Ethyl 3-phenyloxirane-2-carboxylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro bacterial genemutation assay, Ames test (OECD TG 471): Positive

In vitro mammalian genemutation assay, MLA test (HGPRT in CHO cells6 -TG, OECD TG 476): Positive

In vitro micronucleus test using read across from Methyl-betaphenylglycidate (tested in an OECD TG 487): Positive.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 February 2017 - 20 March 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

- S. typhimurium: Histidine gene
- Escherichia coli: Tryptophan gene

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by Aroclor 1254.

Test concentrations with justification for top dose:

Direct plate:
- Dose range finding test:
TA 100 and WP2uvrA (without and with S9): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate
- Experiment 1:
TA 1535, TA 1537 and TA 98 (without and with S9): 52, 164, 512, 1600 and 5000 μg/plate

Pre-incubation:
- Experiment 2:
TA 1535, TA 1537, TA 98, TA 100 and WPruvrA (without and with S9): 17, 52, 164, 512, 1600 and 5000 μg/plate

Direct plate:
- Experiment 3, to verify the mutagenic response observed in the first direct plate assay, an additional experiment was performed:
TA 100 and WP2uvrA (without and with S9): 52, 164, 512, 1600 and 5000 μg/plate

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of vehicle: A solubility test was performed based on visual assessment. The test item was dissolved in dimethyl sulfoxide.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

100 μL/plate DMSO

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191, 2.5 μg/plate in DMSO for TA1537

Remarks:

Without S9-mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

100 μL/plate DMSO

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene in DMSO, 1 μg/plate for TA98 and TA100 (direct plate), 2.5 μg/plate for TA1535 and TA1537, 5 μg/plate for TA100 (pre-incubation), 15 μg/plate for WP2uvrA

Remarks:

With S9

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- Experiment 1 and 3: in agar (plate incorporation)
- Experiment 2: (independent repeat): preincubation

DURATION
- Preincubation period: 30 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain (in all experiments)

NUMBER OF CELLS EVALUATED: 10^8 cells/mL

DETERMINATION OF CYTOTOXICITY
- Method: on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation.

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.
A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Statistics:

Not performed.

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

in the direct plate test only

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see additional information on results

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

in the direct plate test only

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

up to and including 5000 μg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

up to and including 5000 μg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see additional information on results

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see additional information on results

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see additional information on results

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
First experiment: Precipitation of the test item on the plates was observed at the start and at the end of the incubation period at the concentration of 5000 μg/plate, except in tester strain TA100 in the presence of S9-mix, where precipitation could not be determined due to the presence of microcolonies.
Second experiment: Precipitation of the test item on the plates was observed at the start of the incubation period at the top dose of 5000 μg/plate in the absence of S9-mix. No precipitation was observed in the presence of S9-mix and at the end of the incubation period.
Third experiment: Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period.

RANGE-FINDING/SCREENING STUDIES:
The test item precipitated on the plates at the top dose level of 5000 μg/plate in tester strain TA100 in the absence of S9-mix and in tester strain WP2uvrA in the absence and presence of S9-mix. Cytotoxicity, as evidenced by a reduction of the bacterial background lawn and the presence of microcolonies, was observed in tester strain TA100 in the presence of S9-mix at the highest concentration tested.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
TA1535 TA1537 TA98
S9-mix - + - + - +
Range 125 - 1381 78 - 1058 55 – 1311 55 – 1051 410 – 1995 250 - 1907
Mean 828 218 686 376 1270 883
SD 151 109 320 142 338 340
n 1875 1829 1560 1716 1766 1851

TA100 WP2uvrA
S9-mix - + - +
Range 554 – 1848 408 - 2651 112 – 1951 85 - 1359
Mean 892 1352 1165 388
SD 174 342 488 152
n 1820 1857 1506 1557
SD = Standard deviation
n = Number of observations
Historical control data from experiments performed between November 2014 and November 2016.

- Negative (solvent/vehicle) historical control data:
TA1535 TA1537 TA98 TA100 WP2uvrA
S9-mix - + - + - + - + - +
Range 5 - 36 3 - 32 3 – 23 3 – 23 8 - 41 9 - 52 66 - 156 65 - 154 10 – 56 9 - 69
Mean 12 12 6 8 16 23 100 100 25 31
SD 5 4 3 4 5 7 15 16 6 7
n 1865 1862 1740 1715 1852 1912 1853 1877 1571 1583
SD = Standard deviation
n = Number of observations
Historical control data from experiments performed between November 2014 and November 2016.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- First experiment: Cytotoxicity, as evidenced by a decrease in the number of revertants, a reduction of the bacterial background lawn and/or the presence of microcolonies was observed in the tester strains TA1535, TA1537 and TA98 in the absence of S9-mix and in tester strain TA100 in the presence of S9-mix.
- Second experiment: Cytotoxicity, as evidenced by a reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix.
- Third experiment: Cytotoxicity, as evidenced by a reduction of the bacterial background lawn, was observed in tester strain TA100 in the absence and presence of S9-mix at the highest concentration tested.
No toxicity was observed in tester strain WP2uvrA.

Conclusions:

Based on the results of the direct plate assay (but not in the pre-incubation method) the substance is mutagenic in the tester strains TA100 and WP2uvrA of the Salmonella typhimurium and the Escherichia coli reverse mutation assay performed according to OECD 471 (1997) and GLP principles. The test item is not mutagenic in the other Salmonella typhimurium tester strains (TA1535, TA1537 and TA98).

Executive summary:

Ethylphenylglycidate: Ames test (OECD TG 471, Kl 1) Key information and a study record included

Method: The mutagenic activity of the substance was evaluated in accordance with OECD 471 (1997) guideline and according to GLP principles. The test was performed in two independent experiments: at first a direct

plate assay was performed and second a pre-incubation assay in the absence and presence of S9-mix up to and including the concentration of 5000 μg/plate. Acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Results on precipitation and cytotoxicity first test: Precipitation was observed in the first direct plate experiment, no precipitation was observed at the end of the incubation period in the pre-incubation experiment and in the second direct plate experiment. In the direct plate experiment, cytotoxicity, as evidenced by a reduction of the bacterial background lawn and the presence of microcolonies, was observed in tester strain TA100 in the presence of S9-mix at the highest concentration tested. In TA98, TA1535 and TA1537 cytotoxicity, as evidenced by a decrease in the number of revertants and a reduction of the bacterial background lawn was observed in all tester strains in the absence of S9-mix. No toxicity was observed in the presence of S9-mix.

Results on revertants: In the absence of S9-mix, the test item induced dose-related increases in the number of revertant colonies compared to the solvent control in two tester strains (TA100 and WP2uvrA). The increases observed in the tester strains TA100 and WP2uvrA were above the laboratory historical control data range and were up to 3.4- and 2.9-fold the concurrent controls, respectively. In the presence of S9-mix, the test item induced a dose-related increase in the number of revertant colonies compared to the solvent control in the tester strain TA100. The increase observed was above the laboratory historical control data range and was up to 2.2-fold the concurrent control.

Results on precipitation and cytotoxicity in the pre-incubation experiment: Cytotoxicity, as evidenced by a reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix.

Results on revertants: No increase in the number of revertants was observed in this pre-incubation method.

Repeat test: To verify the mutagenic response observed in the direct plate assay, an additional experiment was performed with the tester strains TA100 and WP2uvrA.Cytotoxicity, as evidenced by a reduction of the bacterial background lawn, was observed in tester strain TA100 in the absence and presence of S9-mix at the highest concentration tested. No toxicity was observed in tester strain WP2uvrA.

Result of the repeat test: In the absence of S9-mix, the test item induced up to 2.5- and 3.9-fold dose related, increases in the number of revertant colonies compared to the solvent control in the tester strains TA100 and WP2uvrA, respectively. In the presence of S9-mix, the test item induced an up to 4.0-fold dose related increase in the number of revertant colonies compared to the solvent control in the tester strain TA100.

Conclusion: It is concluded that based on the results of the direct plate assay (but not in the pre-incubation method) the substance is mutagenic in the tester strains TA100 and WP2uvrA of the Salmonella typhimurium and the Escherichia coli reverse mutation assay. The test item is not mutagenic in the other Salmonella typhimurium tester strains (TA1535, TA1537 and TA98).

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No data.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: In this publication a TG test was conducted using a protocol similar to OECDTG 476 without GLP.

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

04 April 1984

GLP compliance:

no

Type of assay:

other: in vitro gene mutation study in mammalian cells

Target gene:

HGPRT locus in CHO-K1-BH4 cells.

Species / strain / cell type:

other: CHO-K1-BH4

Details on mammalian cell type (if applicable):

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
The cells were grown in McCoy's 5a medium supplemented with 10% fetal calf serum and 1% penicillin-streptomycin. They were cultured routinely in plastic culture bottles (Nunc) at 37°C in a water-saturated CO2 incubator.
- Properly maintained: no data
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically 'cleansed' against high spontaneous background: no data

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced with Aroclor 1254.

Test concentrations with justification for top dose:

EPG solution:
Without and with S9-mix, 2 hours treatment: 0.25%, 0.5%, 1%, 5% and 10% (0.125, 0.250, 0.5, 2.5 and 5 mg/mL)
EPG digest:
Without and with S9-mix, 2 hours treatment: 0.3%, 1%, 4%, 8% and 10% (0.123, 0.41, 1.64, 3.28 and 4.1 mg/mL)

The following dose levels were selected to measure mutation frequencies:
EPG solution:
Without and with S9-mix, 2 hours treatment: 0.25% and 0.5% (0.125 and 0.250 mg/mL)
EPG digest:
Without and with S9-mix, 2 hours treatment: 0.3% and 8% (0.123 and 3.28 mg/mL)

Vehicle / solvent:

- Vehicle used: DMSO
- The standard EPG solution contained 50 mg EPG/mL DMSO, appropriate dilutions being prepared for the test.
- The EPG digest was prepared by adding 6 g of the substance to 30 mL distilled water. This solution was treated according to the in vitro digestion procedure described by Phillips et al. (1980). The final concentration of EPG in the digest was 41 mg/mL.

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 2 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): 2 μg/mL Thioguanine (TG)

NUMBER OF REPLICATIONS: Triplicate cultures

STAINING TECHNIQUE USED: Methylene-blue

NUMBER OF CELLS EVALUATED: 2x10^5 cells plated/concentration

DETERMINATION OF CYTOTOXICITY
Cytotoxicity was determined as the percentage of cells surviving after exposure for 2 hours to various concentrations of EPG or EPG digest.

Evaluation criteria:

Results were judged to be positive if the number of induced mutants equaled or exeeded 2.5 times the control value at 50% survival. Hence for dose points without S9 mix any number of mutants exceeding 4.3 mutants/10^6 cells should be regarded as positive. Similarly, for dose points with S9 mix any number of mutants exceeding 20.8 mutants/10^6 cells represents a positive result.

Statistics:

Not performed.

Key result

Species / strain:

other: CHO-K1-BH4

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
No data

RANGE-FINDING/SCREENING STUDIES:
Preliminary investigations were carried out to determine the concentrations of EPG solution and EPG digest that would be tolerated by the CHO cells in culture.

