Benzyl benzoate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

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:

1990

Report date:

1990

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Principles of method if other than guideline:

Study Type; Mutagenicity; Gene Mutation in Cultured Chinese Hamster un Cells (HGPRT).

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Test material information

Constituent 1

Reference substance name:

Benzyl benzoate

EC Number:

204-402-9

EC Name:

Benzyl benzoate

Cas Number:

120-51-4

Molecular formula:

C14H12O2

IUPAC Name:

benzyl benzoate

Test material form:

solid

Details on test material:

generic

Specific details on test material used for the study:

Purity: 99%

Method

Target gene:

(HGPRT)

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9 derived from 8-12 week old male Wistar
Aroclor 1254 induced rat liver

Test concentrations with justification for top dose:

Test Compound Concentrations Used:
(a) Preliminary cytotoxicity assay: Eight doses (0.1, 1.0, 10, 30, 60, 100, 250, and 500 µg/mL) were evaluated with andw ithout S9 activation.
b) Mutation assay:
Without S9 activation: Eight doses 10, 50, 60, 75, 80, 90, 100, and 120 µg/mL) were evaluated in the initial assay, an five doses (50, 90, 100, 120, and 150 µg/mL) were evaluated in the conflrmatory assay.
With S9 activation: Four doses (50, 100, 250, and 500 µg/mL) were evaluated in botht he initial and confirmatory assays.

Vehicle / solvent:

Solvent/volume: Ethanol/1% v/v

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

other: Negative; Dulbecco s minimal essential medium (DMEM)/F12 supplemented wich 10% fetal calf serum (FCS)

Positive control substance:

other: Nonactivation (concentration, solvent): Ethyl methanesulfonate (EMS) was prepared in culture medium to yield a final concentration of 1 mg/ml. Activation concentration, solvent): 7,12-dimethylbenz(a)anthracene (DMBA) was prepared in dimethyl sulfoxide.

Details on test system and experimental conditions:

1. Preliminary Cytotoxicity Assay; Eight doses of the test material (0.1 to 500 µg/mL) were evaluated with and without S9 activation. The solubility limit was 500 µg/mL. Nonactivated benzyl benzoate reduced the relative initial survival (RIS) at 250 µg/mL to 65.4%, and to 5.9% a 500 µg/mL. No cytotoxicity was observed at lower nonactivated doses, or at any S9-activated dose up to the solubility limit.

2. Mutation Assav: Doses for the mutation assays were chosen so that the high dose would reduce the plating efficiency to 20-50%. The study author stated that the first two assays without S9 activation were repeated, since cytotoxicity at levels >100 µg/mL prevented the evaluation of a sufficient number of doses; data from these experiments were not provided. Accordingly, benzyl benzoate was evaluated in the first successful nonactivated mutation assay at six doses ranging from 10 to 120 µg/mL; four S9-activated doses ranging from 50 to 500 µg/mL were also evaluated. The author reported that precipitation of the test material was observed at concentrations above 50 µg/mL. No explanation was provided for the differences in solubility between the preliminary cytotoxicity assay and the mutation assay.
Benzyl benzoate was not cytotoxic at doses <= 50 µg/mL -S9. Cytotoxicity at higher nonactivated concentrations (60-120 µg/mL) was not dose-dependent; RIS was <= 18.6% at all levels. The author attributed the lack of a dose-dependence effect to the insolubility of the compound. In the presence of S9 activation, benzyl benzoate was not cytotoxic at any tested dose. There was no evidence of a mutagenic effect of benzyl benzoate at any assayed concentration with or without S9 activation. In contrast, the positive controls (EMS at 1 mg/mL and DMBA at 15.4 µg/mL) induced marked increases in the number and frequency of mutants.

In the confirmatory assay, the test material was investigated at 50, 90, 100, 120, and 150 µg/mL -S9 and at 50, 100, 250, and 500 µg/mL +S9. RIS for cultures exposed co nonactivated benzyl benzoate ranged from 64.4% at the low dose (50 µg/mL) to 21.8% at the high dose (150 µg/mL); the cytotoxic response was dose-related. Severe cytotoxicity (i.e. <50 cells recovered 7 days postseeding) was reported for the cultures treated with 150 µg/mL -S9 after the expression period. As in the first assay, no cytotoxicity was observed at any S9-activated dose. Also in agreement with the findings of the initial assay, the test material was not mutagenic at any nonactivated or S9-activated dose.
The study author concluded that benzyl benzoate was not mutagenic in this test system.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Any other information on results incl. tables

The US EPA has reviewed the study: REVIEWERS DISCUSSION AND INTERPRETATION OF RESULTS: We assess that the study author’s interpretation of the data was correct. Benzyl benzoate was tested to the limit of solubility, and to cytotoxic doses without S9 activation, but showed no evidence of inducing forward mutations at the HGPRT locus in V79 cells. The response of the test system to the positive controls indicated that the assay was sufficiently sensitive to detect mutagenic response. We, therefore, conclude that benzyl benzoate was not mutagenic in this assay.

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative

Executive summary:

CONCLUSIONS-EXECUTIVE SUMMARY: Under the conditions of the Chinese hamster lung cell HGPRT forward gene mutation assay, doses of non-activated benzyl benzoate (10 to 120 µg/mL), and doses of S9- activated benzyl benzoate (50 to 500 µg/mL) did not induce a mutagenic response in two independent assays. The test material precipitated at levels above 50 µg/mL +/- S9. Marked cytotoxicity was observed at all nonactivated doses >= 60 µg/mL in the first trial, and at 150 µg/mL in the second trial. The S9-activated test material was not cytotoxic. Based on these findings, it was concluded that benzyl benzoate was tested to the limit of solubility, and to cytotoxic levels without S9 activation, with no evidence of a mutagenic effect.