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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
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

Test material

1
Chemical structure
Reference substance name:
Ethyl 3-phenyloxirane-2-carboxylate
EC Number:
204-467-3
EC Name:
Ethyl 3-phenyloxirane-2-carboxylate
Cas Number:
121-39-1
Molecular formula:
C11H12O3
IUPAC Name:
ethyl 3-phenyloxirane-2-carboxylate
Test material form:
liquid

Method

Target gene:
- S. typhimurium: Histidine gene
- Escherichia coli: Tryptophan gene
Species / strainopen allclose all
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.
Controlsopen allclose all
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.

Results and discussion

Test resultsopen allclose all
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.

Applicant's summary and conclusion

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).

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