HISTORICAL CONTROL DATA
No data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxicity of EPG digest and EPG:

Material: EPG (mg/mL) % survival -S9 % survival +S9
Control 100 100
MNNG 48 -
CPA - 53
0.3% EPG digest 0.123 82 94
1% EPG digest 0.41 60 91
4% EPG digest 1.64 79 78
8% EPG digest 3.28 52 69
10% EPG digest 4.1 42 15

0.25% EPG 0.125 50 58
0.5% EPG 0.25 8 7
1% EPG 0.5 0 0
5% EPG 2.5 0 0
10% EPG 5 0 0

Conclusions:

An in vitro gene mutation study in CHO-K1-BH4 cells with the substance was conducted equivalent to OECD 476 guideline without GLP. It is concluded that the substance is positive under the experimental conditions of the study.

Executive summary:

Method: An in vitro gene mutation study in CHO-K1-BH4 cells was conducted equivalent to OECD 476 guideline without GLP. Two types of tests were performed one under normal OECD TG 476 conditions and one with digestive solute. In the first test the substance was tested up to and including concentrations of 10% (5 mg/mL) in the absence and presence of S9 -mix.

Results on cytotoxicity: The cell survival at 0.25% (0.125 mg/mL) was 50% and 58% in the absence and presence of S9 -mix, respectively. In the test with the digestive solute, the test item was tested up to and including concentrations of 10% (4.1 mg/mL) in the absence and presence of S9 -mix. The cell survival at 8% (3.28 mg/mL) was 52% and 69% in the absence and presence of S9 -mix, respectively. The incubation time was 2 hours. Positive control chemicals, MNNG and CPA induced appropriate responses.

Results on mutagenicity: Both in the absence and presence of S9 -mix, the test item induced a significant increase in the mutation frequency (the number of induced mutants exceeded 2.5 times the control value at 50% survival). Based on the results, it is concluded that the substance is positive under normal OECD TG 476 conditions.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 March 2016 - 22 July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The information is used for read across to Ethylphenylglycidate

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2014

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: human peripheral blood

Details on mammalian cell type (if applicable):

Type and identity of media:
- Blood samples
Human venous blood from a healthy, adult donor (nonsmoker without a history of radiotherapy, chemotherapy, or drug usage, and lacking current viral infections) was drawn into sterile, heparinized “vacutainers”.
- Culture medium
The medium was HEPES-buffered RPMI 1640 culture medium supplemented with approximately 20% (v/v) heat-inactivated fetal bovine serum (FBS), penicillin (100 units/mL), streptomycin (100 μg/mL), L-glutamine (2 mM) and 2% phytohemagglutinin M (PHA).
- Lymphocyte cultures
Whole blood cultures were initiated in 15 mL centrifuge tubes by adding approximately 0.6 mL of fresh heparinized blood into a sufficient volume of culture medium so that the final volume was 10 mL in the assay without metabolic activation after the addition of the test article in its chosen vehicle or was 10 mL in the assay with metabolic activation after the addition of the test article in its chosen vehicle and the S9 activation mix.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced with Aroclor 1254

Test concentrations with justification for top dose:

Dose range finding test:
With and without S9-mix, 3 hr exposure; 24 hr fixation: 35.2, 50.3, 71.9, 103, 147, 210, 300, 428, 611, 873, 1247 and 1782 μg/mL
Without S9-mix, 24 exposure; 24 hr fixation: 35.2, 50.3, 71.9, 103, 147, 210, 300, 428, 611, 873, 1247 and 1782 μg/mL
Initial micronucleus assay (B1):
Without 24hr exposure; 24 hr fixation: 17.2, 42.0, 65.6, 103, 128, 135, 142, 150, 157, 166, 174 and 193 μg/mL
Without 3hr exposure; 24 hr fixation: 150, 302, 377, 418, 440, 463, 513, 569, 630 and 698 μg/mL
With 3hr exposure; 24 hr fixation: 65.6, 174, 569, 698, 774, 815, 857, 903, 950 and 1000 μg/mL
The following dose levels were selected for scoring of micronuclei:
Without S9-mix, 24hr exposure; 24 hr fixation: 17.2, 65.6 and 135 μg/mL
Without S9-mix, 3hr exposure; 24 hr fixation: 150, 418 and 569 μg/mL
Repeat micronucleus assay (B3):
With 3hr exposure; 24 hr fixation: 30.4, 96.9, 182, 278, 381, 471, 581, 646, 797, 886, 984, 1094, 1215 and 1350 μg/mL
The following dose levels were selected for scoring of micronuclei:
With S9-mix, 3hr exposure; 24 hr fixation: 581, 646 and 886 μg/mL

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: A solubility test was performed and DMSO was selected as the vehicle for this assay.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hr
- Exposure duration:
Short-term treatment
Without and with S9-mix: 3 hr treatment, 24 hr recovery/harvest time
Continuous treatment
Without S9-mix: 24 hr treatment/harvest time

ARREST OF CELL DIVISION: 6 μg/mL Cytochalasin B
STAIN: Acridine orange

NUMBER OF REPLICATIONS: duplicates

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
All cultures were harvested approximately 24 hours after initiation of treatment. The cultures were centrifuged, the supernatant discarded, and the cells were swollen with 75mM KCl, fixed in methanol: glacial acetic acid (3:1, v/v), dropped onto glass slides, and air dried. The slides were appropriately stained with acridine orange, and analyzed under fluorescent microscopy.

NUMBER OF CELLS EVALUATED: Slides were examined for proportions of mono-, bi- and multinucleate cells, to a minimum of 200 cells per concentration (when available) for the DRF, and 1000 cells per concentration (500 cells per culture, when available) for the Micronucleus Assay.

DETERMINATION OF CYTOTOXICITY
- The cytostasis/cytotoxicity was determined using the cytokinesis-block proliferation index (CPBI index):

CBPI = Number of mononucleate cells + (2 x number binucleate cells) + (3 x number multinucleate cells) / total number of cells scored

Relative CBPI (%) = ( CBPI of treated cultures – 1 / CBPI of vehicle controls – 1) x 100

Cytotoxicity (%) is expressed as (100 – Relative CBPI).

Evaluation criteria:

For valid data, the test article was considered to induce clastogenic and/or aneugenic events if:
1. A statistically significant increase in the frequency of micronucleated binucleate cells (MNBN cells) at one or more concentrations was observed.
2. An incidence of MNBN cells at such a concentration that exceeded the normal range in both replicates was observed.
3. A concentration-related increase in the proportion of MNBN cells was observed.
The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met.

Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Evidence of a concentration-related effect was considered useful but not essential in the evaluation of a positive result. Biological relevance was taken into account, for example consistency of response within and between concentrations, or effects occurring only at very toxic concentrations.

Statistics:

The proportion of MNBN cells for each treatment condition was compared with the proportion in vehicle controls by using Fisher's exact test. A Cochran-Armitage trend test was applied to each treatment condition. Probability values of p≤0.05 were accepted as significant.

Key result

Species / strain:

lymphocytes: human peripheral blood

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: human peripheral blood

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Dose range finding: Precipitate was observed at ≥1247 μg/mL at the time of treatment in all three treatment conditions except the approximate 24 hour treatment without S9, where precipitate was observed at ≥873 μg/mL. At the time of harvest, precipitate was observed at 1782 μg/mL in the 3 and approximate 24 hour treatment without S9.
Micronucleus assay: no precipitate was observed at the end of the treatment or harvest.

RANGE-FINDING/SCREENING STUDIES: In the approximate 24 hour treatment without S9, 41% cytotoxicity was observed at 147 μg/mL. The next higher concentration, 210 μg/mL, was excessively cytotoxic and produced 81% cytotoxicity. In the 3 hour treatment without S9, 55% cytotoxicity was observed at 428 μg/mL. In the 3 hour treatment with S9, 53% cytotoxicity was observed at 873 μg/mL. Based on the DRF results, concentrations were selected for the micronucleus assay.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells:

Without Metabolic Activation - 24-Hour Exposure – B1:
Treatment
(μg/mL) Replicate Mono nucleate Bi nucleate Multi nucleate Total CBPI Cytotoxicity (%)
DMSO, 10.0 μL/mL A 126 380 60 566 1.88
B 120 373 50 543 1.87
Total 246 753 110 1109 1.88 -
17.2 A 135 361 35 531 1.81
B 173 359 31 563 1.75
Total 308 720 66 1094 1.78 11
42.0 A 196 296 20 512 1.66
B 199 341 19 559 1.68
Total 395 637 39 1071 1.67 24
65.6 A 207 300 14 521 1.63
B 241 327 11 579 1.60
Total 448 627 25 1100 1.62 30
103 A 183 387 24 594 1.73
B 270 301 7 578 1.54
Total 453 688 31 1172 1.64 27
128 A 304 244 6 554 1.46
B 295 230 1 526 1.44
Total 599 474 7 1080 1.45 48
135 A 319 196 1 516 1.38
B 298 240 2 540 1.45
Total 617 436 3 1056 1.42 52
142 A 315 232 5 552 1.44
B 350 197 4 551 1.37
Total 665 429 9 1103 1.41 54
150 A 297 221 1 519 1.43
B 317 250 2 569 1.45
Total 614 471 3 1088 1.44 50
157 A 361 217 3 581 1.38
B 342 242 5 589 1.43
Total 703 459 8 1170 1.41 54
166 A 400 159 0 559 1.28
B 392 204 3 599 1.35
Total 792 363 3 1158 1.32 64
174 A 348 192 0 540 1.36
B 351 181 2 534 1.35
Total 699 373 2 1074 1.35 60
193 A 419 126 0 545 1.23
B 344 201 3 548 1.38
Total 763 327 3 1093 1.30 65
MMC, 0.300 A 287 244 3 534 1.47
B 307 289 2 598 1.49
Total 594 533 5 1132 1.48 45

Without Metabolic Activation - 3-Hour Exposure – B1:
Treatment
(μg/mL) Replicate Mono nucleate Bi nucleate Multi nucleate Total CBPI Cytotoxicity (%)
DMSO, 10.0 μL/mL A 86 424 75 585 1.98
B 88 453 47 588 1.93
Total 174 877 122 1173 1.96 -
150 A 104 382 37 523 1.87
B 81 449 48 578 1.94
Total 185 831 85 1101 1.91 5
302 A 171 332 26 529 1.73
B 170 331 13 514 1.69
Total 341 663 39 1043 1.71 26
377 A 201 357 8 566 1.66
B 221 334 10 565 1.63
Total 422 691 18 1131 1.64 33
418 A 202 300 8 510 1.62
B 225 323 15 563 1.63
Total 427 623 23 1073 1.62 35
440 A 258 307 16 581 1.58
B 216 345 12 573 1.64
Total 474 652 28 1154 1.61 36
463 A 202 302 5 509 1.61
B 240 317 8 565 1.59
Total 442 619 13 1074 1.60 37
513 A 261 286 5 552 1.54
B 281 246 2 529 1.47
Total 542 532 7 1081 1.51 47
569 A 301 210 1 512 1.41
B 327 205 3 535 1.39
Total 628 415 4 1047 1.40 58
630 A 309 199 0 508 1.39
B 290 245 6 541 1.48
Total 599 444 6 1049 1.43 55
698 A 335 256 4 595 1.44
B 342 178 2 522 1.35
Total 677 434 6 1117 1.40 58P
MMC, 1.00 A 315 260 1 576 1.45
B 305 215 1 521 1.42
Total 620 475 2 1097 1.44 54

