Phenethyl phenylacetate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-11-11 to 2016-01-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline No. 487 (In vitro Mammalian Cell Micronucleus Test), 2010

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:

Phenethyl phenylacetate

EC Number:

203-013-1

EC Name:

Phenethyl phenylacetate

Cas Number:

102-20-5

Molecular formula:

C16H16O2

IUPAC Name:

2-phenylethyl phenylacetate

Method

Species / strain

Species / strain / cell type:

lymphocytes: human

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

Without and with S9 mix (1st Exp): 13.0, 22.7, 39.8, 69.6, 121.9, 213.2, 373.2, 653.1, 1142.9, 2000 µg/mL
Without S9 mix (2nd Exp): 7.4, 13.0, 22.7, 39.8, 69.6, 121.9, 213.2, 373.2 µg/mL

Vehicle / solvent:

DMSO

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Demecolcin

Remarks:

Without metabolic activation

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours (Exp I), 20 h (Exp II)
- Fixation time: The cultures were harvested by centrifugation 40 hrs after beginning of treatment.

SPINDLE INHIBITOR: Cytochalasin B
STAIN: Gain

NUMBER OF CELLS EVALUATED: At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides.
To describe a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is expressed as % cytostasis.

DETERMINATION OF CYTOTOXICITY: To describe a cytotoxic effect (Cytokinesis-block proliferation index) the CBPI was determined in 500 cells per culture and cytotoxicity is expressed as % cytostasis.

Evaluation criteria:

Negative if, in all of the experimental conditions examined:
− None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− There is no concentration-related increase
− The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data
The test item is then considered unable to induce chromosome breaks and/or gain or loss in this test system.
Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
− At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− The increase is concentration-related in at least one experimental condition
− The results are outside the range of the laboratory historical solvent control data

Statistics:

Statistical significance was confirmed by using the Chi-squared test (α < 0.05) using the validated R Script CHI2.Rnw for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The test item, dissolved in DMSO, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix. Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure period was 20 hours without S9 mix. The cells were prepared 40 hours after start of treatment with the test item. In each experimental group two parallel cultures were analysed. 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. To determine a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is described as % cytostasis. The highest treatment concentration in this study, 2000.0 μg/mL was chosen with respect to the OECD Guideline 487 for the in vitro mammalian cell micronucleus test. No precipitation was observed. In Experiment I, phase separation of the test item in the culture medium was observed at 39.8 μg/mL and above in the absence of S9 mix and at 69.6 μg/mL and above in the presence of S9 mix at the end of treatment. In addition, phase separation occurred in Experiment II in the absence of S9 mix at 69.6 μg/mL and above at the end of treatment.
No relevant influence on osmolarity or pH was observed. In both cytogenetic experiments, in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed phase separation. In the absence and presence of S9 mix, no relevant increase in the number of micronucleated cells was observed after treatment with the test item. However, in Experiment I in the presence of S9 mix after treatment with 39.8 μg/mL the value of 0.70 % micronucleated cells is statistically significant. Since the value is within the laboratory historical control data range (0.15 – 1.45 % micronucleated cells), the finding has to be regarded as biologically irrelevant. In both experiments, either Demecolcin (75.0 ng/mL), MMC (1.0 μg/mL) or CPA (17.5 μg/mL) were used as positive controls and showed distinct increases in cells with micronuclei.

Any other information on results incl. tables

Exp	Preparation invervall	Test item concentration in µg/mL	Proliferation index CBPI	Cytostasis in %	Micronucleated cells in %
		Exposure period 4 h without S9 Mix
I	40 h	Solvent control	1.97		0.35
		Positive control	1.38	61.3	19.60
		13.0	1.96	0.7	0.55
		22.7	1.92	4.8	0.50
		39.8	1.98	n.c.	0.40
		Exposure period 20 h without S9 mix
II	40 h	Solvent control	2.01		0.40
		Positive control	1.69	31.3	2.1
		22.7	1.96	4.6	0.85
		39.8	2.01	n.c.	0.25
		69.6	2.01	n.c.	0.10
		Exposure period 4 h without S9 mix
I	40 h	Solvent control	2.06		0.20
		Positive control	1.52	50.9	6.45
		22.7	2.01	5.1	0.15
		39.8	1.96	9.1	0.70
		69.6	2.04	1.7	0.25

Applicant's summary and conclusion

Conclusions:

It can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, the test item is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to phase separating concentrations.

Executive summary:

The test item, dissolved in DMSO, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in two independent experiments. In each experimental group two parallel cultures were analysed. Per culture 1000 binucleated cells were evaluated for cytogenetic damage. The highest applied concentration in this study (2000.0 μg/mL of the test item) was chosen with respect to the current OECD Guideline 487. Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item phase separation in accordance with OECD Guideline 487. In both cytogenetic experiments, in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed phase separation. In the absence and presence of S9 mix, no relevant increase in the number of micronucleated cells was observed after treatment with the test item. However, in Experiment I in the presence of S9 mix after treatment with 39.8 μg/mL the value of 0.70 % micronucleated cells is statistically significant. Since the value is within the laboratory historical control data range (0.15 – 1.45 % micronucleated cells), the finding has to be regarded as biologically irrelevant. Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei. In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, the test item is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to phase separating concentrations.