1-phenylethyl acetate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- negative, in vitro bacterial reverse mutation (with and without S-9 activation), QSAR and read -across to Benzyl acetate

- negative, in vitro micronucleus (with and without S-9 activation), OECD TG 487 - Peripheral human lymphocyte , 2019     

- negative, in vitro gene mutation test in mammalian cells (with and without S-9 activation), OECD TG 490 - mouse lymphoma cell line - L5178Y thymidine kinase assay, 2019

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

other: Experimental study for structurally similar analogue included as relevant supporting information to strengthen the use of QSAR to address in vitro bacterial reverse mutation test.

Adequacy of study:

key study

Study period:

Not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

A valid study is available for the analogue substance benzyl acetate. It is conducted in compliance with good scientific principles, with no or minor deviations from standard protocols. The read-across is considered to be suitable based on the structural and “mechanistic action” similarities between the target substance (1-phenylethyl acetate) and source substance (benzyl acetate) and their similar physico-chemical properties.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

The methods are comparable to the updated standardised guideline, except that a strain capable of detecting cross linkage was not used, this is comparable to previous versions of this guideline.

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

yes

Remarks:

The methods are comparable to the updated standardised guideline, except that a strain capable of detecting cross linkage was not used, this is comparable to previous versions of this guideline.

Principles of method if other than guideline:

The mutagenicity of benzyl acetate was assessed in a screen of 270 chemicals. The test was performed using six dosing levels (0, 33, 100, 333, 1000, 3333 and 10000 µg/plate), a solvent control and appropriate positive controls in S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 . The test was performed using the preincubation technique.

GLP compliance:

not specified

Remarks:

(pre-dates GLP inception)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine synthesis

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Type and identity of media: Columbia agar slants at 4 °C in Columbia broth, grown overnight for 12-15 hours at 37 °C on a shaker.- Properly maintained: - 70 °C freezerPrior to use, the phenotypes of all strains were analysed.

Metabolic activation:

with and without

Metabolic activation system:

10 % Rat liver S-9 and 10 % Hamster liver S-9 (Arcolor 1254 induced)

Test concentrations with justification for top dose:

0, 33, 100, 333, 1000, 3333 and 10000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

with strains TA 1537 and TA 100 without metabolic acitvation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine

Remarks:

with strain TA 98 without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

with strains TA 97 and TA 1537 without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with all strains with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubationTo each test tube 0.5 mL of S-9 mix or 0.1 M PO4 buffer (pH 7.4), 0.05 mL of the overnight culture, and 0.05 mL of solvent were added. After preincubation, 2.0 mL of molten (45 °C) top agar supplemented with 0.5 mM L-histidine and 0.5 mM D-biotin were added. The contents of the tubes were mixed and poured onto 25 mL of minimal glucose bottom agar in Fisher Scientific Plates. When the top agar had solidified, the plates were inverted and incubated.DURATION- Preincubation period: 20 minutes (at 37 °C)- Expression time (cells in growth medium): 48 hours (at 37 °C)NUMBER OF REPLICATIONS: 3 plates per doseDETERMINATION OF CYTOTOXICITY- Method: Appearance of his- pinpoint colonies, reduced numbers of revertant colonies per plate, or thinning or absence of the bacterial lawn.

Evaluation criteria:

A response was considered mutagenic is a dose-related, reproducible increase in the number of revertants over the spontaneous background was observed even if the increase was less than twofold. No increase in the number of revertants was classified as non-mutagenic. A response was considered questionable when there was an absence of a clear-cut dose-related increase in revertants when the dose-related increases in the number of revertants were not reproducible or when the response was of insufficient magnitude to support a determination of mutagenicity.

Statistics:

The mean response and standard deviation was determined for each strain tested at all dose levels.

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:

cytotoxicity

Remarks:

Complete clearing of the background lawn at 1000 µg/plate in strain TA 1537 without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: A preliminary screen was performed with strain TA 100 in the presence and absence of metabolic activation over a wide dose range with an upper limit of 10 mg/plate unless solubility problems were encountered.ANALOGUE APPROACH JUSTIFICATION:- See attached “Justification for read-across” document for full details.- In summary, important considerations for the use of read-across for acute toxicity are: i) 1-phenylethyl acetate (the target chemical) has similar physico-chemical properties as benzyl acetate (the source substance), ii) there are structural similarities between the two chemicals, iii) the OECD QSAR Toolbox assigns an identical toxicity profiles to both chemicals, and iv) both chemicals have been tested for acute oral toxicity, which demonstrated that neither substance requires classification for acute toxicity and that the benzyl acetate will represent a worst-case scenario, and are adequate for classification and labelling and risk assessment purposes. The information reported in this summary is included to demonstrate comparability between the source (benzyl acetate) and target (1-phenyl-ethyl acetate) substance.

Remarks on result:

other: all strains/cell types tested

Table 1: Results

Dose (µg/plate)	TA 100						TA 1535						TA 1537						TA 98
	-S9		HLI		RLI		- S9		HLI		RLI		- S9		HLI		RLI		- S9		HLI		RLI
	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM
0	97	3.5	102	7.3	105	14.9	8	0.3	7	1.5	6	1.3	2	0.0	6	1.2	7	1.3	12	2.8	16	2.6	20	1.2
33	102	6.7					5	0.9
100	101	4.0	67	2.7	122	2.9	4	0.6	6	1.2	6	1.5	2	1.0	8	2.3	6	1.2	11	1.2	20	1.8	23	2.5
333	85	5.8	97	9.6	110	12.3	5	0.6	8	1.2	6	1.2	5	1.0	4	0.9	9	0.3	13	0.3	20	3.8	24	1.9
1000	91	14.4	93	2.7	99	12.1	4	0.6	2	0.9	5	0.3	1	0.7	4	1.2	7	3.2	15	3.5	16	1.9	20	3.2
3333	58	11.7	104	15.5	78	4.1	3	0.3	3	1.2	8	3.0	5		3	0.3	5	1.5	12	2.3	19	2.3	19	4.8
10000			84	9.0	95	6.5			1	0.7	5	2.3	t		6	1.2	4	1.3	8	1.0	12	1.3	18	4.1
Positive Control	481	29.2	1770	34.1	961	89.2	724	63.9	106	2.6	43	3.5	224	32.4	79	9.2	82	14.2	140	3.7	1169	69.6	524	49.4
RLI = with 10 % Rat liver S9 metabolic activation HLI = with 10 % Hamster liver S9 metabolic activation t = complete clearing of bacterial lawn

 

Conclusions:

Under the conditions of the test, the test substance was found not to be mutagenic in an Ames test using S. typhimurium strains TA1535, TA1537, TA98 and TA100 in the presence and absence of metabolic activation.

Executive summary:

The mutagenicity of benzyl acetate was assessed in an Ames test with S. typhimurium strains TA1535, TA1537, TA98 and TA100 using the preincubation technique. Some toxicity was observed in one strain at the highest dose tested. Under the conditions of the test, the test substance was found to be non-mutagenic in all strains with and without metabolic activation.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 Jan 2019 to 19 May 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

adopted 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

In vitro Micronucleus in Mammalian cells

Species / strain / cell type:

lymphocytes: peripheral human lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: peripheral human lymphocytes
- Suitability of cells: Peripheral human lymphocytes are recommended in the international OECD guideline.

For lymphocytes:
- Sex, age and number of blood donors: 6 donors, age: 25, 27, 26, 31, 26, 25 years,
- Whether whole blood or separated lymphocytes were used: separated lymphocytes
- Whether blood from different donors were pooled or not: No
- Mitogen used for lymphocytes: phytohaemagglutinin

MEDIA USED
Culture medium consisted of RPMI 1640 medium (Life Technologies), supplemented with
20% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum (Life Technologies), L-glutamine
(2 mM) (Life Technologies), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively)
(Life Technologies) and 30 U/mL heparin (Sigma, Zwijndrecht, The Netherlands).
All incubations were carried out in a controlled environment, in which optimal conditions
were a humid atmosphere of 80 - 100% (actual range 26 - 97%), containing 5.0 ± 0.5% CO2
in air in the dark at 37.0 ± 1.0°C (actual range 34.1 - 38.2°C). Temperature and humidity
were continuously monitored throughout the experiment. The CO2 percentage was monitored
once on each working day.

Cytokinesis block (if used):

The cells were re-suspended in 5 mL culture medium with Cytochalasin B (5 μg/mL) and incubated for another 24 hours.

Metabolic activation:

with and without

Metabolic activation system:

Rat S9 homogenate was obtained from Trinova Biochem GmbH, Giessen, Germany and is
prepared from male Sprague Dawley rats that have been dosed orally with a suspension of
phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg).
S9-mix was prepared immediately before use and kept refrigerated. S9-mix components
contained per mL physiological saline: 1.63 mg MgCl2.6H2O (Merck); 2.46 mg KCl
(Merck); 1.7 mg glucose-6-phosphate (Roche, Mannheim, Germany); 3.4 mg NADP (Randox
Laboratories Ltd., Crumlin, United Kingdom); 4 μmol HEPES (Life Technologies).
The above solution was filter (0.22 m)-sterilized. To 0.5 mL S9-mix components 0.5 mL
S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix.
Metabolic activation was achieved by adding 0.2 mL S9-mix to 5.3 mL of a lymphocyte
culture (containing 4.8 mL culture medium, 0.4 mL blood and 0.1 mL (9 mg/mL)
phytohaemagglutinin). The concentration of the S9-fraction in the exposure medium was
1.8% (v/v).

Test concentrations with justification for top dose:

Based on the results of the dose-range finding test the following dose levels were selected for
the first cytogenetic assay:
Without and with S9-mix: 100, 600, 700, 800, 900, 1000, 1100, 1200 μg/mL culture medium (3 hours exposure time, 27 hours harvest time).

Vehicle / solvent:

The vehicle for the test item was dimethyl sulfoxide (DMSO, SeccoSolv, Merck, Darmstadt,
Germany)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

colchicine

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

First Cytogenetic Assay
Lymphocytes were cultured for 46 ± 2 hours and thereafter exposed in duplicate to selected doses of GARDENOL for 3 hours in the absence and presence of S9-mix. After 3 hours exposure, the cells were washed and were re-suspended in 5 mL culture medium with Cytochalasin B (5 μg/mL) and incubated for another 24 hours. To be able to select appropriate dose levels for scoring several repeat assays had to be performed.
Second Cytogenetic Assay
Lymphocytes were cultured for 46 ± 2 hours and thereafter exposed in duplicate to selected
doses of GARDENOL with cytochalasin B (5 μg/mL) for 24 hours in the absence of S9-mix.
Appropriate vehicle and positive controls were included in the second cytogenetic assay. To
be able to select appropriate dose levels for scoring a repeat assay had to be performed. Preparation of Slides
Cell cultures were centrifuged and were re-suspended in 1% Pluronic F68. After centrifugation , the cells in the remaining pellet were swollen by hypotonic 0.56% (w/v) potassium chloride solution. Immediately after, ethanol: acetic acid fixative (3:1 v/v) was added. Fixed cells were dropped onto cleaned slides. At least two slides were prepared per culture. Slides were stained for with 6.7% (v/v) Giemsa solution.

Cytogenetic Assessment/Scoring of Micronuclei
The following criteria for scoring of binucleated cells were used (1 - 2, 6):
 Main nuclei that were separate and of approximately equal size.
 Main nuclei that touch and even overlap as long as nuclear boundaries are able to be
distinguished.
 Main nuclei that were linked by nucleoplasmic bridges.
The following cells were not scored:
 Trinucleated, quadranucleated, or multinucleated cells.
 Cells where main nuclei were undergoing apoptosis (because micronuclei may be gone
already or may be caused by apoptotic process).
The following criteria for scoring micronuclei were adapted from Fenech, 1996 (1):
 The diameter of micronuclei should be less than one-third of the main nucleus.
 Micronuclei should be separate from or marginally overlap with the main nucleus as long
as there is clear identification of the nuclear boundary.
 Micronuclei should have similar staining as the main nucleus.

Rationale for test conditions:

A solubility test was performed based on visual assessment. The test item formed a clear
colourless solution in DMSO.
In order to select the appropriate dose levels cytotoxicity data was obtained in a dose-range finding test in presence and in absence of S9-mix.
The highest tested concentration was the recommended dose level of 1642 μg/mL (= 0.01 M).
Cytotoxicity of GARDENOL in the lymphocyte cultures was determined using the cytokinesis-block proliferation index (CBPI index).
Based on the results of the dose-range finding test an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level showed a cytotoxicity of
55 ± 5% whereas the cytotoxicity of the lowest dose level was approximately the same as the
cytotoxicity of the solvent control.
At least three test concentrations (not including the solvent and positive controls) that meet the acceptability criteria (appropriate cytotoxicity, number of cells, etc) should be evaluated

Evaluation criteria:

A test item is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test
if all of the following criteria are met:
a) At least one of the test concentrations exhibits a statistically significant (Chi-square test,
one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose-related in at least one experimental condition when evaluated with a
Cochran Armitage trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
A test item is considered negative (not clastogenic or aneugenic) in the in vitro micronucleus
test if:
a) None of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a Cochran Armitage trend
test.
c) All results are inside the 95% control limits of the negative historical control data range. The Chi-square test showed that there are statistically significant differences between one or more of the test item groups and the vehicle control group. Therefore a Cochran Armitage trend test (p < 0.05) was performed to test whether there is a significant trend in the induction.

Statistics:

CBPI=(No. mononucleate cells) + (2 x No. binucleate cells) + (3 x No. multinucleate cells)/(Total number of cells)
%Cytostasis = 100-100{(CBPIt – 1)/(CBPIc –1)} ;c=vehicle control culturevehicle control culture; t=test item or control treatment culture.
The incidence of micronucleated cells (cells with one or more micronuclei) for each exposure group was compared to that of the solvent control using Chi-square statistics.

Species / strain:

lymphocytes: peripheral human lymphocites

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

In conclusion, this test is valid and that GARDENOL is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate GARDENOL for its ability to induce micronuclei in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (S9-mix). The possible clastogenicity and aneugenicity of GARDENOL was tested in two independent experiments.

The study procedures described in this report are in compliance with the most recent OECD guideline.

Batch VE00586353 of GARDENOL was a colourless liquid. The vehicle of the test item was dimethyl sulfoxide.

In the first cytogenetic assay, GARDENOL was tested up to 900 and 1000 μg/mL for a 3 hours exposure time with a 27 hours harvest time in the absence and presence of S9-fraction, respectively. Appropriate toxicity was reached at these dose levels. In the second cytogenetic assay, GARDENOL was tested up to 550 μg/mL for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix. Appropriate toxicity was reached at this dose level.

The number of mono- and binucleated cells with micronuclei found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. In addition, the number of mono- and binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

GARDENOL did not induce a statistically significant and biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments.

In conclusion, this test is valid and GARDENOL is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 01 Jul 2019 to 15 Sep 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

adopted 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

TK locus in L5178Y mouse lymphoma cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: American Type Culture Collection, (ATCC, Manassas, USA) (2001).
- Suitability of cells: Recommended test system in international guidelines (e.g. OECD).
- Normal cell cycle time (negative control):

For cell lines:
- Absence of Mycoplasma contamination: yes
- Methods for maintenance in cell culture:
- Cell cycle length, doubling time or proliferation index :
- Modal number of chromosomes:
- Periodically checked for karyotype stability: [yes/no]
- Periodically ‘cleansed’ of spontaneous mutants: [yes/no]

MEDIA USED
All incubations were carried out in a humid atmosphere (80 - 100%, actual range 45 - 99%)
containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.2 - 37.5°C).
Temperature and humidity were continuously monitored throughout the experiment. The
CO2 percentage was monitored once on each working day. Temporary deviations from the
temperature, humidity and CO2 percentage may occur due to opening and closing of the
incubator door. Any variation to these conditions were evaluated and maintained in the raw
data.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Metabolic Activation System
Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and was prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg body weight).

Preparation of S9-Mix
S9-mix was prepared immediately before use and kept refrigerated. S9-mix components contained per mL physiological saline: 1.63 mg MgCl2.6H2O (Merck); 2.46 mg KCl (Merck); 1.7 mg glucose-6-phosphate (Roche, Mannheim, Germany); 3.4 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom); 4 μmol HEPES (Life technologies). The above solution was filter (0.22 μm)-sterilized. To 0.5 mL S9-mix components 0.5 mL S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix.

The concentration of the S9-fraction in the exposure medium was 4% (v/v).

Test concentrations with justification for top dose:

Concentration without metaolic activation 3 h [μg/ml)]: 10, 25, 50, 100, 300, 600 (top dose determined by cytoticity)
Concentration with metaolic activation 3 h [μg/ml)]: 8, 16, 32, 64, 125, 250, 500, 1000 (top dose determined by précipitation in the medium)

Concentration without metaolic activation 24 h [μg/ml)]: 5, 10, 25, 50, 100, 250, 300, 350 (top dose determined by cytoticity)

Vehicle / solvent:

- The vehicle for the test item was dimethyl sulfoxide (DMSO, Merck, Darmstadt, Germany)

The final concentration of the solvent in the culture medium was 1.0% (v/v).

Untreated negative controls:

no

Remarks:

.

Negative solvent / vehicle controls:

yes

Remarks:

The negative control was dimethyl sulfoxide, the vehicle of the test item

True negative controls:

yes

Remarks:

The negative control was dimethyl sulfoxide, the vehicle of the test item

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

Per culture 8 x 106 cells (106 cells/mL for 3 hour treatment) or 6 x 106 cells (1.25 x 105 cells/mL for 24 hour treatment) were used. The cell cultures for the 3 hour treatment were placed in sterile 30 mL centrifuge tubes, and incubated in a shaking incubator at 37.0 ± 1.0°C and 145 rpm. The cell cultures for the 24 hour treatment were placed in sterile 75 cm2 culture flasks at 37.0 ± 1.0°C. Solvent and positive controls were included and the solvent control was tested in duplicate.
In the first experiment, cell cultures were exposed for 3 hours to GARDENOL in exposure medium in the absence and presence of S9-mix. In the second experiment, cell cultures were exposed to GARDENOL in exposure medium for 24 hours in the absence of S9-mix.

For the 3 hour treatment, cell cultures were exposed to the test item in exposure medium in the absence as well as in the presence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were resuspended in 50 mL growth medium (R10-medium).
For the 24 hour treatment, cell cultures were exposed to the test item in exposure medium in the absence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were resuspended in 20 mL growth medium (R10-medium)The cells in the final suspension were counted with the coulter particle counter.

For expression of the mutant phenotype, the remaining cells were cultured for 2 days after the treatment period. During this culture period at least 4 x 106 cells (where possible) were subcultured every day in order to maintain log phase growth. Two days after the end of the treatment with the test item the cells were plated for determination of the cloning efficiency (CEday2) and the mutation frequency (MF).

For determination of the CEday2 the cell suspensions were diluted and seeded in wells of a 96-well dish. One cell was added per well (2 x 96-well microtiter plates/concentration) in non-selective medium.
For determination of the mutation frequency (MF) a total number of 9.6 x 105 cells per concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in
selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 105 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection). The microtiter plates for CEday2 and MF were incubated for 11 or 12 days.
After the incubation period, the plates for the TFT-selection were stained for 1.5-2 hours, by adding 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma) to each well. The plates for the CE day2 and MF were scored with the naked eye or with the microscope.
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120% in order to have an acceptable number of surviving cells analyzed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 106 survivors and ≤ 170 per 106 survivors.
c) The suspension growth (SG) over the 2-day expression period for the solvent controls should be between 8 and 32 for the 3 hour treatment, and between 32 and 180 for the 24 hour treatment.
d) The positive control should demonstrate an absolute increase in the total mutation frequency, that is, an increase above the spontaneous background MF (an induced MF (IMF)) of at least 300 x 10-6. At least 40% of the IMF should be reflected in the small colony MF. And/or, the positive control has an increase in the small colony MF of at least 150 x 10-6 above that seen in the concurrent solvent control (a small colony IMF of
150 x 10-6).
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

Rationale for test conditions:

The highest doses that were tested gave a cell survival of approximately 10-20% and the survival in the lowest doses was approximately the same as the cell survival in the solvent control. Also some intermediate doses were tested.
The highest tested concentration was 1642 μg/mL exposure medium. This concentration was
equal to 0.01 M.
Limited factors are Precipitation in the medium and toxicity.

Evaluation criteria:

The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.

Statistics:

Mutagenicity tests:
The suspension growth (SG) for the 3 hour treatment= [Day 1 cell count/1.6 x 105] x [Day 2 cell count/1.25 x 105]
The suspension growth (SG) for the 24 hour treatment= [Day 0 cell count/1.25 x 105] x [Day 1 cell count/1.25 x 105] x [Day 2 cell count/1.25 x 105]
Relative Suspension Growth (RSG) = SG (test) / SG (controls) x 100
The cloning efficiency was determined by dividing the number of empty wells by the total number of wells. The value obtained is the P(0), the zero term of thePoisson distribution:
P(0) = number of empty wells/total number of wells
The cloning efficiency (CE) was then calculated as follows:
CE = -ln P(0)/number of cells plated per well
The relative cloning efficiency (RCE) at the time of mutant selection =
CE (test) / CE (controls) x 100
The Relative Total Growth (RTG) was also calculated as the product of the cumulative
relative suspension growth (RSG) and the relative survival for each culture:
RTG = RSG x RCE/100
The mutation frequency was expressed as the number of mutants per 106 viable cells. The
plating efficiencies of both mutant and viable cells (CE day2) in the same culture were
determined and the mutation frequency (MF) was calculated as follows:
MF = {-ln P(0)/number of cells plated per well}/ CE day2 x 106
Small and large colony mutation frequencies were calculated in an identical manner.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: The pH at the concentration of 1250 μg/mL was 8.05 compared to 8.06 in solvent control (mesured in another study 20186557)
- Data on osmolality: The osmolarity at the concentration of 1250 μg/mL was 0.386 Osm/kg compared to 0.392 Osm/kg in the solvent control (mesured in another study 20186557)
- Possibility of evaporation from medium:
- Water solubility: No
- Precipitation and time of the determination: Precipitation at 1000 μg/mL and above after 3 hours treatment period and at 1642 μg/mL after the 24 hour treatment period.
- Definition of acceptable cells for analysis:
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES (if applicable):
In the dose-range finding test, L5178Y mouse lymphoma cells were treated with a test item
concentration range of 63 to 1642 μg/mL in the absence of S9-mix with 3 and 24 hour
treatment periods and in the presence of S9-mix with a 3 hour treatment period.
Table 1 shows the cell counts of the cultures after 3 hours of treatment with various
concentrations of the test item and after 24 and 48 hours of subculture, the calculated
suspension growth and the relative suspension growth.
In the absence of S9-mix, the relative suspension growth was 84% at the test item
concentration of 500 μg/mL compared to the relative suspension growth of the solvent
control. No cell survival was observed at test item concentrations of 1000 μg/mL and above.
In the presence of S9-mix, the relative suspension growth was 55% at the test item
concentration of 1000 μg/mL compared to the relative suspension growth of the solvent
control. Hardly any cell survival was observed at the test item concentration of
1642 μg/mL.
Table 2 shows the cell counts of the cultures after 24 hours of treatment with various
concentrations of the test item and after 24 hours of subculture and the calculated suspension
growth and the relative suspension growth.
The relative suspension growth was 46% at the test item concentration of 250 μg/mL
compared to the relative suspension growth of the solvent control. No or hardly any cell
survival was observed at test item concentrations of 500 μg/mL and above.
Since in the dose range finding test in the absence of S9-mix with a 3 hour treatment period, a
very steep toxicity border was observed, an additional experiment was performed. In the
additional dose range finding test, L5178Y mouse lymphoma cells were treated with a test
item concentration range of 500 to 1000 μg/mL in the absence of S9-mix with a 3 hour
treatment period.
Table 3 shows the cell counts of the cultures after 3 hours of treatment with various
concentrations of the test item and after 24 and 48 hours of subculture, the calculated
suspension growth and the relative suspension growth.
In the absence of S9-mix, the relative suspension growth was 45% at the test item
concentration of 600 μg/mL compared to the relative suspension growth of the solvent
control. No cell survival was observed at test item concentrations of 700 μg/mL and above.

STUDY RESULTS
- Concurrent vehicle negative and positive control data

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible
- Statistical analysis; p-value if any
- Any other criteria: e.g. GEF for MLA

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
o Relative total growth (RTG) or relative survival (RS) and cloning efficiency

- Genotoxicity results:
o Number of cells treated and sub-cultures for each cultures
o Number of cells plated in selective and non-selective medium
o Number of colonies in non-selective medium and number of resistant colonies in selective medium, and related mutant frequency
o When using the thymidine kinase gene on L5178Y cells: colony sizing for the negative and positive controls and if the test chemical is positive, and related mutant frequency. For the MLA, the GEF evaluation.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data:
- Negative (solvent/vehicle) historical control data:

Conclusions:

In conclusion, GARDENOL is not mutagenic in the TK mutation test system under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate the mutagenic potential of GARDENOL by testing its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, either in the absence or presence of a metabolic system (S9-mix). The TK mutational system detects base pair mutations, frame shift mutations and small deletions.

The test was performed in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3 hour treatment period.

The study procedures described in this report were based on the most recent OECD guideline.

Batch VE00586353 of the test item was a colourless liquid. The vehicle of the test item was dimethyl sulfoxide.

In the first experiment, the test item was tested up to concentrations of 600 and 1000 μg/mL in the absence and presence S9-mix, respectively. The incubation time was 3 hours. Relative total growth (RTG) was reduced to 13 and 36% in the absence and presence of S9-mix, respectively. The test item precipitated in the culture medium at the concentration of 1000 μg/mL.

In the second experiment, the item was tested up to concentrations of 350 μg/mL in the absence of S9-mix. The incubation time was 24 hours. The RTG was reduced to 14%.

The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical dw negative control database.

Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, GARDENOL did not induce a biologically relevant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment.

In the presence of S9-mix, GARDENOL did not induce a biologically relevant increase in the mutation frequency.

In conclusion, GARDENOL is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Predicted to be negative- based on in vitro genotoxicity studies, QSAR and read-across to Benzyl acetate

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

other: Experimental study for structurally similar analogue included as relevant supporting information to strengthen the use of QSAR to address in vivo chromosome aberration test.

Adequacy of study:

key study

Study period:

Not reported.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

A valid study is available for the analogue substance benzyl acetate. It is conducted in compliance with good scientific principles, with no or minor deviations from standard protocols. The read-across is considered to be suitable based on the structural and “mechanistic action” similarities between the target substance (1-phenylethyl acetate) and source substance (benzyl acetate) and their similar physico-chemical properties.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

not specified

Principles of method if other than guideline:

The test method employed demonstrates basic compliance with the guideline OECD 474.

GLP compliance:

not specified

Type of assay:

micronucleus assay

Species:

mouse

Strain:

not specified

Sex:

male

Details on test animals or test system and environmental conditions:

Not reported

Route of administration:

intraperitoneal

Vehicle:

Corn oil

Details on exposure:

Males were injected intraperitoneally three times at 24 hour intervals with test substance dissolved in corn oil; the total dosing volume was 0.4 mL. Solvent control animals were injected with 0.4 mL of corn oil only. Animals were killed by cervical dislocation 24 hours after the third injection.

Duration of treatment / exposure:

3 single injections

Frequency of treatment:

Injections were 24 hours apart.

Post exposure period:

24 hours post the 3rd injection

Remarks:

Doses / Concentrations:0, 312.5, 625 and 1250 mg/kgBasis:nominal conc.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

The positive control animals received injections of dimethylbenzanthracene.

Tissues and cell types examined:

Bone marrow cells were obtained from the femurs.

Details of tissue and slide preparation:

Smears were prepared from the bone marrow cells obtained from the femurs. Air-dried smears were fixed and stained.

Evaluation criteria:

2,000 polychromatic erythrocytes (PCEs) were scored for the frequency of micronucleated cells in each of five animals per dose group. The results were tabulated as the mean of the pooled results from all animals within a treatment group plus or minus the standard error of the mean.

Statistics:

Analysis of variance (ANOVA)

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No increase in micronucleated polychromatic erythrocytes was observed in bone marrow smears of male mice treated by intraperitoneal injection three times at 24 hour intervals.ANALOGUE APPROACH JUSTIFICATION:- See attached “Justification for read-across” document for full details.- In summary, important considerations for the use of read-across for acute toxicity are: i) 1-phenylethyl acetate (the target chemical) has similar physico-chemical properties as benzyl acetate (the source substance), ii) there are structural similarities between the two chemicals, iii) the OECD QSAR Toolbox assigns an identical toxicity profiles to both chemicals, and iv) both chemicals have been tested for acute oral toxicity, which demonstrated that neither substance requires classification for acute toxicity and that the benzyl acetate will represent a worst-case scenario, and are adequate for classification and labelling and risk assessment purposes. The information reported in this summary is included to demonstrate comparability between the source (benzyl acetate) and target (1-phenyl-ethyl acetate) substance.

