (if applicable):

Primary hepatocytes are isolated from adult male Wistar rats (234 to 335 g) by in situ-collagenase perfusion. Viability of hepatocytes was generally greater than 80%. Hepatocytes were cultured in WILLIAMS' Medium E containing 10% foetal bovine serum, 100 U/ml penicillin, 100 ug/ml streptomycin and 2.5 ug/ml amphotericin and incubated in a humidified atmosphere with 5 % CO2 at 37°C.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

not applicable

Test concentrations with justification for top dose:

A preliminary toxicity test was carried out with concentrations of 4.88, 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500, 5000 ug/ml suspended in DMSO. Based on the preliminary test concentrations of 0.8, 4, 20, 100, 200, 400 µg/ml were used in the main experiment. Concentrations of a confirmatory experiment were: 0.2, 1, 5, 10, 20, 50, 100 and 200 µg/ml.

Vehicle / solvent:

- Vehicle used: DMSO

Controls

Untreated negative controls:

yes

Remarks:

growth medium

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-Aminobiphenyl

Details on test system and experimental conditions:

To assess cytotoxicity cells attached to a cover-slid were incubated for 16 to 18 h, stained with Trypanblue solution and fixed. The percentage of unstained cells was evaluated by counting 100 cells.
To assess DNA-synthesis 3H-thymidine is added to cells after replacement of medium. After treatment (16 to 18 h) nuclei of washed cells were swollen by 1% sodium citrate and fixed with ethanol/acetic acid, 3/1, v/v. The cover-slips were mounted on microscope slides and prepared for autoradiography. The exposure time was 6 days. The autoradiographs were stained with hematoxylin-eosine.
Counting of silver grains was carried out with an electronic counter attached to a microscope at a magnification of 2000x. From each of the treatment groups and from the positive and the negative controls 150 nuclei in altogether three slides (50 cells/slide) were scored, the number of silver grains counted and the mean values and the standard deviations calculated.
The incorporation of radioactive material in the cytoplasm is determined by counting the silver grains in three nucleus-equivalent areas of cytoplasm adjacent to the nucleus. The net values are calculated by subtracting the average grain count over the cytoplasm from the total over the nuclei. The background in the autoradiographs (outside the cells) is determined in cell-free areas microscopically and was found to be negligibly low.

Evaluation criteria:

The test substance is generally considered to be active in the DNA repair test if one of the following conditions is met:
- The mean number of silver grains per nucleus in relation to the vehicle control is more than doubled at any concentration.
-The mean number of silver grains per nucleus in relation to the vehicle control shows a concentration dependent increase and at least at one concentration a statistically significant increase in comparison with the vehicle control is demonstrated.
- The mean value of the net nuclear grain counts at any concentration is 2.0 together with a difference to the value of the vehicle control of 2.0.
- The percentage distribution of silver grains on nuclei reveals a significant difference in comparison with the vehicle control.

Statistics:

Statistical analysis will be carried out as follows:
- the values of silver grains per nucleus were compared using Duncan's multiple range test. Statistical significance was judged to be achieved if the probability was less than 0.01.

Results and discussion

Test results

Species / strain:

hepatocytes: rat

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity was seen at the highest concentration of 400 µg/ml.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

At and above a concentration of 312.5 µg/ml a precipitation was visible in the culture medium.
In the main DNA-repair test the 400 µg/ml group showed toxicity and was not examined. The mean gross number of silver grains per nucleus was 5.39, 5.45, 4.77, 5.72, and 3.47 for concentrations of 0.8, 4, 20, 100 and 200 µg/ml, respectively. The vehicle control revealed a value of 2.97. In the confirmatory DNA-repair test the mean gross number of silver grains per nucleus revealed values of 2.94, 5.42, 4.47, 4.49, 5.53, 5.15, 4.33, 4.03, 4.43 for concentrations of 0, 0.2, 1, 5, 10, 20, 50, 100 and 200 µg/ml, respectively.
Mean gross values of 18.47 and 20.34 as well as 7.90 and 7.81 silver grains per nucleus were seen after treatment with the positive control 4-ABP (50 and 25 µM).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative