Benzyl acetate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Five in vitro mutagenicity studies are available. The in vitro studies include bacterial reverse mutation assay using Salmonella Typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 97, bacterial forward mutation assay using mouse lymphoma L5187Y cells and human lymphoblastoid cells (TK6) and a mammalian chromosome aberration test using Chinese hamster lung fibroblast cell line (CHL/IU).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Benzyl Acetate 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:

not specified

Remarks:

Study is a publication

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): Benzyl Acetate
- Substance type: No information provided
- Physical state: clear liquid
- Analytical purity: 99.1%

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable as the species/strain used are bacterial and not mammalian.

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 97

Details on mammalian cell type (if applicable):

Not applicable as the species/strain used are bacterial and not mammalian.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat and hamster metabolic activation (S-9 mix).

Test concentrations with justification for top dose:

0,10,100,1000 and 10000ug/plate.

Vehicle / solvent:

Distilled water. For chemicals that were not soluble or had low solubility in water, dimethyl sulfoxide (DMSO) was used. Ethanol (95%) or acetone was used for chemicals insoluble in water or DMSO.

Untreated negative controls:

yes

Remarks:

Potassium chloride

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Used with metabolic activation for strains TA 1535 and TA 100

Untreated negative controls:

yes

Remarks:

Potassium chloride

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine

Remarks:

Used with metabolic activation for strain TA 98

Untreated negative controls:

yes

Remarks:

Potassium chloride

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Used with metabolic activation for strains TA 97 and TA 1537

Untreated negative controls:

yes

Remarks:

Potassium chloride

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

Used with all strains with rat and hamster liver metabolic activation systems.

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours
- Expression time (cells in growth medium): Not documented
- Selection time (if incubation with a selection agent): Not documented
- Fixation time (start of exposure up to fixation or harvest of cells): Not documented

SELECTION AGENT (mutation assays): Not documented
SPINDLE INHIBITOR (cytogenetic assays): Not documented
STAIN (for cytogenetic assays): Not documented

NUMBER OF REPLICATIONS: All assays were repeated no less than one week after completion of the initial test.

NUMBER OF CELLS EVALUATED: Not documented

DETERMINATION OF CYTOTOXICITY
- Method: 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.

OTHER EXAMINATIONS:
- Determination of polyploidy: Not documented
- Determination of endoreplication: Not documented
- Other: Not documented

OTHER: At least one toxic dose was incorporated into the first mutagenicity test, the repeat test(s) occasionally had the doses adjusted so that an apparent toxic dose was not reached.

Evaluation criteria:

Mutagenic response: a dose-related, reproducible increase in the number of revertants over background, even if the increase was less than twofold.
Nonmutagenic response: when no increase in the number of revertants was elicited
Questionable response: when there was an absence of a clear cut dose related increase in revertants, when the dose related increase in revertants was not reproducible or when the response was of insufficient magnitude to support a determination of mutagenicity.

Statistics:

No information provided.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

The hamster S-9 mix proved, in general, to be better than the rat S-9 mix in inducing a mutagenic response

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

No additional information provided.

Conclusions:

Based on the results of the study, the test substance, Benzyl Acetate can be considered to be non-mutagenic under the conditions of this study. As a result of this, it does not require any classification according to Regulation EC No. 1272/2008.

Executive summary:

In the study conducted by Mortelmans et al in 1986, Benzyl Acetate was examined for its ability to cause mutagenic changes when tested in five strains of the bacteria Salmonella typhimurium, specifically, TA 1535, TA 1537, TA97, TA 98 and TA 100 through the preincubation assay method. The test was conducted both in the presence and absence of metabolic activation using rat and hamster liver derived S-9 mix, over a range of doses, from 0 to 10000 ug/plate. Based on the results of this study, the test substance Benzyl Acetate was not considered to be mutagenic under the conditions of this test. As a result of this, it does not require any classification according to Regulation EC No. 1272/2008.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

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

Principles of method if other than guideline:

Benzyl acetate was investigated to determine its ability to cause gene mutations when tested in human and mouse lymphoma cells in both the presence and absence of metabolic activation using S9 mix. The target gene was the TK locus.

GLP compliance:

not specified

Remarks:

Study is published

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): Benzyl Acetate
- Physical state: clear liquid

Target gene:

TK gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The mouse lymphoma cells were stored in liquid nitrogen. The laboratory cultures were then maintained in Fischers medium at 37°C on rotary shakers. Fischers medium was supplemented with 2mM L-glutamine 110ug/ml sodium pyruvate, 0.05% pluronic F68, antibiotics and 10% heat activated horse serum (v/v). Less than one week before the start of the experiment, aliquots from the stock cultures were purged of tk¯/tk¯ mutants by exposure for one day to the horse serumcontaining THMG (6 ug/ml thymidine, 5 ug/ml hypoxanthine, 0.1ug/ml methotrexate, and 7.5 ug/ml glycine), then for 3 days to the horse serumcontaining THG only.

Species / strain / cell type:

human lymphoblastoid cells (TK6)

Details on mammalian cell type (if applicable):

Exponentially growing TK6 human Iymphoblasts (grown in RPMI medium 1640 supplemented with 10% v/v horse serum) were treated for 48 h with CHAT (1 X 10-5 M deoxycytidine, 2 X 10-4 M hypoxanthine, 2 X 10-7 M aminopterin, 1.75 X 10-5 M thymidine). Cells were then centrifuged (1000 X g for 5 min) and resuspended in medium containing THC.

Metabolic activation:

with and without

Metabolic activation system:

with and without

Test concentrations with justification for top dose:

preliminary cytotoxicity assays were conducted to determine concentrations for the main mutation assays. Benzyl acetate was tested up to a maximum concentration of 1500 µg/mL

Vehicle / solvent:

DMSO was used as the solvent only in the mouse lymphoma assay.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

1% organic solvent or 10% water

True negative controls:

no

Positive controls:

yes

Remarks:

250 nl/ml

Positive control substance:

ethylmethanesulphonate

Remarks:

Positive control used in the absence of S-9 mix for Mouse lymphoma assay

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

1% organic solvent or 10% water

True negative controls:

no

Positive controls:

yes

Remarks:

5 -10 nl/ml

Positive control substance:

methylmethanesulfonate

Remarks:

Positive control used in the absence of S-9 mix for the Mouse lymphoma assay

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

1% organic solvent or 10% water

True negative controls:

no

Positive controls:

yes

Remarks:

2.5 ug/ml.

Positive control substance:

3-methylcholanthrene

Remarks:

Positive control used in the presence of S-9 mix for the Mouse lymphoma assay

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

1% organic solvent or 10% water

True negative controls:

no

Positive controls:

yes

Remarks:

70 ng/ml

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Positive control without S-9 mix for the Human lymphoma assay

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

1% organic solvent or 10% water

True negative controls:

no

Positive controls:

yes

Remarks:

2.5ug/ml

Positive control substance:

benzo(a)pyrene

Remarks:

Positive control without S-9 mix for the Human lymphoma assay

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: No information
- Exposure duration: For the mouse lymphoma assay, in both the presence and absence of S-9 activation, the exposure duration was 4 hours.
For the human lymphoma assay, in the absence of metabolic activation, the exposure duration was 20 hours. In the presence of metabolic activation, the exposure duration was 3 hours.
- Expression time (cells in growth medium): 2 days for the mouse lymphoma assay and 3 days for the human lymphoma assay
- Selection time (if incubation with a selection agent): No information
- Fixation time (start of exposure up to fixation or harvest of cells): No information

SELECTION AGENT (mutation assays): Trifluorothymidine.
SPINDLE INHIBITOR (cytogenetic assays): No information
STAIN (for cytogenetic assays): No information

NUMBER OF REPLICATIONS: For the mouse lymphoma assay, the study was conducted in triplicate.
For the human lymphoma assay, the study was conducted in duplicate without the selective agent and in triplicate with the selective agent.

NUMBER OF CELLS EVALUATED: 1 x 10 E 6 cells per dish in the mouse lymphoma assay.
30000 cells per well per culture in the human lymphoma assay.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth for the mouse lymphoma assay
other: for the human lymphoma assay, cytotoxicity was determined by multiplying the relative cell growth during expression by the relative cloning efficiency.

OTHER EXAMINATIONS:
- Determination of polyploidy: No information
- Determination of endoreplication: No information
- Other: No information

OTHER: No additional information.

Rationale for test conditions:

Standard methdology available at the time of the study

Evaluation criteria:

Mouse lymphoma:
An experiment was considered positive if the p value was less than 0.05 for at least one of the three highest dose sets (peak response) and if there was a significant trend (p less than 0.05). If there was only a trend response or a single dose increase without a trend, the evaluation was "questionable", A "negative" evaluation was made when there was no trend or peak response. A chemical was considered "positive" only if the positive response was confirmed in a repeat test.

Human lymphoma:
The number of positive wells and the total number of wells scored were recorded for each plate. For each culture, the plate counts were pooled and the plating efficiency (PE) and the mutant fraction (MF) were calculated in accordance with the Poisson distribution.

Statistics:

Mouse Lymphoma:
All data were evaluated for both trend and peak response. The analysis consisted of a trend test and a pairwise comparison of the variance of the mutant fraction of the treated samples against the solvent control. The variance of the mutant fraction was determined by using a series of statistical assumptions associated with a mathematical model of the assay. Three doses were required for an experiment to be evaluated statistically.