Table 1: Induction of Micronuclei in Mouse Bone Marrow Cells

Compound	Dose (mg/kg)	Micronucleated PCEs/1000 cells	PCEs (%)
Dimethylbenzanthracene	12.5	8.6 ± 0.64	51.7 ± 4.61
Benzyl acetate	0	3.0 ± 0.69	69.9 ± 2.37
	312.5	2.9 ± 0.60	65.8 ± 3.18
	625	3.2 ± 0.6	64.3 ± 5.41
	1250	1.8 ± 0.46	60.7 ± 3.08
		Trend test P = -0.076	ANOVA P = 0.0412

Conclusions:

No increase in micronucleated polychromatic erythrocytes was observed in bone marrow smears of male mice treated by intraperitoneal injection three times at 24 hour intervals.

Executive summary:

The study was performed as part of a genetic toxicity review in the NTP (1993) study examining carcinogenicity and toxicity. The study was performed in line with good scientific principles and was performed to a method that was in basic compliance with OECD 474. No information relating to the GLP status of the study was reported. No increase in micronucleated polychromatic erythrocytes was observed in bone marrow smears of male mice treated with benzyl acetate (312.5 to 1250 mg/kg) by intraperitoneal injection three times at 24 hour intervals.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

other: Experimental study for structurally similar analogue included as relevant supporting information to strengthen the use of QSAR to address in vivo chromosome aberration test.

Adequacy of study:

key study

Study period:

Not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

A valid study is available for the analogue substance benzyl acetate. It is conducted in compliance with good scientific principles, with no or minor deviations from standard protocols. The read-across is considered to be suitable based on the structural and “mechanistic action” similarities between the target substance (1-phenylethyl acetate) and source substance (benzyl acetate) and their similar physico-chemical properties.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

not specified

Principles of method if other than guideline:

The test method employed demonstrates basic compliance with the guideline OECD 475.

GLP compliance:

not specified

Type of assay:

chromosome aberration assay

Species:

mouse

Strain:

B6C3F1

Sex:

male

Details on test animals or test system and environmental conditions:

Not reported

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil (injection volume 0.4 mL).

Details on exposure:

Male B6C3F1 mice were injected intraperitoneally with test substance dissolved in corn oil. The animals were subcutaneously implanted with a BrdU tablet (McFee et al 1983) 17 hours before the scheduled harvest (for the standard protocol, the animals were implanted an hour prior to injection with the test substance). The use of BrdU allowed selection of the appropriate cell population for scoring.

Duration of treatment / exposure:

A single exposure

Frequency of treatment:

A single exposure

Post exposure period:

17 hours in the standard protocol36 hours in the extended harvest protocol

Remarks:

Doses / Concentrations:425, 850 and 1700 mg/kgBasis:no data17 hour harvest

Remarks:

Doses / Concentrations:325, 650 and 1300 mg/kgBasis:no data36 hour harvest

No. of animals per sex per dose:

10

Control animals:

yes

Positive control(s):

dimethylbenzanthracene

Details of tissue and slide preparation:

The use of BrdU allowed selection of the appropriate cell population for scoring (abs induced by chemical administration are present in maximum number at the first metaphase following treatment; they decline in number during subsequent nuclear cell divisions due to cell death). Two hours before sacrifice, the animals received an intraperitoneal injection of colchicine in saline. The animals were killed by cervical dislocation 17 or 36 hours post administration (18 hours after BrdU dosing). One or both femurs were removed and the marrow was flushed out with phosphate-buffered saline (pH 7.0). Cells were treated with hypotonic salt solution, fixed and dropped onto chilled slides. After a 24-hour drying period the slides were stained and scored.

Evaluation criteria:

Fifty first-division metaphase cells were scored from each of four animals per treatment. Responses were evaluated as the percentage of aberrant metaphase cells, excluding gaps.

Statistics:

The data were analysed by a trend test (Margolin et al, 1986)

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No induction of chromosome aberrations were observed in the bone marrow cells of animals treated by intraperitoneal injection with benzyl acetate, with either the standard or extended harvest times. The highest dose tested was 1700 mg/kg for the standard harvest protocol and 1300 mg/kg for the extended harvest experiment. In the range finding study, the highest dose (1700 mg/kg) was limited by toxicity and induction of cell cycle delay.ANALOGUE APPROACH JUSTIFICATION:- See attached “Justification for read-across” document for full details.- In summary, important considerations for the use of read-across for acute toxicity are: i) 1-phenylethyl acetate (the target chemical) has similar physico-chemical properties as benzyl acetate (the source substance), ii) there are structural similarities between the two chemicals, iii) the OECD QSAR Toolbox assigns an identical toxicity profiles to both chemicals, and iv) both chemicals have been tested for acute oral toxicity, which demonstrated that neither substance requires classification for acute toxicity and that the benzyl acetate will represent a worst-case scenario, and are adequate for classification and labelling and risk assessment purposes. The information reported in this summary is included to demonstrate comparability between the source (benzyl acetate) and target (1-phenyl-ethyl acetate) substance.

Table 1: Induction of Chromosome Aberrations in Mouse Bone Marrow Cells

Treatment	Dose (mg/kg)	% aberrant cells
		Standard harvest	Extended harvest
Corn oil		2.75 ± 0.65	2.75 ± 1.06
Dimethylbenzanthracene	12.5	17.50 ± 1.40	20.00 ± 2.75
Benzyl acetate	325		1.50 ± 0.82
	425	2.25 ± 0.45
	650		2.00 ± 0.85
	850	1.00 ± 0.53
	1300		2.50 ± 1.24
	1700	4.00 ± 1.07
		P=0.117^a

  ^aOne-tailed trend test (Margolin et al., 1986); significant at P=0.05

Conclusions:

Under the conditions of the test, no induction of chromosome aberrations were observed in the bone marrow cells of male mice treated by intraperitoneal injection with the test substance, with either the standard or extended harvest times. The highest dose tested was 1700 mg/kg for the standard harvest protocol and 1300 mg/kg for the extended harvest experiment

Executive summary:

The study was performed as part of a genetic toxicity review in the NTP (1993) study examining carcinogenicity and toxicity. The study was performed in line with good scientific principles and was performed to a method that was in basic compliance with OECD 475. No information relating to the GLP status of the study was reported. No induction of chromosome aberrations were observed in the bone marrow cells of male mice treated by intraperitoneal injection with benzyl acetate, with either the standard or extended harvest times. The highest dose tested was 1700 mg/kg for the standard harvest protocol and 1300 mg/kg for the extended harvest experiment

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

other: Experimental study for structurally similar analogue included as relevant supporting information to strengthen the use of QSAR to address in vivo chromosome aberration test.

Adequacy of study:

key study

Study period:

30th April 1985 to 14th August 1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

A valid study is available for the analogue substance benzyl acetate. It is conducted in compliance with good scientific principles, with some deviations from standard protocols not thought to affect the validity of the results. The read-across is considered to be suitable based on the structural and “mechanistic action” similarities between the target substance (1-phenylethyl acetate) and source substance (benzyl acetate) and their similar physico-chemical properties.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

13-week dosing regimen

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)

Deviations:

yes

Principles of method if other than guideline:

Peripheral blood samples were obtained from male and female mice at the end of the thirteen week feeding toxicity study.