With Metabolic Activation - 3-Hour Exposure – B3:
Treatment
(μg/mL) Replicate Mono nucleate Bi nucleate Multi nucleate Total CBPI Cytotoxicity (%)
DMSO, 10.0 μL/mL A 159 280 61 500 1.80
B 196 258 46 500 1.70
Total 355 538 107 1000 1.75 -
30.4 A 111 322 67 500 1.91
B 137 311 52 500 1.83
Total 248 633 119 1000 1.87 0
96.9 A 127 320 53 500 1.85
B 155 302 43 500 1.78
Total 282 622 96 1000 1.81 0
182 A 100 323 77 500 1.95
B 160 291 49 500 1.78
Total 260 614 126 1000 1.87 0
278 A 160 315 25 500 1.73
B 167 304 29 500 1.72
Total 327 619 54 1000 1.73 3
381 A 158 315 27 500 1.74
B 200 291 14 505 1.63
Total 358 606 41 1005 1.68 9
471 A 254 242 4 500 1.50
B 186 311 3 500 1.63
Total 440 553 7 1000 1.57 25
581 A 159 337 4 500 1.69
B 216 282 2 500 1.57
Total 375 619 6 1000 1.63 16
646 A 247 247 6 500 1.52
B 244 249 7 500 1.53
Total 491 496 13 1000 1.52 31
797 A 268 230 2 500 1.47
B 320 180 0 500 1.36
Total 588 410 2 1000 1.41 45
886 A 300 195 5 500 1.41
B 348 152 0 500 1.30
Total 648 347 5 1000 1.36 53
984 A 283 213 4 500 1.44
B 333 167 0 500 1.33
Total 616 380 4 1000 1.39 48P
1094 A 335 160 5 500 1.34
B 318 179 3 500 1.37
Total 653 339 8 1000 1.36 53P
1215 A 394 131 1 526 1.25
B 405 93 2 500 1.19
Total 799 224 3 1026 1.22 70P
1350 A 449 50 1 500 1.10
B 451 48 1 500 1.10
Total 900 98 2 1000 1.10 86P
CP, 15.0 A 352 149 1 502 1.30
B 382 118 0 500 1.24
Total 734 267 1 1002 1.27 64

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture:
- Indication whether binucleate or mononucleate where appropriate:
Without Metabolic Activation - 24-Hour Exposure – B1:
Treatment (μg/mL) Replicate Total BN Cells Scored Total MNBN Cells Scored MNBN Cell Frequency (%) Significance Cytotoxicity (%)
DMSO, 10.0 μL/mL A 1000 4 0.40
B 1000 4 0.40
Total 2000 8 0.40 - -
17.2 A 1000 3 0.30
B 1000 5 0.50
Total 2000 8 0.40 NS 11
65.6 A 1000 3 0.30
B 1000 4 0.40
Total 2000 7 0.35 NS 30
135 A 1000 7 0.70
B 1000 9 0.90
Total 2000 16 0.80 NS 52
MMC, 0.300 A 1000 42 4.20
B 1000 35 3.50
Total 2000 77 3.85 p ≤ 0.001 45

Without Metabolic Activation - 3-Hour Exposure – B1:
Treatment (μg/mL) Replicate Total BN Cells Scored Total MNBN Cells Scored MNBN Cell Frequency (%) Significance Cytotoxicity (%)
DMSO, 10.0 μL/mL A 1000 2 0.20
B 1000 1 0.10
Total 2000 3 0.15 - -
150 A 1000 4 0.40
B 1000 8 0.80
Total 2000 12 0.60 p ≤ 0.05 5
418 A 1000 9 0.90
B 1000 9 0.90
Total 2000 18 0.90 p ≤ 0.001 35
569 A 1000 8 0.80
B 1000 5 0.50
Total 2000 13 0.65 p ≤ 0.01 58
MMC, 1.00 A 1000 73 7.30
B 1000 56 5.60
Total 2000 129 6.45 p ≤ 0.001 54

With Metabolic Activation - 3-Hour Exposure – B3:
Treatment (μg/mL) Replicate Total BN Cells Scored Total MNBN Cells Scored MNBN Cell Frequency (%) Significance Cytotoxicity (%)
DMSO, 10.0 μL/mL A 1000 4 0.40
B 1000 2 0.20
Total 2000 6 0.30 - -
581 A 1000 8 0.80
B 1000 11 1.10
Total 2000 19 0.95 p ≤ 0.01 16
646 A 1000 13 1.30
B 1000 10 1.00
Total 2000 23 1.15 p ≤ 0.001 31
886 A 1000 12 1.20
B 1000 11 1.10
Total 2000 23 1.15 p ≤ 0.001 53
CP, 15.0 A 1000 20 2.00
B 1000 18 1.80
Total 2000 38 1.90 p ≤ 0.001 64

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)

Activation % MNBN Cells
Vehicle Controls
3 Hour Exposure With Mean ± SD 0.47 ± 0.23
Observed Range 0.00 to 1.00
95% Reference Range 0.06 to 0.84
Number of cultures 144
3 Hour Exposure Without Mean ± SD 0.43 ± 0.22
Observed Range 0.10 to 1.20
95% Reference Range 0.10 to 0.90
Number of cultures 142
~24 Hour Exposure Without Mean ± SD 0.47 ± 0.26
Observed Range 0.00 to 1.60
95% Reference Range 0.10 to 1.07
Number of cultures 132
Positive Controls
3 Hour Exposure With Mean ± SD 4.13 ± 1.30
(CP, 25 μg/mL) Observed Range 1.90 to 9.00
95% Reference Range 2.16 to 6.77
Number of cultures 144
3 Hour Exposure Without Mean ± SD 10.52 ± 2.63
(MMC, 1.00 μg/mL) Observed Range 5.80 to 17.90
95% Reference Range 6.25 to 16.75
Number of cultures 142
~24 Hour Exposure Without Mean ± SD 5.99 ± 1.72
(MMC, 0.300 μg/mL) Observed Range 1.80 to 10.70
95% Reference Range 2.93 to 9.47
Number of cultures 134
Calculated in January 2017 by Greenfield Genetic Toxicology, for studies started between October 2013 and June 2016
CP = Cyclophosphamide
MMC = Mitomycin C
SD = Standard Deviation;

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: The cytostasis / cytotoxicity was determined by calculating the Cytokinesis-Block Proliferation Index (CBPI).
- In the initial assay (B1) without metabolic activation with a 24-hour treatment, 52% cytotoxicity was observed at 135 μg/mL. This concentration along with two lower concentrations, 17.2 and 65.6 μg/mL, producing 11 and 30% cytotoxicity, respectively, were selected for MN scoring.
- In the initial assay (B1) without metabolic activation with a 3-hour treatment, 58% cytotoxicity was observed at 569 μg/mL. This concentration along with two lower concentrations, 150 and 418 μg/mL, producing 5 and 35% cytotoxicity, respectively, were selected for MN scoring.
- In the repeat assay (B3) with metabolic activation with a 3-hour treatment, 53% cytotoxicity was observed at 886 μg/mL. This concentration along with two lower concentrations, 581 and 646 μg/mL, producing 16 and 31% cytotoxicity, respectively, were selected for MN scoring.

Remarks on result:

other:

Remarks:

The positive result is of questionable biological relevance because the increase was seen at all test concentrations without dose relation.

Conclusions:

An in vitro micronucleus assay with the substance was performed according to OECD 487 guideline and GLP principles, in cultured peripheral human lymphocytes in one experiment. It is concluded that the substance was considered positive with questionable biological relevance for inducing micronuclei in the binucleated cells of human peripheral blood lymphocytes from male donors in the 3 hour treatment with S9 and negative in the 3 and approximate 24 hour treatments without S9, when evaluated up to the limit of cytotoxicity.

Executive summary:

In an in vitro micronucleus assay, cultured peripheral human lymphocytes were exposed to different concentrations of the substance (dissolved in DMSO), in the presence and absence of S9-mix according to OECD 487 guideline and GLP principles.

The test substance was evaluated in the initial micronucleus (MN) assay (B1) in HPBL from a male donor at concentrations ranging from 17.2 to 193 μg/mL in the approximate 24 hour treatment without S9, from 150 to 698 μg/mL in the 3 hour treatment without S9 and from 65.5 to 1000 μg/mL in the 3 hour treatment with S9. No precipitate was observed at the end of the treatment at any tested concentration in any treatment condition with or without S9. In the approximate 24 hour treatment without S9, 52% cytotoxicity was observed at 135 μg/mL. This concentration along with two lower concentrations, 17.2 and 65.6 μg/mL, producing 11 and 30% cytotoxicity respectively were selected for MN scoring. In the 3 hour treatment without S9, 58% cytotoxicity was observed at 569 μg/mL. This concentration along with two lower concentrations, 150 and 418 μg/mL, producing 5 and 35% cytotoxicity, respectively were selected for MN scoring. In the 3 hour treatment with S9, appropriate cytotoxicity could not be achieved and the assay was repeated for this test condition with a new set of concentrations.

In the repeat MN assay (B3), the test article was evaluated in the 3 hour treatment with S9 at concentrations ranging from 30.4 to 1350 μg/mL. No precipitate was observed at the end of treatment at any tested concentration. In this test condition, 53% cytotoxicity was observed at 886 μg/mL. This concentration along with two lower concentrations, 581 and 646 μg/mL, producing 16 and 31% cytotoxicity respectively were selected for MN evaluation.

No statistically significant increase in the frequency of micronucleated binucleated cells (MNBN) was observed at any evaluated concentration in the approximate 24 hour treatment without S9. Statistically significant increases were observed at all evaluated concentrations in the 3 hour treatment without S9. However, the %MNBN frequencies observed at these concentrations were within the historical vehicle control range for this test condition in male donors. Therefore, the statistically significant increase was considered as biologically non-relevant. In the 3 hour treatment with S9, statistically significant increases were observed at all evaluated concentrations. The %MNBN frequencies at the three evaluated concentrations were outside the 95% historical reference range. However, no clear concentration related increase was observed at the evaluated concentrations. Therefore, the statistically significant increase observed in the test condition was considered of questionable biological relevance. The MNBN frequencies of the vehicle and positive controls were within the acceptable range. All acceptance criteria were met and the study was accepted as valid.

The test substance was considered positive with questionable biological relevance for inducing micronuclei in the binucleated cells of human peripheral blood lymphocytes from male donors in the 3 hour treatment with S9 and negative in the 3 and approximate 24 hour treatments without S9, when evaluated up to the limit of cytotoxicity.

Reference 4

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: read-across information

Justification for type of information:

Information for Ethylphenylglycidate is derived from Methyl beta-phenylglycidate using read across. The read across rationale is presented in the Genotoxicity Endpoint summary where also the accompanying files are attached.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

lymphocytes: human peripheral blood

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: human peripheral blood

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Dose range finding: Precipitate was observed at ≥1247 μg/mL at the time of treatment in all three treatment conditions except the approximate 24 hour treatment without S9, where precipitate was observed at ≥873 μg/mL. At the time of harvest, precipitate was observed at 1782 μg/mL in the 3 and approximate 24 hour treatment without S9.
Micronucleus assay: no precipitate was observed at the end of the treatment or harvest.

RANGE-FINDING/SCREENING STUDIES: In the approximate 24 hour treatment without S9, 41% cytotoxicity was observed at 147 μg/mL. The next higher concentration, 210 μg/mL, was excessively cytotoxic and produced 81% cytotoxicity. In the 3 hour treatment without S9, 55% cytotoxicity was observed at 428 μg/mL. In the 3 hour treatment with S9, 53% cytotoxicity was observed at 873 μg/mL. Based on the DRF results, concentrations were selected for the micronucleus assay.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells:

Without Metabolic Activation - 24-Hour Exposure – B1:
Treatment
(μg/mL) Replicate Mono nucleate Bi nucleate Multi nucleate Total CBPI Cytotoxicity (%)
DMSO, 10.0 μL/mL A 126 380 60 566 1.88
B 120 373 50 543 1.87
Total 246 753 110 1109 1.88 -
17.2 A 135 361 35 531 1.81
B 173 359 31 563 1.75
Total 308 720 66 1094 1.78 11
42.0 A 196 296 20 512 1.66
B 199 341 19 559 1.68
Total 395 637 39 1071 1.67 24
65.6 A 207 300 14 521 1.63
B 241 327 11 579 1.60
Total 448 627 25 1100 1.62 30
103 A 183 387 24 594 1.73
B 270 301 7 578 1.54
Total 453 688 31 1172 1.64 27
128 A 304 244 6 554 1.46
B 295 230 1 526 1.44
Total 599 474 7 1080 1.45 48
135 A 319 196 1 516 1.38
B 298 240 2 540 1.45
Total 617 436 3 1056 1.42 52
142 A 315 232 5 552 1.44
B 350 197 4 551 1.37
Total 665 429 9 1103 1.41 54
150 A 297 221 1 519 1.43
B 317 250 2 569 1.45
Total 614 471 3 1088 1.44 50
157 A 361 217 3 581 1.38
B 342 242 5 589 1.43
Total 703 459 8 1170 1.41 54
166 A 400 159 0 559 1.28
B 392 204 3 599 1.35
Total 792 363 3 1158 1.32 64
174 A 348 192 0 540 1.36
B 351 181 2 534 1.35
Total 699 373 2 1074 1.35 60
193 A 419 126 0 545 1.23
B 344 201 3 548 1.38
Total 763 327 3 1093 1.30 65
MMC, 0.300 A 287 244 3 534 1.47
B 307 289 2 598 1.49
Total 594 533 5 1132 1.48 45

Without Metabolic Activation - 3-Hour Exposure – B1:
Treatment
(μg/mL) Replicate Mono nucleate Bi nucleate Multi nucleate Total CBPI Cytotoxicity (%)
DMSO, 10.0 μL/mL A 86 424 75 585 1.98
B 88 453 47 588 1.93
Total 174 877 122 1173 1.96 -
150 A 104 382 37 523 1.87
B 81 449 48 578 1.94
Total 185 831 85 1101 1.91 5
302 A 171 332 26 529 1.73
B 170 331 13 514 1.69
Total 341 663 39 1043 1.71 26
377 A 201 357 8 566 1.66
B 221 334 10 565 1.63
Total 422 691 18 1131 1.64 33
418 A 202 300 8 510 1.62
B 225 323 15 563 1.63
Total 427 623 23 1073 1.62 35
440 A 258 307 16 581 1.58
B 216 345 12 573 1.64
Total 474 652 28 1154 1.61 36
463 A 202 302 5 509 1.61
B 240 317 8 565 1.59
Total 442 619 13 1074 1.60 37
513 A 261 286 5 552 1.54
B 281 246 2 529 1.47
Total 542 532 7 1081 1.51 47
569 A 301 210 1 512 1.41
B 327 205 3 535 1.39
Total 628 415 4 1047 1.40 58
630 A 309 199 0 508 1.39
B 290 245 6 541 1.48
Total 599 444 6 1049 1.43 55
698 A 335 256 4 595 1.44
B 342 178 2 522 1.35
Total 677 434 6 1117 1.40 58P
MMC, 1.00 A 315 260 1 576 1.45
B 305 215 1 521 1.42
Total 620 475 2 1097 1.44 54

With Metabolic Activation - 3-Hour Exposure – B3:
Treatment
(μg/mL) Replicate Mono nucleate Bi nucleate Multi nucleate Total CBPI Cytotoxicity (%)
DMSO, 10.0 μL/mL A 159 280 61 500 1.80
B 196 258 46 500 1.70
Total 355 538 107 1000 1.75 -
30.4 A 111 322 67 500 1.91
B 137 311 52 500 1.83
Total 248 633 119 1000 1.87 0
96.9 A 127 320 53 500 1.85
B 155 302 43 500 1.78
Total 282 622 96 1000 1.81 0
182 A 100 323 77 500 1.95
B 160 291 49 500 1.78
Total 260 614 126 1000 1.87 0
278 A 160 315 25 500 1.73
B 167 304 29 500 1.72
Total 327 619 54 1000 1.73 3
381 A 158 315 27 500 1.74
B 200 291 14 505 1.63
Total 358 606 41 1005 1.68 9
471 A 254 242 4 500 1.50
B 186 311 3 500 1.63
Total 440 553 7 1000 1.57 25
581 A 159 337 4 500 1.69
B 216 282 2 500 1.57
Total 375 619 6 1000 1.63 16
646 A 247 247 6 500 1.52
B 244 249 7 500 1.53
Total 491 496 13 1000 1.52 31
797 A 268 230 2 500 1.47
B 320 180 0 500 1.36
Total 588 410 2 1000 1.41 45
886 A 300 195 5 500 1.41
B 348 152 0 500 1.30
Total 648 347 5 1000 1.36 53
984 A 283 213 4 500 1.44
B 333 167 0 500 1.33
Total 616 380 4 1000 1.39 48P
1094 A 335 160 5 500 1.34
B 318 179 3 500 1.37
Total 653 339 8 1000 1.36 53P
1215 A 394 131 1 526 1.25
B 405 93 2 500 1.19
Total 799 224 3 1026 1.22 70P
1350 A 449 50 1 500 1.10
B 451 48 1 500 1.10
Total 900 98 2 1000 1.10 86P
CP, 15.0 A 352 149 1 502 1.30
B 382 118 0 500 1.24
Total 734 267 1 1002 1.27 64

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture:
- Indication whether binucleate or mononucleate where appropriate:
Without Metabolic Activation - 24-Hour Exposure – B1:
Treatment (μg/mL) Replicate Total BN Cells Scored Total MNBN Cells Scored MNBN Cell Frequency (%) Significance Cytotoxicity (%)
DMSO, 10.0 μL/mL A 1000 4 0.40
B 1000 4 0.40
Total 2000 8 0.40 - -
17.2 A 1000 3 0.30
B 1000 5 0.50
Total 2000 8 0.40 NS 11
65.6 A 1000 3 0.30
B 1000 4 0.40
Total 2000 7 0.35 NS 30
135 A 1000 7 0.70
B 1000 9 0.90
Total 2000 16 0.80 NS 52
MMC, 0.300 A 1000 42 4.20
B 1000 35 3.50
Total 2000 77 3.85 p ≤ 0.001 45

Without Metabolic Activation - 3-Hour Exposure – B1:
Treatment (μg/mL) Replicate Total BN Cells Scored Total MNBN Cells Scored MNBN Cell Frequency (%) Significance Cytotoxicity (%)
DMSO, 10.0 μL/mL A 1000 2 0.20
B 1000 1 0.10
Total 2000 3 0.15 - -
150 A 1000 4 0.40
B 1000 8 0.80
Total 2000 12 0.60 p ≤ 0.05 5
418 A 1000 9 0.90
B 1000 9 0.90
Total 2000 18 0.90 p ≤ 0.001 35
569 A 1000 8 0.80
B 1000 5 0.50
Total 2000 13 0.65 p ≤ 0.01 58
MMC, 1.00 A 1000 73 7.30
B 1000 56 5.60
Total 2000 129 6.45 p ≤ 0.001 54

With Metabolic Activation - 3-Hour Exposure – B3:
Treatment (μg/mL) Replicate Total BN Cells Scored Total MNBN Cells Scored MNBN Cell Frequency (%) Significance Cytotoxicity (%)
DMSO, 10.0 μL/mL A 1000 4 0.40
B 1000 2 0.20
Total 2000 6 0.30 - -
581 A 1000 8 0.80
B 1000 11 1.10
Total 2000 19 0.95 p ≤ 0.01 16
646 A 1000 13 1.30
B 1000 10 1.00
Total 2000 23 1.15 p ≤ 0.001 31
886 A 1000 12 1.20
B 1000 11 1.10
Total 2000 23 1.15 p ≤ 0.001 53
CP, 15.0 A 1000 20 2.00
B 1000 18 1.80
Total 2000 38 1.90 p ≤ 0.001 64

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)

Activation % MNBN Cells
Vehicle Controls
3 Hour Exposure With Mean ± SD 0.47 ± 0.23
Observed Range 0.00 to 1.00
95% Reference Range 0.06 to 0.84
Number of cultures 144
3 Hour Exposure Without Mean ± SD 0.43 ± 0.22
Observed Range 0.10 to 1.20
95% Reference Range 0.10 to 0.90
Number of cultures 142
~24 Hour Exposure Without Mean ± SD 0.47 ± 0.26
Observed Range 0.00 to 1.60
95% Reference Range 0.10 to 1.07
Number of cultures 132
Positive Controls
3 Hour Exposure With Mean ± SD 4.13 ± 1.30
(CP, 25 μg/mL) Observed Range 1.90 to 9.00
95% Reference Range 2.16 to 6.77
Number of cultures 144
3 Hour Exposure Without Mean ± SD 10.52 ± 2.63
(MMC, 1.00 μg/mL) Observed Range 5.80 to 17.90
95% Reference Range 6.25 to 16.75
Number of cultures 142
~24 Hour Exposure Without Mean ± SD 5.99 ± 1.72
(MMC, 0.300 μg/mL) Observed Range 1.80 to 10.70
95% Reference Range 2.93 to 9.47
Number of cultures 134
Calculated in January 2017 by Greenfield Genetic Toxicology, for studies started between October 2013 and June 2016
CP = Cyclophosphamide
MMC = Mitomycin C
SD = Standard Deviation;

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: The cytostasis / cytotoxicity was determined by calculating the Cytokinesis-Block Proliferation Index (CBPI).
- In the initial assay (B1) without metabolic activation with a 24-hour treatment, 52% cytotoxicity was observed at 135 μg/mL. This concentration along with two lower concentrations, 17.2 and 65.6 μg/mL, producing 11 and 30% cytotoxicity, respectively, were selected for MN scoring.
- In the initial assay (B1) without metabolic activation with a 3-hour treatment, 58% cytotoxicity was observed at 569 μg/mL. This concentration along with two lower concentrations, 150 and 418 μg/mL, producing 5 and
35% cytotoxicity, respectively, were selected for MN scoring.
- In the repeat assay (B3) with metabolic activation with a 3-hour treatment, 53% cytotoxicity was observed at 886 μg/mL. This concentration along with two lower concentrations, 581 and 646 μg/mL, producing 16 and 31% cytotoxicity, respectively, were selected for MN scoring.

