1-phenylethyl acetate

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

1-phenylethyl acetate

EC Number:

202-288-5

EC Name:

1-phenylethyl acetate

Cas Number:

93-92-5

Molecular formula:

C10H12O2

IUPAC Name:

1-phenylethyl acetate

Test material form:

liquid

Method

Target gene:

In vitro Micronucleus in Mammalian cells

Species / strain

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)

Controls

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.

Results and discussion

Test results

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

Applicant's summary and conclusion

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.