Benzyl benzoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The mutagenicity of benzyl benzoate was investigated in two Ames tests. Additional an in vitro gene mutation study in mammalian cells and an in vitro cytogenicity / chromosome aberration study in mammalian cells with benzyl benzoate are available.

 

In the MSCA's Proposal for amendments (PfAs) from 29/08/2019 until 30/09/2019 (MSC Code: CCH-133/2019) an ‘In vitro gene mutation study in bacteria (Annex VII, Section 8.4.1; test method: Bacterial reverse mutation test, EU B.13/14. / OECD TG 471) using S. typhimurium TA102 or E. coli WP2 uvrA or E. coli WP2 uvrA (pKM101) with the registered substance’ (PfAs1) and a vivo follow-up test regarding the mutagenic potential of benzyl benzoate (PfAs2) was required.

Lanxess as the lead registrant used the OPPORTUNITY TO COMMENT ON PROPOSAL(S) FOR AMENDMENT OF A DRAFT DECISION ON A COMPLIANCE CHECK – Communication No. CCH-D-2114486744-35-01/D 

Lanxess explained that “the Lead Registrant very recently became aware of a study which would adequately fill this perceived data gap. While the full study was never made public, it was evaluated in the US Environmental Protection Agency’s (EPA’s) Reregistration Eligibility Decision (RED) for Benzyl Benzoate (RED Case No. 4013)”.

“As this study was only recently discovered by the Lead Registrant (after receipt of the MSCA PfAs), it was obviously not included in the registration dossier”.
“Once we have been able to establish the right to refer to the study, the dossier will be updated in a timely fashion”.

The study is now included in the IUCLID Dataset.

Link to relevant study records

Reference

Reference 1

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

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

Specific details on test material used for the study:

Purity: 99%

Target gene:

(HGPRT)

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

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.

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

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.

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration 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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Principles of method if other than guideline:

Chromosome Aberration Assay in Human Lymphocytes In Vitro with Benzyl Benzoate

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Purity: 99%

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

human

Metabolic activation:

with and without

Metabolic activation system:

derived from Wistar, strain WU, male (8-12 weeks of age)
Aroclor 1254 induced rat liver

Test concentrations with justification for top dose:

(a) Preliminary cytotoxicity assay: Cytotoxicity as assessed in parallel with the cytogenetic assay.
(1) Nonactivated conditions: Eight doses (0.3, 1.0, 3.0, 10.0, 30.0, 100.0, 250.0, and 500.0 µg/mL) with a 24-hour cell harvest and six dose (3.0, 10.0, 30.0, 100.0, 250.0, and 500.0 µg/mL) with a 48-hour cell harvest.
(2) S9-activated conditions; As above.

(b) Cytogenetic assay:
(1) Non activated conditions: Cultures in the cytotoxicity phase of testing that were exposed to 10, 100, and 250 µg/mL (24-hour harvest) and to 250 µg/mL (8-hour harvest) were scored for chromosome aberrations.
(2) S9-activated conditions: Cultures in the cytotoxicity phase of testing that were exposed to 30, 250, and 500 µg/mL (24-hour harvest) and to 500 µg/mL (48-hour harvest) were scored for chromosome aberrations.

Vehicle / solvent:

Solvent/final concentration: ETOH/1%

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: Nonactivation (concentrations, solvent): Ethyl methane sulfonate (EMS was prepared in DMEM/F12 to yield a concentration of 720 µg/mL. Activation (concentrations, solvent): Cyclophosphamide (CP) was prepared in DMEM/F12 to yield a concentration of 60 µg/mL

Details on test system and experimental conditions:

(a) Treatment: Forty-eight hours after Initiation, duplicate cultures were exposed to the selected test material doses, the solvent control (ETOH), or the positive Controls (EMS or CP) in both the presence and absence of S9 activation. At the end of the 4-hour treatment, cells were centrifuged, refed culture medium, and reincubated. Colcemid (final concentration, 0.2 µg/mL was added 3 hours before the cultures were harvested (2 and 8 hours posttreatment). Metaphase cells were collected, swollen in 0.0375 M KCl, and fixed in glacial acetic acid: absolute methanol (1:3). Slides were stained with Giemsa and coded.
(b) Metaphase analysis: Two hundred metaphase plates (100 cells/culture) from each selected dose group and the negative, solvent, and positive control groups were scored for chromosome aberrations; gaps were recorded but not included in the aberration frequencies. The mitotic index (MI) was determined by counting 1000 cells per culture. Polyploid cells per 100 scored cells were also determined.
(c) Statistical methods: The data from the experimental groups were evaluated for statistical significance (p<0.05) by the Chi-square test.