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

B6C3F1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: Frederick Cancer Research Facility (Frederick, MD)- Age at study initiation: 42 days- Housing: Polycarbonate cages, individually- Diet: ad libitum- Water: ad libitum- Acclimation period: 13 daysENVIRONMENTAL CONDITIONS- Temperature (°C): 22.2 ± 2 °C- Humidity (%): 39 to 59 %- Air changes (per hr): Minimum of 10 per hour- Photoperiod (hrs dark / hrs light): 12 hours/dayIN-LIFE DATES: From: 14 May 1985 To: 12-14 August 1985

Route of administration:

oral: feed

Vehicle:

Unchanged, no vehicle

Details on exposure:

DIET PREPARATION- Rate of preparation of diet (frequency): Weekly- Mixing appropriate amounts with (Type of food): NIH-07 open formula meal rat and mouse diet (Zeigler Brothers, Inc., Gardners, PA)- Storage temperature of food: -20 °C in the dark

Duration of treatment / exposure:

Food available ad libitum for 13 weeks

Frequency of treatment:

Food available ad libitum for 13 weeks

Post exposure period:

None

Remarks:

Doses / Concentrations:0, 3130, 6250, 12500, 25000 and 50000 ppmBasis:nominal in diet

Remarks:

Doses / Concentrations:0, 425, 1000, 2000, 3700 and 7900 mg/kg bw/day (males); 0, 650, 1280, 2980, 4300 and 9400 mg/kg (females)Basis:nominal in diet

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Positive control(s):

No positive control

Tissues and cell types examined:

Peripheral blood cells. 2000 polychromatic erythrocytes (PCE) and 10000 nonchromatic erythrocytes (NCE)

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION:Peripheral blood samples were obtained at the end of the 13 week study. Smears were immediately prepared and fixed in absolute methanol, stained with a chromatin-specific fluorescent dye mixture of Hoechst 33258/pyronin Y and coded. Slides were scanned to determine the frequency of micronuclei in 2000 PCE and 10,000 NCE in each animal per dose group.

Evaluation criteria:

The criteria of Schmid (1976) were used to define micronuclei, with the additional requirement that the micronuclei exhibit the characteristic fluorescent emissions of DNA (blue with 360 nm orange with 540 nm UV illumination); the minimum size limit was approximately one-twentieth the diameter of the NCE cell. In addition, the percentage of PCEs among the total erythrocyte population was determined.

Statistics:

Log transformation of the NCE data and testing for normality by the Shapiro-Wilk test and for heterogeneity of variance by Cochran's test were performed before statistical analyses. The frequency of micronucleated cells among NCEs was analysed by analysis of variance using the SAS GLM procedure. The NCE data for each dose group was compared with the concurrent solvent control using Student's t-test. The frequency of micronucleatd cells among PCEs was analysed by the Cochran-Armitage trend test, individual dose groups were compared to the concurrent solvent control by Kastenbaum-Bowman's binomial test. The percentage of PCEs among total erythrocytes was analysed by an analysis of variance on ranks (classed by sex), and individual dose groups were compared with the concurrent solvent control using a t-test on ranks.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Additional information on results:

ANALOGUE APPROACH JUSTIFICATION:- See attached “Justification for read-across” document for full details.- In summary, important considerations for the use of read-across for acute toxicity are: i) 1-phenylethyl acetate (the target chemical) has similar physico-chemical properties as benzyl acetate (the source substance), ii) there are structural similarities between the two chemicals, iii) the OECD QSAR Toolbox assigns an identical toxicity profiles to both chemicals, and iv) both chemicals have been tested for acute oral toxicity, which demonstrated that neither substance requires classification for acute toxicity and that the benzyl acetate will represent a worst-case scenario, and are adequate for classification and labelling and risk assessment purposes. The information reported in this summary is included to demonstrate comparability between the source (benzyl acetate) and target (1-phenyl-ethyl acetate) substance.

Table 1: Frequency of micronuclei in mouse peripheral blood erythrocytes following treatment for 13 weeks

		Micronucleated cells/1000 cells
	Dose (ppm)	PCE	NCE	PCE (%)	Number of mice
Male	0	1.87 ± 0.33	1.67 ± 0.18	1.91 ± 0.09	9
	3130	1.54 ± 0.25	1.32 ± 0.11	1.86 ± 0.11	8
	6250	1.63 ± 0.34	1.24 ± 0.08	1.63 ± 0.10	9
	12500	1.64 ± 0.31	1.45 ± 0.15	1.82 ± 0.11	9
	25000	1.38 ± 0.17	1.52 ± 0.12	2.03 ± 0.21	8
	50000	1.98 ± 0.30	1.71 ± 0.11	2.02 ± 0.09	8
Female	0	1.16 ± 0.14	1.02 ± 0.09	1.77 ± 0.09	7
	3130	0.92 ± 0.19	0.93 ± 0.09	1.78 ± 0.11	7
	6250	1.17 ± 0.15	0.99 ± 0.06	1.87 ± 0.12	9
	12500	1.25 ± 0.17	0.98 ± 0.06	1.64 ± 0.12	9
	25000	1.52 ± 0.34	0.98 ± 0.06	1.88 ± 0.09	8
	50000	1.54 ± 0.32	1.28 ± 0.10	1.65 ± 0.06	8

Conclusions:

Under the conditions of the test, no increases in the frequencies of micronucleated PCEs or NCEs were seen in peripheral blood smears prepared from male and female mice at the termination of the 13-week toxicity study, where the test substance was administered in feed at 3130 to 50,000 ppm.

Executive summary:

In a GLP study investigating the sub-chronic toxicity of benzyl acetate in feed, extra observations investigating the cytogenicity of the test substance were performed. The method showed basic compliance with OECD 408 concerning the dosing and animal maintenance during the course of the study, the observations for cytogenicity were similar to those described in OECD 474. No increases in the frequencies of micronucleated PCEs or NCEs were seen in peripheral blood smears prepared from male and female mice at the termination of the 13-week toxicity study, where the test substance was administered in feed at 3130 to 50,000 ppm.

Reference 4

Endpoint:

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

Type of information:

other: Experimental study for structurally similar analogue included as relevant supporting information to strengthen the use of QSAR to address in vivo chromosome aberration test.

Adequacy of study:

supporting study

Study period:

Not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

A valid study is available for the analogue substance benzyl acetate. It is conducted in compliance with good scientific principles, with some deviations from standard protocols not thought to affect the validity of the results. The read-across is considered to be suitable based on the structural and “mechanistic action” similarities between the target substance (1-phenylethyl acetate) and source substance (benzyl acetate) and their similar physico-chemical properties.

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.39 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells In Vivo)

Deviations:

yes

Remarks:

No positive control was included and a lower top dose than the suggested limit dose of the assay was used

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Deviations:

yes

Remarks:

No positive control was included and a lower top dose than the suggested limit dose of the assay was used

Principles of method if other than guideline:

Benzyl acetate was dosed via oral gavage in corn oil at doses of 50, 200 and 1000 mg/kg in male Fischer 344 rats. After 2 or 12 hours, livers were harvested, and hepatocytes were prepared for assessment. Cells were cultured, then incubated in media containing 10 µCi/mL 3H-(methyl)-thymidine (s.a. approx. 80 Ci/mmol).