Human Lymphoma:
A mutagenic response was defined on the basis of 2 statistical tests on the pooled data from 2 or 3 independent experiments and on checks of the assay performance. For a test substance to be defined as mutagenic, a mean mutant fraction for a given test substance concentration had to be both greater than the 99% upper confidence limit of all historical negative control values and greater than the concurrent negative controls at the 95% confidence level (Dunnett's t-test, one tail). If no mean mutant fraction fulfilled these criteria, the response was defined as nonmutagenic.

Species / strain:

other: mouse lymphoma and human lymphoma

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 500 µg/mL and higher

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

other: mouse lymphoma and human lymphoma

Metabolic activation:

with

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 500 µg/mL and higher

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Benzyl acetate was mutagenic in the presence of S9, but only in the presence of cytotoxicity. The lowest concentrations at which positive responses were seen were 500 µg/mL and 1500 µg/mL in the mouse and human lymphoma assays, respectively. Benzyl acetate was not mutagenic int he absence of S9; cytotoxicity was also observed.

When comparing the results from the two systems the following topics are considered: the analytical procedures used; the degree of concordance between the two systems; the possible influence of gene locus, metabolic activation systems, and differences in assay procedures; and finally, the possibility of species-specificity in the responses observed.

Conclusions:

Under the conditions of this study, the test substance was considered to be mutagenic in the presence of metabolic activation in both the mouse and human lymphoma assays, but only in the presence of cytotoxicity. In the absence of metabolic activation, the test substance was toxic but was not considered to be mutagenic.

Executive summary:

In the study conducted by Caspary et al. (1988) benzyl acetate was investigated for its ability to cause gene mutations at the TK locus using a forward mutation assay. Mouse lymphoma cells (L5178Y) and human lymphoma cells (TK6) were used, and were tested both in the presence and absence of metabolic activation using S9 mix. Under the conditions of this study, the test substance was considered to be mutagenic in the presence of metabolic activation in both the mouse and human lymphoma assays, but only in the presence of cytotoxicity.  In the absence of metabolic activation, the test substance was toxic but was not considered to be mutagenic.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Not documented

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Principles of method if other than guideline:

No information

GLP compliance:

not specified

Remarks:

Study is published data

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

- Name of test material (as cited in study report): Benzyl Acetate
- Molecular weight (if other than submission substance): 150.18
- Analytical purity: ≥98%
- Lot/batch No.: KCR 3596

Target gene:

TK locus.

Species / strain / cell type:

other: Chinese hamster lung fibroblast cell line (CHL/IU)

Details on mammalian cell type (if applicable):

Eagle's minimum essential medium supplemented with 10% heat inactivated calf or foetal bovine serum. The doubling of cells was approximately 15 hours and the modal chromosome number was 25.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix prepared from male Sprague Dawley rats.

Test concentrations with justification for top dose:

The maximum concentration, determined by preliminary cytotoxicity tests, was the concentration showing more than 50% inhibition of cell growth regardless of solubility. For the non-toxic chemicals, the upper limit concentration was 10 mM or 5 mg/ml, whichever was lower.
Concentrations tested for benzyl acetate were: 0, 0.15, 0.3, 0.6 and 0.9 mg/mL for the 24 hour treatment and 0, 0.3, 0.6, 0.9 and 1.2 mg/mL for the 48 hour treatment both without metabolic activation. For the 6 hour treatment with 18 hour recovery the tested concentrations were 0, 0.3, 0.6, and 1.2 mg/mL without activation and 0, 0.6, 1.2, 1.8 and 2.4 with activation.

Vehicle / solvent:

Dimethyl sulfoxide (DMSO)

Untreated negative controls:

yes

Remarks:

treated with DMSO alone at 1% or 0.5%

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Treated with the above substance in the presenc and absence of metabolic activation for 24 and 48 hours. Migrated to IUCLID6: 0.03 ug/ml

Untreated negative controls:

yes

Remarks:

treated with DMSO alone at 1% or 0.5%

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Treated with the above substance in the presenc and absence of metabolic activation for 6 hours. Migrated to IUCLID6: 10 ug/ml

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: Not documented
- Exposure duration: Not documented
- Expression time (cells in growth medium): 18hours
- Selection time (if incubation with a selection agent): 15 minutes at 37°C
- Fixation time (start of exposure up to fixation or harvest of cells): Not documented

SELECTION AGENT (mutation assays): Not documented
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa stain

NUMBER OF REPLICATIONS: No information provided.

NUMBER OF CELLS EVALUATED: 100 well-spread metaphases were analyzed for each dose group

DETERMINATION OF CYTOTOXICITY
- Method: other: A treatment was positive when the frequency of structurally aberrant cells or polyploidy was 10% or more; marginal when it was 5% to less than 10%; and negative when it was less than 5%. An overall positive evaluation was made when structural aberrations or polyploidy was shown for one or more treatments, regardless of the presence of an exogenous metabolic activation system.