Evaluation criteria:

(1) Assay validity: The assay was considered acceptable if
(a) the frequency of chromosome aberrations in the negative and/or solvent control cultures was within the performing laboratory’s historical range (not provided) and
(b) the positive controls induced significant increases in the frequency of aberrations.

Positive response: The test material was considered positive if least one dose caused a significant increase in the chromosome aberrations frequency compared to the negative control.

Statistics:

Statistical methods: The data from the experimental groups were evaluated for statistical significance (p<0.05) by the Chi-square test.

Key result

Species / strain:

lymphocytes: human

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

Additional information on results:

REPORTED RESULTS:
Cytotoxicity Assay. Initially doses ranging from 0.3 to 500.0 µg/mL +/-S9 were assessed for cytotoxic effects 24 and 48 hours post-treatment.
Slight compound precipitation was reported at concentrations >100.0 µg/mL +/-S9.
In the absence of S9 activation, the MI or cells sampled 24 hours post exposure to 500 µg/mL as markedly reduced (-75%) compared to the solvent value; a slight reduction was also seen at 250 µg/mL.
Below 250 µg/mL, the MI as not adversely affected by compound treatment.
There was an ca. 40% reduction in mitotic cell recovery 48 hours following treatment with 500 µg/mL -S9; no convincing evidence of cytotoxicity was seen at lower nonactivated doses. No adverse effects on the number of mitotic cells were seen with the S9-activated test material at either harvest time. Based on these preliminary findings, cultures exposed to nonactivated 10,0, 100.0, and 250.0 µg/mL (24-hour cell harvest) and 250.0 µg/mL (48-hour cell harvest) were examined for chromosome aberrations. No significant increases in the frequency of structural chromosome aberrations were found . Similarly, the incidence of numerical aberrations in treated groups were generally comparable to the negative and solvent control values.

In the presence of S9 activation, the test material was not cytotoxic at any level; accordingly, cultures exposed to 30, 250, 500 µg/mL (24-hour sampling time) and 500 µg/mL (48-hour sampling time) were examined for abnormal chromosome morphology. In agreement with the nonactivated findings, there were no significant increases in either structural or numerical chromosome aberrations at any S9-activated dose.

Based on the results the study author concluded that benzyl benzoate was not clastogenic in this in vitro human lymphocyte cytogenetic assay.

The US EPA has reviewed the study: REVIEWERS' DISCUSSION/CONCLUSIONS: We conclude that there was no evidence of a clastogenic response induced by nonactivated benzyl benzoate in human lymphocytes derived from a single donor. However, the biological significance of the rare complex aberrations at all S9-activated levels following the 2 -hour harvest illustrates the rationale for conduction human lymphocyte cytogenetic assays with replicate cultures from different donors or performing independent experiments. We believe that the relevance, if any, of these results could have been clearly established either by the
use of donor cells from a second source or the performance of a repeat test. We assess, therefore, that the data from the S9-activated phase of testing are inconclusive and that the study should be repeated.

Conclusions:

Interpretation of results: negative

Executive summary:

Human lymphocytes derived from a single donor were evaluated for chromosome aberrations 24 hours postexposure to three nonactivated doses (10.0, 100.0, and 250.0 µg/mL) and three S9-activated doses (30.0, 250.0, and 500.0 µg/mL of benzyl benzoate. Chromosome aberrations were also scored in cultures exposed to the high dose with and without S9 activation 8 hours posttreatment. Results indicated that levels >100.0 µg /mL +/-S9 were partially insoluble in culture medium and that nonactivated 500 µg/mL was cytotoxic. No evidence of a clastogenic effect was seen in the absence of S9 activation. However, no conclusions can be reached for the S9- activated phase of testing.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Published review of data not including information normally presented in regulatory guideline compliant studies

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

adapted to investigate photochemical reactions and interactions of photochemically induced products from commonly used fragrances elements

Principles of method if other than guideline:

Essential oil components, including benzyl benzoate, were irradiated to mimic UV/sunlight induced photochemical reactions and then tested in a standard Ames test for bacterial reverse mutation effects.

