Benzyl butyrate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed publication

Data source

Reference

Reference Type:

publication

Title:

Gene mutation toxicity study of the test chemical

Author:

Mortelmans et al

Year:

1986

Bibliographic source:

Environ. Mutagen

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Similar to OECD 471

Principles of method if other than guideline:

The test chemical was examined alongside 269 other chemicals for its ability to induce mutagenic changes when tested in Salmonella typhimurium bacterial strains in the presence and absence of metabolic activation with rat and hamster S-9 mix using the preincubation assay method.

GLP compliance:

no

Type of assay:

bacterial gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Benzyl acetate

EC Number:

205-399-7

EC Name:

Benzyl acetate

Cas Number:

140-11-4

Molecular formula:

C9H10O2

IUPAC Name:

Benzyl acetate

Test material form:

liquid

Details on test material:

- Name of test material: Benzyl acetate
- Molecular formula: C9H10O2
- Molecular weight: 150.176 g/mol
- Substance type:Organic
- Physical state:Liquid
- Purity: 99.1%
- Impurities (identity and concentrations): No data

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: Male Sprague-Dawley rats and male Syrian hamsters were routinely used for the preparation of the liver fractions.
- method of preparation of S9 mix : Aroclor 1254 (200 mg/ml in corn oil) was administered ip at 500 mg/kg 5 days prior to decapitation (EGG, SRI) or cervical dislocation (CWR). The animals were deprived of food 12-24 hr immediately preceding death; otherwise food and water were provided ad libitum. The livers were removed aseptically, washed in ice-cold 0.15 M KCl, and minced and homogenized (3 ml of 0.15 M KC1 per gm of wet tissue) in a Potter-Elvehjem apparatus with a
Teflon pestle. CWR initially used a Waring blender, but switched to a Potter-Elvehjem apparatus. The S-9 fraction was obtained by centrifugation of the liver homogenate for 10 min at 9,000 g at 4°C. The S-9 fraction was dispensed into freezing ampules and stored in a -70°C freezer, or in liquid nitrogen.
- source of S9:
- concentration or volume of S9 mix and S9 in the final culture medium :The S-9 mix was prepared immediately prior to each assay and consisted of the following, per milliliter: S-9 fraction, 0.10 ml; 0.04 M MgC12,0.02 ml; 1.65 M KCl,0.02 ml; 0.04 M P-nicotinamide adenine dinucleotide phosphate (NADP), 0.10 ml;0.05 M glucose-6-phosphate, 0.10 ml; 1.0 M NaH2P04, (pH 7.4), 0.10 ml; and distilled water, 0.56 ml. Other levels of S-9 in the S-9 mix were used for some
Aliquots.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability)

Test concentrations with justification for top dose:

0, 33, 100, 333, 1000, 3333 or 10000 ug/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used [none; no data; acetone; arachis oil; beeswax; carbowaxe; castor oil; cetosteryl alcohol; cetyl alcohol; CMC (carboxymethyl cellulose); coconut oil; corn oil; cotton seed oil; DMSO; ethanol; glycerol ester; glycolester; hydrogenated vegetable oil; lecithin; macrogel ester; maize oil; olive oil; paraffin oil; peanut oil; petrolatum; physiol. saline; poloxamer; polyethylene glycol; propylene glycol; silicone oil; sorbitan derivative; soya oil; theobroma oil; vegetable oil; aqueous solvents (water or saline or culture medium)]: DMSO
- Justification for choice of solvent/vehicle: The chemical was soluble in DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 4-nitro-o-phenylenediamine, 2-aminoanthracene

Details on test system and experimental conditions:

2.NUMBER OF REPLICATIONS:

- Number of cultures per concentration (single, duplicate, triplicate): All assays were repeated in duplicate one week after completion of the initial test. At least five dose levels of the chemicals were tested, with three plates per dose level.
- Number of independent experiments : 3

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk: pre-incubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 minutes
- Exposure duration/duration of treatment: 48 hours
- Harvest time after the end of treatment (sampling/recovery times):

Rationale for test conditions:

No data

Evaluation criteria:

1) mutagenic response: a dose-related, reproducible increase in the number of revertants over background, even if the increase was less than twofold;
2) nomutagenic response: when no increase in the number of revertants was elicited by the chemical;
3) questionable response: when there was an absence of a clear-cut dose-related increase in revertants; when the dose-related increases in the number of revertants were not reproducible; or when the response was of insufficient magnitude to support a determination of mutagenicity.