Conclusions:

It is concluded that the substance was considered positive with questionable biological relevance for inducing micronuclei in the binucleated cells of human peripheral blood lymphocytes from male donors in the 3 hour treatment with S9 and negative in the 3 and approximate 24 hour treatments without S9, when evaluated up to the limit of cytotoxicity.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Ethylphenylglycidate result in the in vivo micronucleus using read across from Methyl beta-phenylglycidate tested in OECD TG 474: Negative

Ethylphenylglycidate result in the Comet assay based on read across from Methyl beta-phenylglycidate tested in OECD TG 489: Negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 January 2017 - 05 April 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

other: Mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

other: Hsd:ICR (CD-1)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS, Inc., Frederick, MD
- Age at study initiation: 6 weeks
- Weight at study initiation: males: 30.6 - 36.1g; females: 24.3 - 26.7g
- Assigned to test groups randomly: yes
- Fasting period before study: No data.
- Housing: Animals of the same sex were housed up to three per Micro-Barrier cage.
- Diet: Free access to a certified laboratory rodent chow (Envigo 2018C Teklad Global 18% Protein Rodent Diet).
- Water: Free access to tap water.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS (set to maintain)
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 07 February 2017 To: 05 April 2017

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: corn oil
- Justification for choice of vehicle: Corn oil was the vehicle of choice based on the solubility of the test substance and compatibility with the test system.
- Concentration of test material in vehicle: The test substance formed a solution in corn oil at a concentration of approximately 200 mg/mL, the maximum concentration tested.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VOLUME: 10 mL/kg bw

Duration of treatment / exposure:

24 hours/dose

Frequency of treatment:

dosed on four consecutive days

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

250 mg/kg bw/day

Dose / conc.:

500 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide and methylmethanesulfonate
- Route of administration: oral gavage
- Doses / concentrations: 20 mg/kg/day / 2 mg/mL (cyclophosphamide), dosed on study days 1 and 2; 40 mg/kg/day / 4 mg/mL (methylmethanesulfonate), dosed on study day 4

Tissues and cell types examined:

The frequency of micronucleated reticulocytes in peripheral blood was analyzed after flow cytometry calibration using Malaria infected biostandard and CD71-negative control standards provided in the Litron kit. Up to 20,000 RETs per animal, when possible, were analyzed.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: In the dose range finding test, clinical signs of toxicity were observed at all dose levels in male mice and at 1000 and 2000 mg/kg/bw day in female mice. Mortality was observed in all animals at 2000 mg/kg/bw day. Due to the clinical observations and mortality observed, both sexes were used in the definitive assay.

Peripheral Blood Collection for Flow Cytometry:
Approximately 3 to 4 hours after the last dose, peripheral blood (>120 μL) was collected for the detection of micronuclei using flow cytometry. MicroFlow Anticoagulant, supplied by Litron Labs (stored at 2-8 ºC) was used as Anticoagulant.

Collection Location
Retro-orbital Sinus:
Animals were anesthetized prior to blood collection by exposure to 70% CO2/30% O2. Each sample was collected into a blank micro-centrifuge tube; then, 100 μL was transferred into a pre-labeled tube with anticoagulant, pipetted up and down several times to mix and placed on wet ice until all of the samples were collected. Blood samples in anticoagulant were kept on wet ice for less than two hours until fixation was complete. Approximately 180 μL of the blood mixture with anticoagulant was transferred into 2 sets of tubes containing fixative (freezing cold methanol). After fixation, samples were stored at -80 ± 5°C for 15 days prior to flow cytometric analysis.

METHOD OF ANALYSIS:
Detection of Micronucleated Reticulocytes with Flow Cytometry:
Peripheral blood samples were washed with ice cold buffer solution to remove the fixative. The cells were then pelleted by centrifugation, and the supernatant was poured off leaving a small amount of supernatant with the pellet. The cells were re-suspended and 20 μL of suspension was added to 80 μL of staining solution containing RNase, FITC-conjugated anti-CD 71 antibodies (mouse antibody) and PE-conjugated anti-CD 61 antibodies (platelet antibody). The samples were incubated at 2-8ºC for 30 minutes, re-suspended, and then incubated at room temperature for an additional 30 minutes. 1.5 mL of DNA staining solution (propidium iodide) was added; then, the samples were placed on wet ice for 5 minutes prior to flow cytometric analysis.

Calculation of Flow Cytometric Analysis
The proportion of reticulocytes to total number of cells scored (% RETs) was determined for each animal and treatment group. This calculation was carried out as indicated below:
% RET = (UL + UR) X 100 / (UL + UR + LL + LR)
UL: The number of events in the upper-left quadrant
UR: The number of events in the upper-right quadrant
LL: The number of events in the lower-left quadrant
LR: The number of events in the lower-right quadrant

The % RETs served as a parameter of the test substance cytotoxicity in peripheral blood. A decrease in this ratio in the test substance groups, as compared to the vehicle (negative) control, would indicate a toxic effect of the test substance, while an increase would represent a sign of recovery from earlier toxic insult. The animal with a <5% %RET of the vehicle control was considered excessively cytotoxic and was excluded from evaluation.
The quantization of the MnRETs in peripheral blood was expressed as percentage of MnRETs per total number of cells evaluated. The % MnRETs was presented for each animal and the mean ± standard deviation was calculated and presented for each treatment group as follows:
% MnRET = (UR) X 100 / (UL + UR)

Evaluation criteria:

A test substance was considered to have induced a positive response if:
a) at least one of the test substance doses exhibited a statistically significant increase when compared with the concurrent negative control (p ≤ 0.05), and
b) when multiple doses were examined at a particular sampling time, the increase was dose-related (p ≤ 0.01), and
c) results of the group mean or of the individual animals in at least one group were outside the 95% control limit of the historical negative control data.
A test substance was considered to have induced a clear negative response if none of the criteria for a positive response were met and there was evidence that the peripheral blood was exposed to the test substance (unless intravenous administration was used).

Statistics:

Statistical analysis was performed on the micronucleus frequency (%MnRET) and RET% using the animal as the unit. The mean and standard deviation of %MnRET and RET% were presented for each treatment group.
The use of parametric or non-parametric statistical methods in the evaluation of data was based on the variation between groups. The group variances for micronucleus frequency for the vehicle and test substance groups at the respective sampling time were compared using Levene’s test (significant level of p ≤ 0.05). Since the variation between groups was found not to be significant, a parametric one-way ANOVA was performed followed by a Dunnett’s post-hoc analysis to compare each dose group to the concurrent vehicle control.
A linear regression analysis was conducted to assess dose responsiveness in the test substance-treated groups (p ≤ 0.01).
A pair-wise comparison (Student’s T-test, p ≤ 0.05) was used to compare the positive control groups to the concurrent vehicle control groups.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
In the dose range finding test, clinical signs of toxicity were observed at all dose levels in male mice and at 1000 and 2000 mg/kg/bw day in female mice. Mortality was observed in all animals at 2000 mg/kg/bw day. No considerable reductions in mean group body weights were observed in the test substance-treated groups during the course of the study.
The following clinical signs were observed:
Dose Level (mg/kg/bw day) Males Females
500 Piloerection Normal
1000 Piloerection Piloerection
2000 Piloerection, ataxia and Piloerection and ataxia
irregular breathing

RESULTS OF DEFINITIVE STUDY
Mortality occurred in 1/6 female mice at 1000 mg/kg/bw day in the definitive assay. No mortality occurred in male mice. No considerable reductions in mean group body weights were observed in the test substance-treated groups during the course of the study.
The following clinical signs were observed:
Dose Level (mg/kg/bw day) Males Females
Vehicle Normal Normal
250 Piloerection Piloerection
500 Piloerection Piloerection
1000 Piloerection Piloerection
Positive control Normal Normal

The scoring results and a statistical analysis of data indicated the following:
• No appreciable reductions in %RETs were observed in the test substance-treated groups compared to the vehicle control groups, indicating the test substance did not induce cytotoxicity.
• Group variances for mean of means of the micronucleus frequency in the vehicle and test substance groups were compared using Levene’s test. The test indicated that there was no significant difference in the group variance (p > 0.05); therefore, the parametric approach, ANOVA followed by Dunnett’s post-hoc analysis, was used in the statistical analysis of data.
• No statistically significant increases in the incidence of MnRETs were observed in the test substance-treated groups relative to the vehicle control groups (ANOVA followed by Dunnett’s post-hoc analysis, p > 0.05).
• The positive control, CP, induced a statistically significant increase in the incidence of MnRETs (Student’s t-test, p ≤ 0.05).
• The number of MnRETs in the vehicle control groups did not exceed the historical control range

Conclusions:

A micronucleus study with the substance was performed according to OECD 474 guideline and GLP principles, in male and female mice. It is concluded that the substance is not mutagenic in the mouse micronucleus assay.

Executive summary:

Method: In an in vivo micronucleus study (as part of the Comet assay), 6 male and 6 female mice per dose were exposed to 250, 500 and 1000 mg/kg bw day of the substance for four consecutive days, performed according to OECD 474 guideline and GLP principles. The maximum tolerated dose for the definitive Micronucleus assay was set at 1000 mg/kg/bw day based on mortality observed in all animals at 2000 mg/kg/bw day in a dose range finding assay.

Results on toxicity: Mortality occurred in 1/6 female mice at 1000 mg/kg/bw day in the definitive assay. No mortality occurred in male mice. No considerable reductions in mean group body weights were observed in the test substance-treated groups during the course of the study. Clinical signs were observed at all dose levels and included piloerection.

Results on genotoxicity: No appreciable reductions could also be increase in %RETs (reticulocytes) were observed in the test substance-treated groups compared to the vehicle control groups, indicating the test substance did not induce cytotoxicity in the red blood cells. In view of the systemic effects seen (mortality) the substance (and its metabolites) have reached the systemic circulation. No statistically significant increases in the incidence of MnRETs (reticulocytes containing micronuclei) were observed in the test substance-treated groups relative to the vehicle control groups (ANOVA followed by Dunnett’s post-hoc analysis, p > 0.05).

Conclusion: The substance is negative in the in vivo micronucleus assay.