Fragrance materials have wide uses in consumer products including cosmetics, detergents, soaps and other houselhold products. natural and synthetic cemicals are used in the production of these fragrances and interactions can be expected based on the large number of chemicals involved. In addition there is a need for restrictions in fragrance use since photochemical reactions may result in toxic products and the widespread use of these fragrances in skin care products means the potential effects of photochemical reaction products should be investigated.

In this assay three major essential oil ingredients with uses in skin care products were investigated.
The results for benzyl benzoate are pertinent and generally only results for this substance are summarised in this dossier.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

reverse mutations in two standard tester strains, TA98 and TA100.

Species / strain / cell type:

S. typhimurium, other: TA98 and TA100

Metabolic activation:

with and without

Metabolic activation system:

S-9

Test concentrations with justification for top dose:

5000, 2000, 500, 50 and 5 µg/plate with and without S-9 metabolic activation. Two tester strains, TA98 and TA100, used in a plate incorporation and a pre-incubation assays.

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

furylfuramide

Remarks:

The test substances were administered as supplied and also as irradiated reaction mixtures to investigate possible UV degradation effects, and to assess safety of fragrances used on skin in personal care products exposed to sunlight

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

Salmonella typhimurium tester strains TA98 and TA100 were obtained from Dr BN Ames and examined for genetic properties before use. S-9 was prepared according to standard methods from polychlorinated biphenyl induced male Sprague-Dawley rats. The test substances were obtained as reagent grade materials and mixed with isopropyl alcohol for the irradiation step in either a photochemical reactor model or by exposure to UV or natural sunlight (for benzyl benzoate UV exposure was completed with or without the presence of a photosensitiser). Irradiated reaction mixtures were stored at 5°C until used.
Bacterial cultures were prepared according to standard Ames test methods and two standard positive controls, serially diluted 10 -, 100- or 1000 -fold in DMSO were also prepared. Plate incorporation and pre-incubation assays were set up and plates prepared in triplicate.

In each assay, the test substance was administered as supplied, as the irradiated reaction mixture and as the irradiated reaction mixture in the presence of one of two known photosensitsers, benzophenone or acetophenone. The four test substances were administered in a dose range of 5 to 5000 µg/plate, with or without metabolic activation, in both a pre-incubation assay or a plate incorporation assay. Replicates plates were prepared at each test concentration.

Benzo(a)pyrene and furylfuramide were included as positive controls to confirm validity of methods and optimum activity of the metabolic activation system

Evaluation criteria:

Microscopic examination of the bacterial lawn was used to determine cytotoxic effects. The presence of pin-point colonies was one criterion for determining a toxic effect

Statistics:

No data

Species / strain:

S. typhimurium, other: TA98 and TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Benzyl benzoate, benzyl acetate and eugenol were tested as irradiated reaction mixtures and as non-irradiated substances. Results for the benzyl benzoate and acetate assays are tabulated below. None of the three essential oil constituents were found to increase the frequency of reverse mutations in the two tester strains and were not mutagenic in the plate incorporation or pre-incubation assays.

The cytotoxicity determinations for each substance and its reactions products following UV irradiation confirmed that a large number of potential photochemical reactions could occur resulting in a diverse range of potentially cytotoxic products and this could be exacerbated by addition of photosensitising agents. The assay did not identify specific reaction products responsible for particular mechanisms of cytotoxicity but concluded that overall the essesntial oils in irradiated or non-irradited form, exhibited bacteriacidal activity at concentrations of 2000 µg/plate or higher.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

benzyl acetate

 

Sample	Concentration (µg/plate)	sensitiser	Plate incorporation assay				Pre-incubation assay
			TA98		TA100		TA98		TA100
			-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
1	5000	--	28	37	T	89	--	--	--	--
	2000	--	32	36	95	105	T	--	T	--
	500	--	31	31	101	95	T	30	65	120
	50	--	30	32	90	90	25	24	101	125
	5	--	30	34	90	89	--	26	96	121
2	5000	--	32	38	83	89	0	--	--	--
	2000	--	37	33	88	96	T	--	T	--
	500	--	34	35	91	94	T	27	74	120
	50	--	32	35	90	86	25	24	101	125
	5	--	32	36	92	92	--	26	96	121
3	5000	A	32	36	T	87	--	--	--	--
	2000	A	35	35	T	93	T	--	T	--
	500	A	34	35	91	94	T	--	T	--
	50	A	32	37	93	90	28	103	128	121
	5	A	31	38	87	92	--	27	104	122
4	5000	B	33	31	T	88	--	--	--	--
	2000	B	38	33	94	104	T	--	T	--
	500	B	32	35	108	105	T	23	89	118
	50	B	37	35	92	89	28	34	105	107
	5	B	29	37	97	91	--	26	96	125