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes - positive when polyploid was 10% or more.
- Determination of endoreplication: Not documented
- Other: No additional information

OTHER: Cells were treated for 24 or 48 h continuously until cell harvest without an exogenous metabolic activation system. Cells were also treated wifor 6 h in the presence or absence of S9 mix.

Rationale for test conditions:

Standard methodology at the time of publication

Evaluation criteria:

A treatment was positive when the frequency of structurally aberrant cells or polyploidy was 10% or more; marginal when it was 5% to less than 10%; and negative when it was less than 5%. An overall positive evaluation was made when structural aberrations or polyploidy was shown for one or more treatments, regardless of the presence of an exogenous metabolic activation system.

Statistics:

No information provided.

Species / strain:

other: Chinese hamster lung fibroblast cell line (CHL/IU)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

Benzyl acetate showed a marginal induction of structural aberrations, predominantly chromatid exchanges but only at the highest dose in 24 hour continuous treatment without S9 mix. Because there was no structural aberration induction with any other treatment and the dose range used, it was still considered to be negative.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

No additional information

Conclusions:

Under the conditions of this study, the test substance, benzyl acetate was not considered to be mutagenic and as a result, does not require classification according to Regulation EC No. 1272/2008.

Executive summary:

In the study conducted by Matsuoka et al 1996, benzyl acetate was investigated for its ability to induce chromosome aberrations when tested on the mammalian cell line, Chinese hamster lung fibroblast cell line (CHL/IU). The study was conducted both in the presence and absence of metabolic activation using S9 mix. Under the conditions of this study, the test substance was not considered to cause chromosomal aberrations and as such, does not require classification according to Regulation EC No. 1272/2008.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1985

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

The test substance was examined in a reverse mutagenicity study using bacterial strains TA 98 and TA 100 of the bacteria Salmonella typhimurium.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): Benzyl Acetate
- Analytical purity: Minimum of 99%

Target gene:

Not documented

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

Not applicable as bacterial strains used

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

Not applicable as bacterial strains used

Additional strain / cell type characteristics:

not specified

Metabolic activation:

not specified

Metabolic activation system:

No information provided

Test concentrations with justification for top dose:

No information provided

Vehicle / solvent:

No information provided

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation). The compound, buffer and bacteria were mixed, molten agar was added, and the suspension was immediately plated.

DURATION
- Preincubation period: Not documented
- Exposure duration: Not documented
- Expression time (cells in growth medium): 48hours
- Selection time (if incubation with a selection agent): Not documented
- Fixation time (start of exposure up to fixation or harvest of cells): Not documented

SELECTION AGENT (mutation assays): Not documented
SPINDLE INHIBITOR (cytogenetic assays): Not documented
STAIN (for cytogenetic assays): Not documented

NUMBER OF REPLICATIONS: At least two separate experiments were done, each run in triplicate.

NUMBER OF CELLS EVALUATED: Not documented

DETERMINATION OF CYTOTOXICITY
- Method: other: The number of revertant colonies was scored.

OTHER EXAMINATIONS:
- Determination of polyploidy: Not documented
- Determination of endoreplication: Not documented
- Other: Not documented

OTHER: Not documented

Evaluation criteria:

Toxicities were determined by counting the number of surviving colonies.

Statistics:

No information provided.

Species / strain:

S. typhimurium TA 98

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 100

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

No additional information provided

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Meso-anthracenic benzylic acetates were mutagenic, whereas benzylic acetate esters attached to other carbon atoms were inactive.

Conclusions:

Under the conditions of this study, Benzyl Acetate was not considered to be mutagenic.

Executive summary:

In a study conducted by Rogan et al in 1986, benzyl acetate was investigated in an in vitro reverse mutation assay using bacterial strains TA 98 and TA 100 from Salmonella typhimurium. Based on the results of this study, the test substance was not considered to be mutagenic.

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

The test substance was examined in a reverse mutagenicity study using 4 strains of bacterial Salmonella typhimurium, specifically, TA 98, TA 100, TA 1535 and TA 1537.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): Benzyl Acetate

Target gene:

No information provided

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable as bacterial strains used.

Additional strain / cell type characteristics:

other: histidine-requiring mutants of S. typhimurium LT-2.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 or methylcholanthrene induced S9 mix.

Test concentrations with justification for top dose:

No information provided

Vehicle / solvent:

No information provided

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

positive control in the absence of metabolic activation

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

positive control in the presence of metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation). Initially cultures were grown in Difco nutrient broth, however, this medium was suspected to have a weak mutagenic activity and was later substituted with Oxoid nutrient broth No. 2. Revertants were scored on glucose minimal salts medium supplemented with 0.05umol histidine and 0.05umol biotin.