Reference 2

Endpoint:

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

Remarks:

Mammalian Alkaline Comet Assay

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 January 2017 - 05 April 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This information is used for read across to Ethylphenylglycidate.
For Methyl beta-phenylglycidate the micronucleus assay and the Comet assay were combined in a single study using one set of animals. This combined four daily dose study design allowed for the assessment of the test substance at two genotoxicity end points and markedly reduced the number of animals, the time for generation of the relevant data, as well as the amount of the test substance required.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: See under "Principles of method if other than guideline"

GLP compliance:

yes

Type of assay:

mammalian comet assay

Species:

mouse

Strain:

other: Hsd:ICR (CD-1)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS, Inc., Frederick, MD
- Age at study initiation: 6 weeks
- Weight at study initiation: males: 30.6 - 36.1; females: 24.3 - 26.7
- Assigned to test groups randomly: yes
- Fasting period before study: No data.
- Housing: Animals of the same sex were housed up to three per Micro-Barrier cage.
- Diet: Free access to a certified laboratory rodent chow (Envigo 2018C Teklad Global 18% Protein Rodent Diet).
- Water: Free access to tap water.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS (set to maintain)
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 07 February 2017 To: 05 April 2017

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: corn oil
- Justification for choice of vehicle: Corn oil was the vehicle of choice based on the solubility of the test substance and compatibility with the test system.
- Concentration of test material in vehicle: The test substance formed a solution in corn oil at a concentration of approximately 200 mg/mL, the maximum concentration tested.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
Dose formulations were prepared prior to use on each day of dose administration as follows:
An appropriate amount of test substance was weighed and transferred to a suitable sized amber glass vial with a PTFE stir bar, which was then calibrated to the target batch size. Approximately 70% of the total volume of vehicle was added to the vial and the mixture was stirred magnetically. The remaining volume of vehicle was added until the final target volume was achieved. The mixture was stirred magnetically for 5 to 28 minutes until uniform. The final formulation was stored at room temperature until used for dosing.

VOLUME: 10 mL/kg bw

Duration of treatment / exposure:

four days

Frequency of treatment:

four consecutive days

Post exposure period:

Animals were euthanized 3-4 hours after last treatment.

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

250 mg/kg bw/day

Dose / conc.:

500 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

6 (test substance groups);
3 (positive control group)

Control animals:

yes, concurrent vehicle

Positive control(s):

Methylmethanesulfonate
- Route of administration: oral gavage
- Dose / concentration: 40 mg/kg bw/day (10 mL/kg bw/day), dosed on study day 4 only.
- The positive control was prepared in 0.9% sodium chloride. The dosing formulation was prepared at a concentration of 4 mg/mL for MMS, just prior to use.

Tissues and cell types examined:

- Animals were dissected and the liver was extracted (removed) and collected.
- Another section of the liver was placed in chilled mincing solution (Hanks’ balanced salt solution with EDTA and DMSO) and was used in preparation of cell suspensions and Comet slides.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: In a dose range-finder assay, 3 animals/sex were exposed to 2000, 1000 and 500 mg/kg/day at a dose volume of 10 mL/kg bw/dose for four consecutive days. The second and third doses occurred approximately 24 hours after the prior dose. The fourth dose occurred approximately 21 hours after the third dose. At least three hours following the final dose, all surviving animals were euthanized by CO2 inhalation and discarded without further examination. Clinical signs of toxicity were observed at all dose levels in male mice and at 1000 and 2000 mg/kg bw/day in female mice. Mortality was observed in all animals at 2000 mg/kg bw/day. Due to the clinical observations and mortality observed, both sexes were used in the definitive assay. Based on these results, 1000 mg/kg bw/day was estimated to be the maximum tolerated dose (MTD), which was chosen as the high dose for the definitive assay.

DETAILS OF SLIDE PREPARATION: From each liver suspension, an aliquot of 2.5 μL was mixed with 75 μL (0.5%) of low melting agarose. The cell/agarose suspension was applied to commercially available pre-treated multi-well slides. The slides were kept at 2 to 8°C for at least 15 minutes to allow the gel to solidify. At least two 3-well slides were prepared per animal per tissue. Three wells were used in scoring and the other wells were designated as a backup. Following solidification of agarose, the slides were placed in jars containing lysis solution. Following solidification of the agarose, the slides were submerged in a commercially available lysis solution supplemented with 10% DMSO on the day of use. The slides were kept in this solution at least overnight at 2 to 8°C. After cell lysis, slides/wells were washed with neutralization buffer (0.4 M tris hydroxymethyl aminomethane in purified water, pH 7.5) and placed in the electrophoresis chamber. The chamber reservoirs were slowly filled with alkaline buffer composed of 300 mM sodium hydroxide and 1 mM EDTA (disodium) in purified water. The pH was > 13. All slides remained in the buffer for 20 minutes at 2 to 10°C and protected from light, allowing DNA to unwind. Using the same buffer, electrophoresis was conducted for 30 minutes at 0.7 V/cm, at 2 to 10°C and protected from light. The electrophoresis time was constant for all slides. After completion of electrophoresis, the slides were removed from the electrophoresis chamber and washed with neutralization buffer for at least 10 minutes. The slides (gels) were then dehydrated with 200-proof ethanol for at least 5 minutes, then air dried for at least 4 hours and stored at room temperature with desiccant. Slides were stained with a DNA stain (i.e., Sybr-goldTM) prior to scoring. The stain solution was prepared by diluting 1 μL of Sybr-goldTM stain in 15 mL of 1xTBE (tris-boric acid EDTA buffer solution).

METHOD OF ANALYSIS: Three slides/wells per organ/animal were used. Fifty randomly selected cells per slide/well were scored resulting in a total of 150 cells evaluated per animal.
The following endpoints of DNA damage were assessed and measured:
- Comet Tail Migration; defined as the distance from the perimeter of the Comet head to the last visible point in the tail;.
- % Tail DNA (also known as % tail intensity or % DNA in tail); defined as the percentage of DNA fragments present in the tail.
- Tail Moment (also known as Olive Tail moment); defined as the product of the amount of DNA in the tail and the tail length [(% Tail DNA x Tail Length)/ 100]
Each slide/well was also examined for indications of cytotoxicity (scoring of "clouds"/ "hedgehogs" in 150 cells per animal). The “clouds”, also known as
“hedgehogs,” are a morphological indication of highly damaged cells often associated with severe genotoxicity, necrosis or apoptosis. A “cloud” is produced when almost the entire cell DNA is in the tail of the Comet and the head is reduced in size, almost nonexistent. “Clouds” with visible gaps between the nuclei and the Comet tail were excluded from Comet image analysis.

Evaluation criteria:

A test substance was considered to have induced a positive response if:
a) at least one of the test substance doses exhibited a statistically significant increase when compared with the concurrent vehicle control (p ≤ 0.05), and
b) when multiple doses were examined at a particular sampling time, the increase was dose-related (p ≤ 0.01) and
a) results of the group mean or of the individual animals of at least one group were outside the distribution of the historical negative control database for that tissue.
The test substance was considered to have induced a clear negative response if none of the criteria for a positive response were met and direct or indirect evidence supportive of exposure of, or toxicity to, the target tissue was demonstrated.

Statistics:

The median value of 150 counts of % Tail DNA, Tail moment and Tail migration were determined and presented for each animal in each treatment group for each organ. The mean and standard deviation of the median values only for % Tail DNA were presented for each treatment group. Statistical analysis was performed only for % Tail DNA. The use of parametric or non-parametric statistical methods in evaluation of data was based on the variation between groups. The group variances for % tail DNA generated for the vehicle and test substance groups were compared using Levene’s test (significant level of p ≤ 0.05). Since the differences and variations between groups were found not to be significant, a parametric one-way ANOVA followed by a Dunnett’s post-hoc test was performed (significant level of p < 0.05). A linear regression analysis was conducted to assess dose responsiveness in the test substance treated groups (p ≤ 0.01). A pair-wise comparison (Student’s T-test, p ≤ 0.05) was used to compare the positive control group to the concurrent vehicle control group.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
Mortality occurred in all animals at 2000 mg/kg bw/day in the dose range-finding assay. No considerable reductions in mean group body weights were observed in the test substance-treated groups during the course of the study. In males, piloerection was seen at all dose levels (at 2000 mg/kg bw/day, in addition ataxia and irregular breathing was seen). In females piloerection was seen at 1000 and 2000 mg/kw bw/day, at the highest dose also ataxia was noted. Based on these data the maximum tolerated dose for the definitive Comet assay was set at 1000 mg/kg bw/day.

RESULTS OF DEFINITIVE STUDY
No statistically significant increases in % tail DNA were observed in the test substance-treated groups relative to the vehicle control groups. The positive control induced a statistically significant increase in % tail DNA compared to the vehicle control. The vehicle control % tail DNA was within the Testing Facility’s historical range.

Mortality occurred in 1/6 female mice at 1000 mg/kg bw/day in the definitive assay. No mortality occurred in male mice. Piloerection was noted in all treated animals. No considerable reductions in mean group body weights were observed in the test substance-treated groups during the course of the study.

Table 1: % Tail DNA in male liver cells following four dose administrations

Treatment	Number of animals	Group mean % of clouds	Tail DNA (%), mean±SD
Vehicle control	6	1.3	0.23	0.22
250 mg/kg bw	6	1.7	0.16	0.14
500 mg/kg bw	6	1.8	0.09	0.08
1000 mg/kg bw	6	1.8	0.07	0.07
Positive control	3	6.7	13.87*	7.76

*p ≤ 0.05

Table 2: % Tail DNA in female liver cells following four dose administrations

Treatment	Number of animals	Group mean % of clouds	Tail DNA (%), mean±SD
Vehicle control	6	1.8	0.10	0.10
250 mg/kg bw	6	1.5	0.08	0.06
500 mg/kg bw	6	2.7	0.13	0.08
1000 mg/kg bw	6	1.6	0.08	0.08
Positive control	3	2.7	9.67*	3.56

*p ≤ 0.05

Conclusions:

Based on the results of a Comet assay performed equivalent to OECD 489 guideline and GLP principles, it is concluded that the test substance does not increase DNA damage in the liver.

Executive summary:

Method: An in vivo Comet assay was performed according to OECD guideline 489 and GLP principles, in which male and female mice (6/sex/group) were exposed to 250, 500 and 1000 mg/kg bw for four consecutive days. The maximum dose represents the maximum tolerated dose based on a dose range finder study.

Results on toxicity: Mortality occurred in 1/6 female mice at 1000 mg/kg bw/day in the definitive assay. No mortality occurred in male mice. No considerable reductions in mean group body weights were observed in the test substance-treated groups during the course of the study. Clinical signs were observed at all dose levels and included piloerection indicating systemic exposure of the substance and its metabolites.

Results on genotoxicity: In this assay, DNA damage was determined in liver cells only. No statistically significant increases in % tail DNA were observed in the test substance-treated groups relative to the vehicle control groups. The positive control induced a statistically significant increase in % tail DNA compared to the vehicle control. The vehicle control % tail DNA was within the Testing Facility’s historical range.

Conclusion: Based on these results it is concluded that the substance does not increase DNA damage in the liver.

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: read-across information

Justification for type of information:

Information is derived from Methyl betaphenylglycidate using read across. The rationale is presented in the Genotoxicity Endpoint summary and the accompanying files are also attached there.

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Ethylphenylglycidate is negative in the in vivo micronucleus test based on read across from Methyl beta-phenylglycidate tested in OECD TG 474)

Reference 4

Endpoint:

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

Remarks:

Mammalian Alkaline Comet Assay

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: read-across information

Justification for type of information:

The information for Ethylphenylglycidate is derived from Methyl beta-phenylglycidate, tested in a Comet assay, using read across.

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
Mortality occurred in all animals at 2000 mg/kg bw/day in the dose range-finding assay. No considerable reductions in mean group body weights were observed in the test substance-treated groups during the course of the study. In males, piloerection was seen at all dose levels (at 2000 mg/kg bw/day, in addition ataxia and irregular breathing was seen). In females piloerection was seen at 1000 and 2000 mg/kw bw/day, at the highest dose also ataxia was noted. Based on these data the maximum tolerated dose for the definitive Comet assay was set at 1000 mg/kg bw/day.