Statistics:

Mutagenic responses of Salmonella tester strains TA100, TA1535, TA1537,TA97, and TA98 (mean SEM) to test chemicals

Results and discussion

Test results

Species / strain:

S. typhimurium, other: Salmonella tester strains TA100, TA1535, TA1537, TA97, and TA98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: The chemical was tested initially with strain TA100 in the presence and the absence of the metabolic activation systems, over a wide dose range with an upper limit of 10 mgjplate, or less when solubility problems were encountered. Toxicity was evidenced by one or more of the following phenomena: appearance of his+ pinpoint colonies, reduced numbers of revertant colonies per plate, or thinning or absence of the bacterial lawn. Nontoxic chemicals were tested in the initial experiment up to the 10 mg/plate dose level, or to a level determined by their solubility. Toxic chemicals were tested up to a high dose which exhibited some degree of toxicity.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: The positive controls used generated a positive response.

Ames test: The test chemical did not induce mutation in the Salmonella typhimurium strain TA98, TA100, TA1535 or TA1537 both in the presence and absence of S9 metabolic activation system
- Signs of toxicity
- Individual plate counts
- Mean number of revertant colonies per plate and standard deviation

Remarks on result:

other: No mutagenic potential

Applicant's summary and conclusion

Conclusions:

The test chemical did not induce mutation in the Salmonella typhimurium strain TA98, TA100, TA1535 or TA1537 both in the presence and absence of S9 metabolic activation system and hence is not likely to be mutagenic under the conditions of this study.

Executive summary:

A bacterial cell gene mutation assay was performed using Salmonella strains TA1535, TA1537, TA98, and TA100 with and without Aroclor 1254-induced rat and hamster metabolic activation systems to assess the mutagenic potential of the test chemical. Since the test chemical was insoluble in water, DMSO was used as the solvent.Salmonella strains TA1535, TA1537, TA97, TA98, and TA100 were obtained by the individual laboratories from Dr. Bruce Ames and were stored according to the protocol described in AMES et.al, 1975. The test chemical was assayed for mutagenicity in the preincubation assay [Haworth et al, 1983]. To each of 13 X 100-mm test tubes maintained at 37°C were added in the following order: 0.5 ml of S-9 mix or 0.1 M PO4 buffer (pH 7.4), 0.05 ml of the overnight culture, and 0.05 ml of solvent or chemical dilution. The mixture was mixed and allowed to incubate without shaking at 37°C for 20 min, at which time 2.5 ml or 2.0 ml of molten (45°C) top agar supplementedwith 0.5 mM L-histidine and 0.5 mM D-biotin were added. The contents of the tubeswere mixed and poured onto 25 ml of minimal glucose bottom agar [Vogel andBonner, 19561 in 15 X 100-mm plastic petri dishes. When the top agar had solidified, theplates were inverted and incubated at 37°C for 48 hr. The doses of the test chemical tested were 0, 33, 100, 333, 1000, 3333 or 10000 ug/plate. The following mutagens were used as concurrent positive controls: sodium

azide for TA1535 and TA 100,4-nitro-o-phenylenediaminef or TA98, and 9-aminoacridine for TA97 and TA1537; 2-aminoanthracene was used with all strains with hamster and rat liver metabolic activation systems. The criteria for a positive response was as described: 1) mutagenic response: a dose-related, reproducible increase in the number of revertants over background, even if the increase was less than twofold;2) nomutagenic response: when no increase in the number of revertants was elicited by the chemical; 3) questionable response: when there was an absence of a clear-cut dose-related increase in revertants; when the dose-related increases in the number of revertants were not reproducible; or when the response was of insufficient magnitude to support a determination of mutagenicity. The test chemical did not induce mutation in the Salmonella typhimurium strain TA98, TA100, TA1535 or TA1537 both in the presence and absence of S9 metabolic activation system and hence is not likely to be mutagenic under the conditions of this study.