DURATION
- Preincubation period: Not documented
- Exposure duration: Not documented
- Expression time (cells in growth medium): Not documented
- Selection time (if incubation with a selection agent): Not documented
- Fixation time (start of exposure up to fixation or harvest of cells): Not documented

SELECTION AGENT (mutation assays): Not documented
SPINDLE INHIBITOR (cytogenetic assays): Not documented
STAIN (for cytogenetic assays): Not documented

NUMBER OF REPLICATIONS: Not documented

NUMBER OF CELLS EVALUATED: Not documented

DETERMINATION OF CYTOTOXICITY
- Method: other: viable count and the number of spontaneous revertants was measured.

OTHER EXAMINATIONS:
- Determination of polyploidy: Not documented
- Determination of endoreplication: Not documented
- Other: Not documented

OTHER: Not documented

Evaluation criteria:

No information provided

Statistics:

No information provided

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

No additional information

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

No additional information .

Conclusions:

Under the conditions of this study, the test susbtance was considered to be non-mutagenic both in the presence and absence of metabolic activation.

Executive summary:

In a study conducted by Florin et al. (1980), the test susbtance, Benzyl Acetate, was investigated for its ability to induce mutagenic activity when tested in an in vitro reverse mutagenicity test using four strains of the bacteria Salmonella typhimurium, specifically TA 98, TA 100, TA 1535 and TA 1537. The study was conducted both in the presence and absence of metabolic activation using S9 mix from Aroclor 1254 or methylcholanthrene induced rats.

Under the conditions of this study, benzyl acetate was not considered to be mutagenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Two in vivo mutagenicity studies are available.

The in vivo studies included an unscheduled DNA synthesis test in male Fischer 344 rats and a micronucleus assay in male B6C3F1 mice.

Link to relevant study records

Reference

Reference 1

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Benzyl Acetate was administered as a single dose by oral gavage to Fischer-344 rats. Hepatocytes were prepared from the livers of these rats and assessed for the incidence of DNA repair. The assessment of toxicty was evaluated based on the number of nuclear grains and the percentage of cells undergoing repair.

GLP compliance:

not specified

Remarks:

Data is published literature

Type of assay:

unscheduled DNA synthesis

Specific details on test material used for the study:

- Name of test material (as cited in study report): Benzyl acetate
- Molecular formula (if other than submission substance): C9H10O2
- Molecular weight (if other than submission substance): 150.18
- Physical state: clear liquid

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Simonsen Laboratories (Gilroy, CA) and Hilltop Laboratory Animals (Chatsworth CA).
- Age at study initiation: no data
- Weight at study initiation: 180 - 300g
- Assigned to test groups randomly: no data
- Fasting period before study: no data
- Housing: Three rats per cage in polypropylene cages with hardwood chip bedding.
- Diet (e.g. ad libitum): Purina Rodent Chow # 5001 (Ralston Purina Co., St. Louis) ad libitum
- Water (e.g. ad libitum): Deionized 0.5um charcoal filtered tap water ad libitum.
- Acclimation period: Not documented

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Not documented
- Humidity (%): Not documented
- Air changes (per hr): Not documented
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle.

IN-LIFE DATES: From: To: Not documented

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Animals were administered the test substance by oral gavage as a single bolus dissolved or suspended in corn oil or water. Doses were selected based approximately on the oral LD50 of the compound and were generally selected as 80%, 40% and 10% of the LD50. The acute LD50 was never exceeded and 1000mg/kg was selected as the highest dose if hte LD50 exceeded this value.

Duration of treatment / exposure:

Animals were exposed to a single dose of the test substance.

Frequency of treatment:

Single exposure

Post exposure period:

No information provided

Dose / conc.:

50 mg/kg bw (total dose)

Dose / conc.:

200 mg/kg bw (total dose)

Dose / conc.:

1 000 mg/kg bw (total dose)

No. of animals per sex per dose:

3 male rats were used at each dose level.

Control animals:

not specified

Positive control(s):

corn oil

Tissues and cell types examined:

Hepatocyte cultures prepared from perfused liver.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Doses were selected based approximately on the oral LD50 of the compound and were generally selected as 80%, 40%, and 10% of the LD50. The acuteLD50 was never exceeded, and 1000 mg/kg was selected as the highest dose if the LD50 exceeded this value.
If hepatocyte cultures showed very poor viability and attachment at nonlethal doses, the top dose was reduced to achieve acceptable hepatocyte attachment.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
A single-cell suspension of hepatocytes was obtained by combing out cells from the perfused liver into a petri dish containing 37°C collagenase solution. Cells were collected by 5 min centrifugation at 50g, resuspended in cold medium, and filtered through sterile gauze. Viability was determined using Trypan blue exclusion. In general, hepatocyte viability was not adversely affected by chemical treatment; i.e., viability generally exceeded 70%, and attachment did not vary. In cases where attachment or viability were poor, lower doses were selected for testing.
Approximately 6 x 10E5 cells were seeded into each well of a Falcon 9.6 cm2, 6-well culture plate. Each well contained a 25 mm round Thermanox coverslip in Williams' medium E (WE) supplemented with 2 mM I-glutamine, 50 ug/ml gentamycin sulfate, and 10% fetal bovine serum. After 1.5-2.0 hr incubation in a humidified atmosphere at 37°C, 5% CO2 the cultures were washed to remove nonviable cells (those not attached to the coverslips). All washes and subsequent culturing were done using serum-free WE medium.