RESULTS OF DEFINITIVE STUDY
No statistically significant increases in % tail DNA were observed in the test substance-treated groups relative to the vehicle control groups. The positive control induced a statistically significant increase in % tail DNA compared to the vehicle control. The vehicle control % tail DNA was within the Testing Facility’s historical range.

Mortality occurred in 1/6 female mice at 1000 mg/kg bw/day in the definitive assay. No mortality occurred in male mice. Piloerection was noted in all treated animals. No considerable reductions in mean group body weights were observed in the test substance-treated groups during the course of the study.

Table 1: % Tail DNA in male liver cells following four dose administrations

Treatment	Number of animals	Group mean % of clouds	Tail DNA (%), mean±SD
Vehicle control	6	1.3	0.23	0.22
250 mg/kg bw	6	1.7	0.16	0.14
500 mg/kg bw	6	1.8	0.09	0.08
1000 mg/kg bw	6	1.8	0.07	0.07
Positive control	3	6.7	13.87*	7.76

*p ≤ 0.05

Table 2: % Tail DNA in female liver cells following four dose administrations

Treatment	Number of animals	Group mean % of clouds	Tail DNA (%), mean±SD
Vehicle control	6	1.8	0.10	0.10
250 mg/kg bw	6	1.5	0.08	0.06
500 mg/kg bw	6	2.7	0.13	0.08
1000 mg/kg bw	6	1.6	0.08	0.08
Positive control	3	2.7	9.67*	3.56

*p ≤ 0.05

Conclusions:

It is concluded that the test substance does not increase DNA damage in the liver.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The executive summaries of the substance are presented below followed by the executive summaries of the source substance and the read-across rationale.

 

Ethylphenylglycidate: Ames test (OECD TG 471, Kl 1) Key information and a study record included

Method: The mutagenic activity of the substance was evaluated in accordance with OECD 471 (1997) guideline and according to GLP principles. The test was performed in two independent experiments: at first a direct

plate assay was performed and second a pre-incubation assay in the absence and presence of S9-mix up to and including the concentration of 5000 μg/plate. Acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Results on precipitation and cytotoxicity first test: Precipitation was observed in the first direct plate experiment, no precipitation was observed at the end of the incubation period in the pre-incubation experiment and in the second direct plate experiment. In the direct plate experiment, cytotoxicity, as evidenced by a reduction of the bacterial background lawn and the presence of microcolonies, was observed in tester strain TA100 in the presence of S9-mix at the highest concentration tested. In TA98, TA1535 and TA1537 cytotoxicity, as evidenced by a decrease in the number of revertants and a reduction of the bacterial background lawn was observed in all tester strains in the absence of S9-mix. No toxicity was observed in the presence of S9-mix.

Results on revertants: In the absence of S9-mix, the test item induced dose-related increases in the number of revertant colonies compared to the solvent control in two tester strains (TA100 and WP2uvrA). The increases observed in the tester strains TA100 and WP2uvrA were above the laboratory historical control data range and were up to 3.4- and 2.9-fold the concurrent controls, respectively. In the presence of S9-mix, the test item induced a dose-related increase in the number of revertant colonies compared to the solvent control in the tester strain TA100. The increase observed was above the laboratory historical control data range and was up to 2.2-fold the concurrent control.

Results on precipitation and cytotoxicity in the pre-incubation experiment: Cytotoxicity, as evidenced by a reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix.

Results on revertants: No increase in the number of revertants was observed in this pre-incubation method.

Repeat test: To verify the mutagenic response observed in the direct plate assay, an additional experiment was performed with the tester strains TA100 and WP2uvrA.Cytotoxicity, as evidenced by a reduction of the bacterial background lawn, was observed in tester strain TA100 in the absence and presence of S9-mix at the highest concentration tested. No toxicity was observed in tester strain WP2uvrA.

Result of the repeat test: In the absence of S9-mix, the test item induced up to 2.5- and 3.9-fold dose related, increases in the number of revertant colonies compared to the solvent control in the tester strains TA100 and WP2uvrA, respectively. In the presence of S9-mix, the test item induced an up to 4.0-fold dose related increase in the number of revertant colonies compared to the solvent control in the tester strain TA100.

Conclusion: It is concluded that based on the results of the direct plate assay (but not in the pre-incubation method) the substance is mutagenic in the tester strains TA100 and WP2uvrA of the Salmonella typhimurium and the Escherichia coli reverse mutation assay. The test item is not mutagenic in the other Salmonella typhimurium tester strains (TA1535, TA1537 and TA98).

 

Ethylphenylglycidate Ames test (supporting information from literature: Tilch and Elias, 1984), study record not included:

The Salmonella test was performed according to Ames et al. (1975) using strains TA98, TA100, TA1535, TA1537 and TA1538 with and without S9-mix. Preliminary tests for cytotoxicity revealed that the substance doses exceeding 1000 μg/plate were toxic to the tester strains used. The substance was tested in the main experiment with and without S9-mix at 80, 400 and 2000 μg/plate. In TA100 with S9-mix the substance showed a positive response at the concentration of 2000 μg/plate.Reference: Tilch, C.; Elias, P.S.; Investigation of the mutagenicity of ethylphenylglycidate: Tilch, C.; Elias, P.S.; 1984; Mutation Research, 138 (1984) 1-8.

 

Ethylphenylglycidate Ames test (OECD TG 471) supporting information from literature Von der Hude et al, 1990), study record not included:It seems that in this publication only the results of the Ames test are reported from other sources (e.g.Wild et al. 1983 and Tilch and Elias, 1984) and are therefore not considered further.Reference: Von der Hude, W.; Seelbach, A.; Basler, A.; Epoxides: comparison of the induction of SOS repair in Escherichia coli PQ37 and the bacterial mutagenicity in the Ames test: Von der Hude, W.; Seelbach, A.; Basler, A.; 1990; Mutation Research, 231 (1990) 205 – 218.

 

Ethylphenylglycidate in an in vivo micronucleus test (OECD TG 474, Wild et al (1982), no record included

Method: A micronucleus test was performed similar to OECD 474. Male and female NMRI mice (total of 4 mice for each group) were treated intraperitoneally once with 0, 577, 961 and 1538 mg/kg test substance in olive oil. The following deviations are diminishing the reliability of the study: not a minimum of 5 analysable animals, samples of bone marrow are not taken at least twice after treatment with appropriate intervals, no positive control animals to demonstrate the sensitivity and reliability of the test. EFSA considered the test insufficient reliable.

Result: Mean numbers of micronucleated PE/1000 PE was 2.7, 3.2, 2.7 and 2.7 for the tested doses, respectively, indicating absence of toxicity. No deaths occurred. Bone marrow smears were prepared 30 hours after treatment. The substance is considered to be not clastogenic up to and including 1538 mg/kg, which is likely the maximum tolerable dose because LD50 rat is 2300 mg/kg bw and mice are often slightly more sensitive. In view of its molecular weight in physico-chemical properties full absorption via the gut is likely.

 

Ethylphenylglycidate in vitro genemutation assay in mammalian cells TG test (Tilch and Elias, 1984), study record included.

Method: An in vitro gene mutation study in CHO-K1-BH4 cells was conducted equivalent to OECD 476 guideline without GLP. Two types of tests were performed one under normal OECD TG 476 conditions and one with digestive solute. In the first test the substance was tested up to and including concentrations of 10% (5 mg/mL) in the absence and presence of S9 -mix.

Results on cytotoxicity: The cell survival at 0.25% (0.125 mg/mL) was 50% and 58% in the absence and presence of S9 -mix, respectively. In the test with the digestive solute, the test item was tested up to and including concentrations of 10% (4.1 mg/mL) in the absence and presence of S9 -mix. The cell survival at 8% (3.28 mg/mL) was 52% and 69% in the absence and presence of S9 -mix, respectively. The incubation time was 2 hours. Positive control chemicals, MNNG and CPA induced appropriate responses.

Results on mutagenicity: Both in the absence and presence of S9 -mix, the test item induced a significant increase in the mutation frequency (the number of induced mutants exceeded 2.5 times the control value at 50% survival). Based on the results, it is concluded that the substance is positive under normal OECD TG 476 conditions.

Reference: Tilch, C.; Elias, P.S.; 1984; Mutation Research, 138 (1984) 1-8.

 

Methyl beta-phenylglycidate used for read across to Ethylphenylglycidate

For Methyl beta-phenyl glycidate an Ames teste, an in vitro micronucleus test, in vivo micronucleus test and Comet assay are available. The Ames test of this study supports the read across because similar positive results were seen. The in vitro micronucleus test performed together with the Comet assay can be used for read across to Ethylphenylglycidate.

 

First the executive summaries of these genotoxicity studies of Methyl beta-phenylglycidate are presented below followed by the read-across rationale.

 

Methyl beta-phenylglycidate: Ames test (OECD TG 471), supporting information, study record not included

Method: The mutagenic activity of the substance was evaluated in accordance with OECD 471 (1997) guideline and according to GLP principles. The test was performed in two independent experiments: at first a direct

plate assay was performed and second a pre-incubation assay in the absence and presence of S9-mix up to and including the concentration of 5000 μg/plate. All positive and vehicle control values were within acceptable ranges, and all criteria for a valid study were met.

Results: A greater than 2 fold increase was observed in the mean number of revertant colonies at 5000 μg/plate in TA100 and WP2uvrA in the absence of S9 -mix in the direct plate assay. No increase in the mean number of revertant colonies was observed in the pre-incubation assay at any tested dose level in the presence or absence of S9 -mix. In the repeat pre-incubation assay, should this be the direct plate assay, a ≥2 fold increase in the mean number of revertant colonies was observed at 5000 μg/plate in TA100 and WP2uvrA in the absence of S9 –mix at (non) toxic and absence of precipitation. These results indicate that Methyl beta-phenylglycidate is positive in TA100 and WP2uvrA in the absence of S9 and negative in all other tester strains in the presence and absence of S9 -mix, in the Salmonella typhimurium mutation assay and Escherichia coli reverse mutation assay.

 

Methyl beta-phenylglycidate: In vitro micronucleus test (OECD TG 487), supporting information, study record not included

Method: In an in vitro micronucleus assay, cultured peripheral human lymphocytes were exposed to different concentrations of the substance (dissolved in DMSO), in the presence and absence of S9-mix according

to OECD 487 guideline and GLP principles. The test substance was evaluated in the initial micronucleus (MN) assay (B1) in HPBL from a male donor at concentrations ranging from 17.2 to 193 μg/mL in the approximate 24 hour treatment without S9, from 150 to 698 μg/mL in the 3 hour treatment without S9 and from 65.5 to 1000 μg/mL in the 3 hour treatment with S9. No precipitate was observed at the end of the treatment at any tested concentration in any treatment condition with or without S9. In the approximate 24 hour treatment without S9, 52% cytotoxicity was observed at135 μg/mL. This concentration along with two lower concentrations, 17.2 and 65.6 μg/mL, producing 11 and 30% cytotoxicity respectively were selected for MN scoring. In the 3 hour treatment without S9, 58% cytotoxicity was observed at569 μg/mL. This concentration along with two

lower concentrations, 150 and 418 μg/mL, producing 5 and 35% cytotoxicity, respectively were selected for MN scoring. In the 3 hour treatment with S9, appropriate cytotoxicity could not be achieved and the assay was repeated for this test condition with a new set of concentrations. The repeat micronucleus assay with S9 was not reported because the shelf-life of the test material lot was expired. In the repeat MN assay (B3), the test article was evaluated in the 3 hour treatment with S9 at concentrations ranging from 30.4 to 1350 μg/mL. No precipitate was observed at the end of treatment at any tested concentration. In this test condition, 53% cytotoxicity was observed at 886 μg/mL. This concentration along with two lower concentrations, 581 and 646 μg/mL, producing 16 and 31% cytotoxicity respectively were selected for MN evaluation.