GLP compliance:

not specified

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- Weight at study initiation: 180 to 300 g- Housing: 3 rats per cage in polypropylene cages- Diet: ad libitum- Water: deionised, 0.5 µm charcoal filtered tap water ad libitumENVIRONMENTAL CONDITIONS- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark

Route of administration:

oral: gavage

Vehicle:

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

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:The test substance was suspended in corn oil at the determined concentration levels.

Duration of treatment / exposure:

A single bolus dose

Frequency of treatment:

1 dose

Post exposure period:

2 or 12 hours

Remarks:

Doses / Concentrations:50, 200 or 100 mg/kgBasis:actual ingested

No. of animals per sex per dose:

3

Control animals:

yes, concurrent vehicle

Tissues and cell types examined:

50 isolated and cultured hepatocyte cells were counted from each slide, with three slides per animal

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Doses were selected based on the known LD50 of the test substance, as 80, 40 and 10 % of the LD50. 1000 mg/kg was selected as the highest dose if the LD50 was known to be greater than 1000 mg/kgTREATMENT AND SAMPLING TIMES: From the treated and control rats, primary hepatocyte cultures were prepared. Livers were perfused in situ with 0.5 mM ethyleneglycolbis (β-amino ethyl ether)N,N'-tetraacetic acid in Hank's balanced salt solution without Ca⁺² or Mg⁺² for 2 minutes, then for 12 minutes in type I collegenase in William's Medium E at 37 °C.The hepatocytes were prepared as a single-cell suspension through combing out the perfused liver. Cells were collected by centrifugation (50 g for 5 minutes), resuspended in cold medium and filtered. Viability was determined using Trypan blue exclusion. 6 x 10⁵ cells (approx.) were seeded into each well of a 6 well culture plate. Each well contained a 25 mm round coverslip in William's medium E supplemented with 2 mM l-glutamine, 50 µg/mL gentamycin sulphate and 10 % foetal bovine serum. After 1.5 to 2 hours incubation at 37 °C in a humidified atmosphere with 5 % CO₂, non viable cells were removed by washing with serum free WE medium.DETAILS OF SLIDE PREPARATION: Cultures were then incubated in Williams Medium E (WE) with 10 µCi/mL ³H-(methyl)-thymidine (spec. act. 80 Ci/mmol approximately) for 4 hours at 37 °C and 5 % CO₂, followed by incubation with 0.25 mM unlabelled thymidine in WE for 14-18 hours, washed, then incubated for 10 minutes with 1 % sodium citrate and fixed in glacial acetic acid:ethanol for 30 minutes and washed with deionised water. After drying the coverslips were mounted, and dipped in Kodak NTB-2 nuclear track emulsion diluted 1:1 with deionised water and exposed for 7-14 days at - 20 °C and then developed and stained for assessment.METHOD OF ANALYSIS:Slides were assessed by quantitative autoradiographic grain counting. An area of each slide was randomly selected and 50 morphologically unaltered cells were counted using an AR-TEK Model 880 counter interfaced with a VAX 9900 computer. The highest of the two nuclear-sized areas over the cytoplasm and adjacent to the nucleus was subtracted from the nuclear count to determine net grains/nucleus (NG). Three slides were scored for each animal or concentration for a total of 150 cells per animal.

Evaluation criteria:

A response was considered negative if the NG (net grain count) of all dose groups was a negative number and the %IR (percent of cells in repair) was less than 10 %. A response was considered positive if the average NG of any dose group exceeded 0 NG. Negative NG values but %IR greater than 10 % were considered equivocal.

Sex:

male

Genotoxicity:

negative

Remarks:

The test substance failed to induce a positive response

Toxicity:

no effects

Remarks:

The test substance was dosed at the highest dosing level used in the study

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not examined

Additional information on results:

ANALOGUE APPROACH JUSTIFICATION:- See attached “Justification for read-across” document for full details.- In summary, important considerations for the use of read-across for acute toxicity are: i) 1-phenylethyl acetate (the target chemical) has similar physico-chemical properties as benzyl acetate (the source substance), ii) there are structural similarities between the two chemicals, iii) the OECD QSAR Toolbox assigns an identical toxicity profiles to both chemicals, and iv) both chemicals have been tested for acute oral toxicity, which demonstrated that neither substance requires classification for acute toxicity and that the benzyl acetate will represent a worst-case scenario, and are adequate for classification and labelling and risk assessment purposes. The information reported in this summary is included to demonstrate comparability between the source (benzyl acetate) and target (1-phenyl-ethyl acetate) substance.

Table 1: UDS results with benzyl acetate

	Dose (mg/kg)	Time (hr)	NG	(n)	%IR
Control (corn oil)	-	2	-6.4 ± 2.9	(2)	1 ± 0
	-	12	-5.6 ± 0.4	(52)	2 ± 0
Benzyl acetate	50	2	-4.1 ± 1.4	(3)	3 ± 1
		12	-2.2 ± 0.2	(3)	1 ± 0
	200	2	-5.3 ± 0.3	(3)	1 ± 1
		12	-4.8 ± 1.0	(3)	2 ± 1
	1000	2	-4.5 ± 1.1	(3)	1 ± 0
		12	-4.6 ± 0.3	(3)	1 ± 1
NG net grains/nucleus (± SD) % IR percentage of cells with at least 5 NG ± SD (n) No. of animals treated

 

Conclusions:

Under the conditions of the test, benzyl acetate failed to induce a positive response in a rat unscheduled DNA synthesis assay when dosed up to 1000 mg/kg.

Executive summary:

The measurement of excision repair of DNA after treatment with the test substance was assessed in an unscheduled DNA synthesis test in rats in a method similar to that of OECD guideline 486 and EU method B.39. Benzyl acetate was dosed via oral gavage in corn oil at doses of 50, 200 and 1000 mg/kg in male Fischer 344 rats. After 2 or 12 hours, livers were harvested, and hepatocytes were prepared for assessment. Cells were cultured, then incubated in media containing 10 µCi/mL ³H-(methyl)-thymidine (s.a. approx. 80 Ci/mmol). Under the conditions of the test, benzyl acetate failed to induce a positive response in a rat unscheduled DNA synthesis assay when dosed up to 1000 mg/kg.

Reference 5

Endpoint:

in vivo mammalian germ cell study: gene mutation

Type of information:

other: Experimental study for structurally similar analogue included as relevant supporting information to strengthen the use of QSAR to address in vivo chromosome aberration test.

Adequacy of study:

supporting study

Study period:

Not reported

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The information presented in the report demonstrates basic compliance with OECD 477, the information presented in the NTP report is a review of the findings of an earlier study. Information presented as a review of genetic toxicology relating to benzyl acetate which is a structurally similar substance to 1-phenylethyl acetate and is anticipated to be toxicologically more active. The results presented for benzyl acetate are considered to be representative of the effects of 1-phenylethyl acetate.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 477 (Genetic Toxicology: Sex-linked Recessive Lethal Test in Drosophila melanogaster)

Deviations:

not specified

Principles of method if other than guideline:

From the limited level of detail in this review, the study presents some basic compliance with guideline OECD 477.