A = acetophenone                   T = toxic

B =benzophenone

Sample 1 = non-irradiated benzyl acetate

Samples 2-4 = irradiated benzyl acetate

Benzyl benzoate

 

Sample	Concentration (µg/plate)	sensitiser	Plate incorporation assay				Pre-incubation assay
			TA98		TA100		TA98		TA100
			-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
1	5000	-	T	35	T	T	--	--	--	--
	500	-	T	36	T	90	T	--	T	--
	250	-	T	34	T	89	T	27	T	119
	50	-	34	39	92	105	29	50	50	121
	5	-	34	37	97	97	36	24	113	118
2	5000	-	T	35	T	94	--	--	--	--
	500	-	T	35	T	93	T	--	T	--
	250	-	T	34	T	92	T	29	T	115
	50	-	36	34	94	87	T	28	94	116
	5	-	35	32	91	93	33	27	98	--
3	5000	A	T	35	T	T	--	--	--	--
	500	A	T	37	T	90	T	--	T	--
	250	A	T	35	T	96	T	29	T	116
	50	A	35	36	93	95	T	29	47	121
	5	A	33	34	103	106	21	29	97	113
4	5000	B	T	T	T	92	--	--	--	--
	500	B	T	33	T	98	T	--	T	--
	250	B	T	37	T	94	T	31	T	105
	50	B	36	37	103	105	T	30	48	116
	5	B	34	36	104	100	24	29	98	119

 

A = acetophenone                   T = toxic

B =benzophenone

Sample 1 = non-irradiated benzyl benzoate

Samples 2-4 = irradiated benzyl benzoate

Conclusions:

Interpretation of results: negative.

Benzyl benzoate, irradiated or unchanged, in the presence or absence of photosensitising agents and in the presence or absence of metabolic activation gave negative responses for mutagenic activity in two tester strains, TA98 and TA100 of S.typhimurium, when evaluated in a plate incorporation or pre-incubation bacterial reverse mutation assay.

Executive summary:

The three essential oil constituents - eugenol, benzyl acetate and benzyl benzoate - were tested for mutagenic potential in the Salmonella typhimurium tester strains TA98 and TA100 after being irradiated by low pressure mercury lamp or exposed to UV/sunlight in an isopropyl alcohol solution in the presence of oxygen. Test mixtures were prepared without irradiation, with irradiation and irradiated in the presence of one of two photosensitisers, benzophenone or acetophenone. The four test mixtures were then evaluated in a plate incorporation assay with and without metabolic activation, three replicates per tested concentration over a range of 5 -5000 µg/plate, and also in a pre-incubation assay with and without metabolic activation, three replicates per tested concentration over a range of 5 -5000 µg/plate.

None of the test reaction mixtures gave any indication of mutagenic activity although all samples showed a degree of toxicity, bacteriacidal activity, at doses of 2000 µg/plate or higher.

The major photochemical reaction products were identified for each of the three essesntial oil constituents. For benzyl benzoate these were benzoic acid, benzyl alcohol and 2-methyl-1-phenyl-1,2-propanediol. The addition of a photosensitising agent promoted the formation of photochemical reaction products including solvent addition products.

One of the three essential oil constituents were found to increase the frequency of reverse mutations in the two tester strains and were not mutagenic in the plate incorporation or pre-incubation assays. The cytotoxicity determinations for each substance and its reactions products following UV irradiation confirmed that a large number of potential photochemical reactions could occur resulting in a diverse range of potentially cytotoxic products and this could be exacerbated by addition of photosensitising agents. The assay did not identify specific reaction products responsible for particular mechanisms of cytotoxicity but concluded that overall the essesntial oils in irradiated or non-irradited form, exhibited bacteriacidal activity at concentrations of 2000 µg/plate or higher.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Published guideline-comparable study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

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

Target gene:

histidine dependent bacterial strains

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 for concentrations administered in each assay.

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 0.05 µmol histidine and 0.05 µmol 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, the standard criteria of a two or three fold increase in revertant colony numbers would apply.