Negative results: No statistically significant increase in the frequency of micronucleated binucleated cells (MNBN) was observed at any evaluated concentration in the approximate 24 hour treatment without S9. Statistically significant increases were observed at all evaluated concentrations in the 3 hour treatment without S9. However, the %MNBN frequencies observed at these concentrations were within the historical vehicle control range for this test condition in male donors. Therefore, the statistically significant increase was considered as biologically non-relevant.

Positive results: In the 3 hour treatment with S9, statistically significant increases were observed at all evaluated concentrations. The %MNBN frequencies at the three evaluated concentrations were outside the 95% historical reference range. However, no clear concentration related increase was observed at the evaluated concentrations. The maximum increase was 1.15% versus 0.30% in the control. Therefore, the statistically significant increase observed in the test condition was considered of questionable biological relevance. The MNBN frequencies of the vehicle and positive controls were within the acceptable range. All acceptance criteria were met and the study was accepted as valid.

In summary: The test substance, Methyl beta-phenylglycidate, was considered positive with questionable biological relevance for inducing micronuclei in the binucleated cells of human peripheral blood lymphocytes from male donors in the 3 hour treatment with S9 and negative in the 3 and approximate 24 hour treatments without S9, when evaluated up to the limit of cytotoxicity.

Methyl beta-phenylglycidate: in vivo micronucleus test (OECD TG 474, Kl. 1) Key information, study record included

Method: In an in vivo micronucleus study (as part of the Comet assay), 6 male and 6 female mice per dose were exposed to 250, 500 and 1000 mg/kg bw day of the substance for four consecutive days, performed according to OECD 474 guideline and GLP principles. The maximum tolerated dose for the definitive Micronucleus assay was set at 1000 mg/kg/bw day based on mortality observed in all animals at 2000 mg/kg/bw day in a dose range finding assay.

Results on toxicity: Mortality occurred in 1/6 female mice at 1000 mg/kg/bw day in the definitive assay. No mortality occurred in male mice. No considerable reductions in mean group body weights were observed in the test substance-treated groups during the course of the study. Clinical signs were observed at all dose levels and included piloerection.

Results on genotoxicity: No appreciable reductions could also be increase in %RETs (reticulocytes) were observed in the test substance-treated groups compared to the vehicle control groups, indicating the test substance did not induce cytotoxicity in the red blood cells. In view of the systemic effects seen (mortality) the substance (and its metabolites) have reached the systemic circulation. No statistically significant increases in the incidence of MnRETs (reticulocytes containing micronuclei) were observed in the test substance-treated groups relative to the vehicle control groups (ANOVA followed by Dunnett’s post-hoc analysis, p > 0.05).

Conclusion: The substance is negative in the in vivo micronucleus assay.

 

Methyl beta-phenylglycidate: in vivo Comet assay (OECDTG 489, Kl .1) Key information, study record included.

Method: An in vivo Comet assay was performed according to OECD guideline 489 and GLP principles, in which male and female mice (6/sex/group) were exposed to 250, 500 and 1000 mg/kg bw for four consecutive days. The maximum dose represents the maximum tolerated dose based on a dose range finder study.

Results on toxicity: Mortality occurred in 1/6 female mice at 1000 mg/kg bw/day in the definitive assay. No mortality occurred in male mice. No considerable reductions in mean group body weights were observed in the test substance-treated groups during the course of the study. Clinical signs were observed at all dose levels and included piloerection indicating systemic exposure of the substance and its metabolites.

Results on genotoxicity: In this assay, DNA damage was determined in liver cells only. No statistically significant increases in % tail DNA were observed in the test substance-treated groups relative to the vehicle control groups. The positive control induced a statistically significant increase in % tail DNA compared to the vehicle control. The vehicle control % tail DNA was within the Testing Facility’s historical range.

Conclusion: Based on these results it is concluded that the substance does not increase DNA damage in the liver.

Mutagenic potential of Ethylphenylglycidate (CAS 121-39-1) using read across from Methyl beta-phenylglycidate (CAS 37161-74-3).

Introduction and hypothesis for the read across

Ethylphenylglycidate has an oxirane ring (epoxide) with a phenyl at C1 and a carboxylic acid ethyl ester at C2. For Ethylphenylglycidate gene mutation data in bacteria (Ames) and mammalian cells (CHO cells) are available, which show positive results. For this substance no data on in vivo mutagenicity is available and further information is needed.

In accordance with Article 13 of REACH, lacking information should be generated whenever possible by means other than vertebrate animal tests, i.e. applying alternative methods such as in vitro tests, QSARs, grouping and read-across. For assessing the mutagenic potential ofEthylphenylglycidatethe analogue approach is selected because in vivo mutagenicity information on a closely related analogue is available which can be used for read across.

Hypothesis:Ethylphenylglycidateis not expected to have mutagenic potential in vivo based on the negativein vivomutagenicity test results of the analogue Methyl beta-phenylglycidate resulting in absence of classification for mutagenicity.

Target and Source chemical(s):The information on substance target and the analogue information from substance source are presented in the data matrix.

Purity / Impurities:

Ethylphenylglycidate is a mono-constituent >=80%, which is the trans-form. The key impurity is the cis-form of this substance. One other impurity is ethyl -2-chloro-3-phenylprop-2-enoate / 26880-33-1, which is considered to have similar reactivity compared to the epoxide functional group. The similarity in reactivity between the oxirane/epoxide group and the double bond with the chloride is derived from the pKa of the acid (after ester cleavage). The pKa of the oxirane/epoxide and the chloride substituent is 3.99 and 3.57, respectively. The impurity is similarly reactive and therefore does not need to be considered further.

Analogue justification

According to REACH Annex XI 1.5 read across can be used to replace testing when the similarity can be based on a common backbone and a common functional group. When using read across the result derived should be applicable for C&L and/or risk assessment and it should be presented with adequate and reliable documentation.

Analogue selection: From analogues identified in the RIFM database,Methyl beta-phenylglycidatewas selected because it was a close analogue with in vivo mutagenicity information. In view of the close analogy betweenMethyl beta-phenylglycidateand Ethylphenylglycidate, the difference is a methyl ester instead of an ethyl ester of the carboxylic acid which is close. The substance Ethyl 3-methylphenylglycidate has also been considered but the methyl group attached to the epoxide may slightly stabilize the epoxide group and therefore the Methyl beta-phenylglycidate was considered the better analogue. For this latter substance also more in vivo genotoxicity information is available.

Available information:The source chemical, Methyl beta-phenylglycidate, has been tested in an in vitro Ames test (OECD 471) and an in vitro micronucleus (OECD 487), which were both positive. Subsequently, in an in vivo test combining micronucleus (OECD 474) and Comet assay (OECD 489) Methyl beta-phenylglycidate tested negative.

Structural similarities and differences:Ethylphenylglycidate and Methyl beta-phenylglycidate have a similar backbone and an oxirane (epoxide) ring with a phenyl at C1 and a carboxylic acid ester on the C2 position.The structural difference between Methyl beta-phenylglycidate and Ethylphenylglycidate is the methyl ester instead of an ethyl ester of the carboxylic acid group, for Methyl beta-phenylglycidate and Ethylphenylglycidate, respectively. The difference is only one carbon atom and it is not in the neighbourhood of the reactive group (oxirane/epoxide). Therefore, this difference in alkyl length is not expected to influence the reactivity of the substance and thus its mutagenic potential.

Toxico-kinetic features: Ethylphenylglycidate and Methyl beta-phenylglycidate have similar water solubilities and log Kow values indicating similar bioavailability. The metabolic pathways will also be similar: The ester will be cleaved into an acid (pKa 3.99), which will be fully dissociated at physiological pH.

Toxico-dynamic features: The conjugated oxirane/epoxide group with the ester will be the reactive group.

Remaining uncertainties:In view of the reasoning above, there are no remaining uncertainties.

Conclusions per endpoint for hazard

For Ethylphenylglycidate insufficient genotoxicity information is available. Therefore analogue information of Methyl beta-phenylglycidate is used. Based on the in vitro and in vivo test results Methyl beta-phenylglycidate is concluded to be non-mutagenic. In view of the high similarity in chemical structure, considering backbone and functional group Ethylphenylglycidate is non-mutagenic.

Final conclusion: Ethylphenylglycidate does not need to be classified for genotoxicity, according to EU CLP (EC No. 1272/2008 and its amendments).

Data matrix: Information on Ethylphenylglycidate and its analogue Methyl beta-phenylglycidate

Important for the assessment of genotoxicity.

Chemical name	Ethylphenylglycidate	Methyl beta-phenyl glycidate
Molecular structure
Cas No.	121-39-1	37161-74-3
Einecs number	204-467-3	253-370-2
REACH registration	To be registered under Annex VII	Pre-registered
Empirical	C11H12O3	C10H10O3
Molecular weight	192.21	178.19
Physico-chemical properties
Appearance	Liquid	Liquid
Melting point (˚C) EpiSuite Measured	47 <-20 (IFF, 2015)	36.70
Vapour pressure at 25˚C(Pa) EpiSuite Measured	1.16 0.12 (IFF, 2015)	2.18
Water solubility at 0˚C(mg/L) EpiSuite Measured	320 748.4 (IFF, 2015)	985.9
Log Kow EpiSuite Measured	2.55 2.0 and 2.4 (IFF, 2015)	2.06
Human health
Ames (OECD 471)	Mutagenic in the tester strains TA100, in the absence of S9, and WP2uvrA, without and with S9 (direct plate assay only) (OECD TG 471, IFF, 2017)	Mutagenic in TA100 and WP2uvrA in the absence of S9 (direct plate assay only) (OECD TG 471, RIFM, 2017)
In vitro mammalian cell gene mutation test	Mutagenic in CHO-K1-BH4 cells both with and without S9 (equivalent to OECD TG 476, Tilch and Elias, 1984) )	Not available
In vitro micronucleus assay	Not available	Questionable biological relevance for inducing micronuclei in the 3 hour treatment with S9 only (OECD TG 487, RIFM, 2017)
In vivo micronucleus assay	Read across	Non-clastogenic (OECD TG 474, RIFM, 2017)
In vivo COMET assay	Read across	Non-DNA damaging (OECD TG 489, RIFM, 2017)

 

Justification for classification or non-classification

Based on the in vitro and in vivo test results available on the source and target substance, it is concluded that the substance does not need to be classified for mutagenicity, according to EU CLP (EC No. 1272/2008 and its amendments).