The sampling for the UDS test was conducted at 2 and 12 hours.

DETAILS OF SLIDE PREPARATION:
Cultures were incubated in Williams medium E (WE) containing 10 uCi/ml 3H-(methyl)-thymidine (specific activity, approximately 80 Ci/mmol) for 4 hr at 37°C, 5% C02, followed by 14-18 hr in WE containing 0.25 mM unlabeled thymidine. The cultures were then washed twice with WE, followed by 10 min in 1% sodium citrate to swell cells, fixed in 1:3 glacial acetic acid:ethanol for 30 min, and washed 3 to 6 times with deionized water. The dried coverslips were mounted to glass slides using Permount. The slides were dipped in Kodak NTB-2 nuclear track emulsion diluted 1: 1 with deionized water, and exposed at -20°C for 7-14 days and then developed and stained

METHOD OF ANALYSIS:
UDS Measurement:
An area of a slide was randomly selected, and 50 morphologically unaltered cells were counted using an ARTEK Model 880 colony counter interfaced to a VAX 8800 computer. The highest of two nuclear-sized areas over the cytoplasm and adjacent to the nucleus was subtracted from the nuclear count to determine the net grains/nucleus (NG). The percentage of cells undergoing repair (%IR) was determined as the percent of those cells exhibiting 5 or more NG. Three slides \vere scored for each animal or concentration for a total of 150 cells per animal.
The test substance was considered negative if the NG of all dose groups was a negative number and the %IR was less than 10%. It was considered positive if the average NG of any dose group exceeded 0 NG. If the test compound had negative NG values, but %IR values greater than 10%, it was considered equivocal.

Evaluation criteria:

UDS Synthesis:
Compounds were considered negative if the NG of all dose groups was a negative number and the %IR was less than 10%. Compounds were considered positive if the average NG of any dose group exceeded 0 NG. Compounds with negative NG values, but %IR values greater than 10% were considered equivocal.

Statistics:

No information provided.

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

The test substance was considered to be negative as the net grain/nucleus was always a negative number and the percentage of cells undergoing repair was always less than 10% for each dose level tested at each timepoint examined.

Measurement of UDS in Male Rat Hepatocytes Following In Vivo Treatment:

			Male rat
	Dose (mg/kg)	Time (hr)	NG	(n)	%IR
Control / corn oil	-	2 12	-6.4±2.9 -5.6± 0.4	(2) (52)	1± 0 2±0
Benzyl Acetate	50     200     1000	2 12   2 12   2 12	-4.1± 1.4 -2.2 ±0.2   -5.3±0.3 -4.8±1.0   -4.5±1.1 -4.6±0.3	(3) (3)   (3) (3)   (3) (3)	3±1 1±0   1± 1 2 ± 1   1± 0 1 ± 1

Conclusions:

Based on the results of this study, the test material, Benzyl Acetate was considered to be negative under the conditions of this study. As a result of this, it does not need to be classified under Regulation EC No. 1272/2008.

Executive summary:

In the study conducted by Mirsalis et al (1989), Benzyl Acetate was investgated for its ability to unscheduled DNA synthesis in Fischer -344 rats following in vivo treatment. Hepatocytes, isolated by liver perfusion, were used to assess the mutagenicity potential of Benzyl Acetate. Rats received a single dose of the test substance dissolved or suspended in corn oil administered by oral gavage. The dose levels used were 50, 200 and 1000 mg/kg. The responses were examined at the 2 and 12 hour timepoints. The results of this study indicate that the test substance, Benzyl Acetate, was negative under the conditions employed in this study as the net grain/nucleus count was always negative and the percentage of cells undergoing repair was less than 10%. As a result of this, Benzyl Acetate does not require classification according to Regulation EC no. 1272/2008.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Benzyl acetate was tested in a mouse bone marrow micronucleus test that employed three daily exposures by intraperitoneal injection. Bone marrow samples were obtained 24 hr following the final exposure. This study was conducted as part of an effort to assess the ability of the micronucleus test to discriminate between rodent carcinogens and noncarcinogens and to determine its potential role, in combination with other short term tests, in identifying genotoxic chemicals that present a carcinogenic hazard.