GLP compliance:

not specified

Type of assay:

Drosophila SLRL assay

Species:

Drosophila melanogaster

Strain:

other: Canton S males Basc females

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Age at study initiation: 24 to 72 hours

Route of administration:

other: Via feed or Injection into the thorax

Vehicle:

- Vehicle(s)/solvent(s) used: 5% sucrose (feed) or saline (injection)

Details on exposure:

Injection: To administer the test substance by injection, a glass Pasteur pipette was drawn out in a flame to a microfine filament and the tip broken off to allow delivery. Injection was performed either manually, by attaching a rubber bulb to the other end of the pipette and forcing through sufficient solution (0.2 to 0.3 µL) to slightly distend the abdomen of the fly or by attaching the pipette to a microinjector, which automatically delivers a calibrated volume. Flies were anaesthetised with ether and immobilised on a strip of tape. Injection into the thorax, under the wind was performed with the aid of a dissecting microscope.Oral: Oral exposure was achieved by allowing Canton-S males to feed for 72 hours on a solution of benzyl acetate in 5 % sucrose.

Duration of treatment / exposure:

Feeding: 3 daysInjection: Single exposure

Frequency of treatment:

Feeding: daily

Post exposure period:

Injection: 24 hours

Remarks:

Doses / Concentrations:300 ppm feedingBasis:no data

Remarks:

Doses / Concentrations:20000 ppm injectionBasis:no data

No. of animals per sex per dose:

Not reported

Control animals:

yes, concurrent vehicle

Tissues and cell types examined:

Not applicable

Evaluation criteria:

SLRL mutation data were analysed by simultaneous comparison with the concurrent and historical controls using a normal approximation to the binomial test (Margolin et al, 1983). A test result was considered positive if the P value was less than 0.01 and the mutation frequency in the tested group was greater than 0.1%, or if the P value was less than 0.05 and the frequency in the treatment group was greater than 0.15%. A test was considered to be inconclusive if (a) the P value was between 0.05 and 0.01 but the frequency in the treatment group was greater than 0.1% and 0.15% or (b) the P value was between 0.1 and 0.05 but the frequency in the treatment group was greater than 0.1%. A test was considered negative if the P value was greater than 0.1 or if the frequency in the treatment groups was less than 0.1%.

Sex:

male

Genotoxicity:

negative

Toxicity:

not examined

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Additional information on results:

No induction of SLRL mutations was observed in germ cells of male D. melanogaster by feeding or by injection.

Table 1: Induction of Sex-Linked Recessive Lethal Mutations in Drosophila melanogaster^a

Route of exposure	Dose (ppm)	Incidence of deaths (%)	Incidence of sterility (%)	No. of lethals/No. of X chromosomes tested			Total^b
				Mating 1	Mating 2	Mating 3
Feeding	300	2	0	1/3303	4/3317	2/3232	17/9852 (0.17%)
	0			2/3255	4/2783	4/2865	10/8903 (0.11%)
Injection	20,000	3	17	2/1814	6/1605	6/1398	14/4817 (0.29%)
	0			1/2053	8/1711	4/1897	13/5661 (0.23%)

^a Results were not significant at the 5 % level (Margolin et al, 1983) (P<0.001)

^bCombined total number of lethal mutations/number of X chromosomes tested for three mating trials.

Conclusions:

Under the conditions of the test, no induction of SLRL mutations was observed in germ cells of male D. melanogaster administered benzyl acetate by feeding or by injection.

Executive summary:

A summary of a sex linked recessive lethal (SLRL) assay assay performed in Drosophila melanogaster was presented as part of a genotoxic review of benzyl acetate in the NTP (1993) report on carcinogenicity and toxicity. The method employed had some basic compliance with OECD Guideline 477 but no information was provided relating to GLP. No induction of SLRL mutations was observed in germ cells of male D. melanogaster administered benzyl acetate by feeding or by injection.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Due to the lack of Ames data and in vivo genotoxicity studies on 1-phenylethyl acetate, and the wealth of data available on benzyl acetate, the Apotential of 1-phenylethyl acetate was assessed by reading-across between the two substances. The reported results with benzyl acetate were considered representative of the effects of 1-phenylethyl acetate. The substances are structurally very similar, the only difference being, benzyl acetate has no branching from the acetate. Both molecules have very similar physical-chemical properties and are anticipated to undergo the same metabolic pathway, and the simpler structure of benzyl acetate is likely to make it more toxicologically available and active and is therefore potentially a worse case of the effects of 1-phenylethyl acetate.

All studies presented, with the exception of the in vitro mammalian cell gene mutation studies, indicated that benzyl acetate was not mutagenic or clastogenic. When re-evaluating the results presented as recommended by the mouse lymphoma assay (MLA) Workgroup of the International Workshop on Genotoxicity Testing and taking into account the initial considerations and advice for evaluating the data produced in these assays detailed in the guideline OECD 476, the data are not indicative of a mutagenic effect. The in vivo data indicated that benzyl acetate was not mutagenic or clastogenic, this was therefore considered to be the most accurate reflection of the substance.

 

IN VITRO GENETIC MUTATION STUDY IN BACTERIA

 

The key study for this endpoint, Mortelmans et al (1986), was performed to a method comparable to the standardised guideline OECD 471 prior to the recommendation of strains able to detect cross-linking. Although the study was a screen of 270 chemicals, the reporting of the performance of the study and the results was sufficient to assess the quality of the information presented and its suitability for use in assessing the mutagenic potential of the test substance. The study was performed on benzyl acetate. The results are therefore considered representative of the effects of 1-phenylethyl acetate and can be read-across between the two substances. The study was assigned a reliability score of 2 in accordance with Klimisch (1997). Under the conditions of the test, the test substance was found to be non-mutagenic in S. typhimurium strains TA100, TA98, TA1535 and TA1537 in both the presence and absence of metabolic activation.

 

IN VIVO GENOTOXICITY STUDIES

In the key study for this endpoint, NTP (1993), the in vivo genetic toxicity of benzyl acetate was investigated in the following studies:

Sister chromatid exchange assay: Negative

Chromosome aberration assay: Negative

Bone marrow micronucleus assay: Negative

13 week peripheral blood micronucleus assay: Negative

All of the in vivo tests were performed and reported as original studies in this report; all studies were assigned a reliability score of 2 in accordance with Klimisch (1997). The studies were performed to methods which were in basic compliance with their respective OECD guideline, with the exception of the 13 week micronucleus test which was performed to a method which followed the dosing procedures of a 13 week subchronic toxicity study, but the examinations performed were in line with those reported in the OECD guideline 474 (Mammalian Erythrocyte Micronucleus Test).

The study Mirsalis et al (1989) assessed the excision repair of DNA after treatment with the benzyl acetate in an unscheduled DNA synthesis assay. The method used in the study was comparable to that of the standardised guidelines (OECD 489 and EU Method B.39). Although benzyl acetate was studied as part of a screening study of multiple chemicals, and therefore was briefly reported in areas, the level of detail provided in the study was sufficient to assess the quality of the data and presented conclusions. In accordance with Klimisch (1997), the study was assigned a reliability score of 2. Under the conditions of the test, benzyl acetate failed to induce a positive response in rat hepatocytes.

IN VIVO GENOTOXICITY STUDIES

NTP (1993), in vitro and in vivo review of mutagenicity and cytogenicity: Negative (benzyl acetate)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The overall conclusion based on several genetic toxicity studies presented, indicates that the test substance is not mutagenic or cytogenic. Therefore the substance does not require classification in line with Directive 67/548/EEC and Regulation 1272/2008.