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:

Of the 239 compounds examined in this screening study, only 9 were found to be mutagenic. The large number of substances made it necessary to screen for potential mutagens using spot tests (which are less sensitive than quantitative experiments). For technical reasons, strain TA 100 is difficult to use in spot tests because of its high background (150 to 200 colonies/plate) and the potential for poor solubility, instability and/or toxicity of the test substance to influence results.
Benzyl acetate and benzyl benzoate were allocated to the same chemical grouping and gave the same response in the mutagenicity assay.

Remarks on result:

other: all strains/cell types tested

No additional information provided.

Conclusions:

Interpretation of results: negative in the presence and absence of metabolic activation.

Benzyl benzoate ws not found to be mutagenic in this assay.

Executive summary:

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 benzoate was not considered to be mutagenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

 

The mutagenicity of benzyl benzoate was investigated in two Ames tests. In both tests benzyl benzoate was negative. No evidence of clastogenicity was seen in the chromosome aberration test. Under the conditions of the Chinese hamster lung cell HGPRT forward gene mutation assay benzyl benzoate did not induce a mutagenic response in two independent assays.

 

In vitro data are also available for the read-across substance benzyl acetate (Ames test, mammalian cell mutation, chromosomal aberration). A study of UDS in vivo is also available for benzyl acetate. Since the in vitro assays gave both positive and negative indications of mutagenic activity, the results of the UDS in vivo study were considered key to interpretation of overall genotoxicity. Benzyl acetate was investigated for its ability to induce unscheduled DNA synthesis (UDS) in male F344 rats following in vivo treatment. Hepatocytes, isolated by liver perfusion, were used to assess the DNA-damaging 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 bw. The responses were examined at the 2 and 12 hour timepoints. The results of this study indicate that 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%.

Link to relevant study records

Reference

Endpoint:

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

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:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Published study.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Induction of UDS in the rat liver in vivo

GLP compliance:

not specified

Remarks:

Data is published literature

Type of assay:

unscheduled DNA synthesis

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

Remarks:

Doses / Concentrations:
50, 200, 1000 mg/kg bw
Basis:
no data

No. of animals per sex per dose:

3 male rats

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:
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 applicable

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:

Interpretation of results: negative
No evidence for the induction of UDS was seen in this assay

Executive summary:

The read-across substance benzyl acetate was investigated for its ability to induce unscheduled DNA synthesis (UDS) in male F344 rats following in vivo treatment. Hepatocytes, isolated by liver perfusion, were used to assess the DNA-damaging 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 bw. The responses were examined at the 2 and 12 hour timepoints. The results of this study indicate that 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%.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

No evidence of mutagenicity was seen in a Ames test performed with Salmonella typhimurium strains TA98 and TA100 using benzyl benzoate; additional assays performed with uv irradiation in the presence of a photosensitiser also gave negative results. Cytotoxicity was observed in these assays at concentrations of 2000 µg/plate or higher (Schunk et al, 1986). Benzyl benzoate and the read-across substance benzyl acetate also gave negative results in four strains of S. typhimurium, with or without metabolic activation (Florin et al, 1980).

 

No evidence of clastogenicity was seen in a study performed with benzyl acetate using CHL cells (Matsuoka et al, 1996). Benzyl acetate gave a negative result for mammalian cell mutation in mouse lymphoma cells (L5178Y) and human lymphoma cells (TK6) in the absence of metabolic activation; a positive result was seen in both cell types in the presence of metabolic activation (Caspary et al, 1988). 

 

Benzyl acetate was investigated for its ability to induce unscheduled DNA synthesis (UDS) in the hepatocytes of F-344 rats following in vivo treatment.  Rats received a single dose of the test substance dissolved or suspended in corn oil and administered by oral gavage at dose levels of 5, 200 and 1000 mg/kg bw (Mirsalis et al, 1989). The responses were examined at the 2 and 12 hour time points.  A negative response was seen in this assay.

 

The weight of evidence from studies in vitro and in vivo therefore indicate that benzyl benzoate is not genotoxic in vivo.

Justification for selection of genetic toxicity endpoint
A battery of in vitro and in vivo tests with the test substance or read-across candidates, indicated no significant mutagenic activity. 

Justification for classification or non-classification

Results from a battery of genotoxicity tests combine to indicate no mutagenic activity for benzyl benzoate and no classification is justified for genotoxicity in accordance with the requirements of CLP Regulation 1272/2008