GLP compliance:

not specified

Type of assay:

micronucleus assay

Specific details on test material used for the study:

- Name of test material (as cited in study report):Benzyl acetale

Species:

mouse

Strain:

B6C3F1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: National Toxicology Program production facility
- Age at study initiation: between 9 and 14 weeks
- Weight at study initiation: 25 - 33 g

Note: reference within report to further data on animal husbandry to be found in Tice et al 1991a

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: water insoluble substance
- Concentration of test material in vehicle: 0, 312, 625, 1250 mg/kg
- Amount of vehicle (if gavage or dermal): 0.4 ml

Details on exposure:

Benzyl acetate was prepared in corn oil and suspended using Tek-Mar Tissumizer. The test substance was administered within 30 minutes of preparation.

Duration of treatment / exposure:

Dose determination study: IP injection on three consecutive days
MN Induction: IP injection on three consecutive days

Frequency of treatment:

Dose determination study: Daily
MN Induction: Daily

Post exposure period:

Dose determination study: 48 hours after the third treatment
MN Induction: 24 hours after the third treatment

Dose / conc.:

312 mg/kg bw/day (nominal)

Dose / conc.:

625 mg/kg bw/day (nominal)

Dose / conc.:

1 250 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Dose determination study: 5 mice

Control animals:

yes, concurrent vehicle

Positive control(s):

7,12-dimethylbenzanthracene
- Route of administration: intraperitoneal injection
- Doses / concentrations: weakly active dose

Tissues and cell types examined:

Dose determination study: Bone marrow and peripheral blood smears (two slides/tissue/mouse)

Details of tissue and slide preparation:

Dose determination study:
CRITERIA FOR DOSE SELECTION: The selection of the maximum dose to be tested was based on either mortality, administration characteristics, depression in percentage of bone marrow, or arbitrary maximum dose of 2000 mg/kg/day.

TREATMENT AND SAMPLING TIMES: Groups of 5 mice were administered the test chemical by intraperitoneal injection on three consecutive days. Animals were monitored twice daily and 48 hours after the third treatement the survivng mice were euthanized by CO2 asphyxiation. Bone marrow and peripheral blood smears (two slides/tissue/mouse) by a direct technique.

DETAILS OF SLIDE PREPARATION: Air-dried smears were fixed using absolute methanol and stained with acridine orange.

METHOD OF ANALYSIS:Bone marrow smears from each animal were evaluated at 1000x magnification using epi-illuminated Fluorescence microscopy (450-490 nm excitation, 520 nJ;ll emission) for determination of the percentage of PCE among 200 erythrocytes.

MN Induction:
CRITERIA FOR DOSE SELECTION: Based on the dose selection study.

TREATMENT AND SAMPLING TIMES: Groups of 5-7 mice were administered the test chemical by intraperitoneal inhection on three consecutive days. 24 hours after the third treatement mice were euthanized by CO2 asphyxiation. Bone marrow and peripheral blood smears (two slides/tissue/mouse) were prepared.

DETAILS OF SLIDE PREPARATION: Bone marrow smears (two slides mouse) were prepared, fixed in absolute methanol, and stained with acridine orange.

METHOD OF ANALYSIS:For each animal, slides were evaluated at I,000 X magnification for the number of MN-PCE among 2,000 PCE and for the percentage of PCE among 200 erythrocytes.

Evaluation criteria:

The test conclusions are based on the statistical analysis of trend and of pairwise comparisons of the solvent control with individual doses and the absolute increases in MN-PCE frequency.

Statistics:

The data were analyzed using the Micronucleus Assay Data Management and Statistical software package (version 1.4), which was designed specifically for in vivo micronucleus data. The level of significance was set at an alpha level of 0.05. To determine whether a specific treatment resulted in a significant increase in MN-PCE, the number of MN-PCE were pooled within each dose group and analyzed by a one-tailed trend test. In the software package used, the trend test incorporates a variance innation factor to account for excess animal variability. In the event that the increase in the dose response curve is nonmonotonic, the software program allows for the data to be analyzed for a significant positive trend after data at the highest dose only has been excluded. However, in this event, the alpha level is adjusted to 0.01 to protect against false positives. The %PCE data were analyzed by an analysis of variance (ANOVA) test based on pooled data. Pairwise comparisons between each group and the concurrent solvent control group was by an unadjusted one-tailed Pearson chisquared test which incorporated the calculated variance innation factor for the study.

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

No further data

Dose (mg/kg)	MN-PEC/1000 (no of animals)	Pairwise	Survival	%PCE
0	3.00+0.69		5/5	69.9
312	2.90+0.60	0.5519	5/5	65.8
625	3.20+0.60	0.3396	5/5	64.3
1250	1.80+0.46	0.9586	6/6	60.7

Conclusions:

The initial test was negative to 1,250 mg/kg and was not repeated. According to Regulation (EC) No. 1272/2008, no classification is warranted.

Executive summary:

Benzyl acetate was tested in a mouse bone marrow micronucleus test that employed three daily exposures by intraperitoneal injection. Bone marrow samples were obtained 24 hr following the final exposure. This study was conducted as part of an effort to assess the ability of the micronucleus test to discriminate between rodent carcinogens and noncarcinogens and to determine its potential role, in combination with other short term tests, in identifying genotoxic chemicals that present a carcinogenic hazard. The initial test was negative to 1,250 mg/kg and was not repeated. According to Regulation (EC) No. 1272/2008, no classification is warranted.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic Toxicity in Vitro:

In the study conducted by Mortelmans et al in 1986, Benzyl Acetate was examined for its ability to cause mutagenic changes when tested in five strains of the bacteria Salmonella typhimurium, specifically, TA 1535, TA 1537, TA97, TA 98 and TA 100 through the preincubation assay method. The test was conducted both in the presence and absence of metabolic activation using rat and hamster liver derived S-9 mix, over a range of doses, from 0 to 10000ug/plate. Based on the results of this study, the test substance Benzyl Acetate was not considered to be mutagenic under the conditions of this test. As a result of this, it does not require any classification according to Regulation EC No. 1272/2008 or Directive 67/548/EEC.

In the study conducted by Caspary et al. (1988) benzyl acetate was investigated for its ability to cause gene mutations at the TK locus using a forward mutation assay. Mouse lymphoma cells (L5178Y) and human lymphoma cells (TK6) were used, and were tested both in the presence and absence of metabolic activation using S9 mix. The results of this study indicate that in the absence of metabolic activation, benzyl acetate was considered to be mutagenic in the presence of metabolic activation in both the mouse and human lymphoma cell assays. In the absence of metabolic activation, the result was slightly ambiguous as, only at higher dose levels was the test substance toxic, however, it was not considered to be mutagenic.

In the study conducted by Matsuoka et al 1996, benzyl acetate was investigated for its ability to induce chromosome aberrations when tested on the mammalian cell line, Chinese hamster lung fibroblast cell line (CHL/IU). The study was conducted both in the presence and absence of metabolic activation using S9 mix. Under the conditions of this study, the test substance was not considered to cause chromosomal aberrations and as such, does not require classification according to Regulation EC No. 1272/2008 and Directive 67/548/EEC.

In a study conducted by Rogan et al in 1986, benzyl acetate was investigated in an in vitro reverse mutation assay using bacterial strains TA 98 and TA 100 from Salmonella typhimurium. Based on the results of this study, the test substance was not considered to be mutagenic. As a result of this, benzl acetate does not require classification according to Directive 67/548/EEC or Regulation EC No. 1272/2008.

In a study conducted by Florin et al.(1980), the test susbtance, Benzyl Acetate, was investigated for its ability to induce mutagenic activity when tested in an in vitro reverse mutagenicity test using four strains of the bacteria Salmonella typhimurium, specifically TA 98, TA 100, TA 1535 and TA 1537. The study was conducted both in the presence and absence of metabolic activation using S9 mix from Aroclor 1254 or methylcholanthrene induced rats.

Under the conditions of this study, benzyl acetate was not considered to be mutagenic. Based on these results, the test substance did not require classification according to Regulation EC No. 1272/2008 or Directive 64/548/EEC.

Genetic Toxicity - In Vivo:

In the study conducted by Mirsalis et al (1989), Benzyl Acetate was investgated for its ability to unscheduled DNA synthesis in Fischer -344 rats following in vivo treatment. Hepatocytes, isolated by liver perfusion, were used to assess the mutagenicity potential of Benzyl Acetate. Rats received a single dose of the test substance dissolved or suspended in corn oil administered by oral gavage. The dose levels used were 5, 200 and 1000 mg/kg. The responses were examined at the 2 and 12 hour timepoints. The results of this study indicate that the test substance, Benzyl Acetate, was negative under the conditions employed in this study as the net grain/nucleus count was always negative and the percentage of cells undergoing repair was less than 10%. As a result of this, Benzyl Acetate does not require classification according to Regulation EC no. 1272/2008 or Directive 67/548/EEC.

In a study conducted by Shelby (1993), Benzyl acetate was tested in a mouse bone marrow micronucleus test that employed three daily exposures by intraperitoneal injection. Bone marrow samples were obtained 24 hr following the final exposure. This study was conducted as part of an effort to assess the ability of the micronucleus test to discriminate between rodent carcinogens and noncarcinogens and to determine its potential role, in combination with other short term tests, in identifying genotoxic chemicals that present a carcinogenic hazard. The initial test was negative to 1,250 mg/kg and was not repeated. According to Directive 67/548/EEC, no classification is warranted. According to Regulation (EC) No. 1272/2008, no classification is warranted.

Short description of key information:


Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the results of the in vivo studies, it is not necessary to classify the test substance according to Regulation EC No. 1272/2008.