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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-09-02 to 2015-09-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethylene carbonate
EC Number:
202-510-0
EC Name:
Ethylene carbonate
Cas Number:
96-49-1
Molecular formula:
C3H4O3
IUPAC Name:
1,3-dioxolan-2-one
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): Ethylene carbonate
- Purity: > 99%
- Physical state: fine crystals
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: LC61650513
- Expiration date of the lot/batch: no data
- Purity test date: 16-04-2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from light
- Stability under test conditions: The stability to define the test substance has not been determined.
- Solubility and stability of the test substance in the solvent/vehicle: no data
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance dilutions were prepared immediately before use and delivered to the test system at room temperature under filtered light.

Method

Target gene:
tryptophan locus of Escherichia coli strain WP2 uvrA
histidine locus of Salmonella strains
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
Preliminary toxicity assay: 6.67, 10, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 µg/plate
Mutagenicity assay: 50, 150, 500, 1500 and 5000 µg/plate

The maximum dose of 5000 µg/plate was achieved using a concentration of 50 mg/ml and a 100 µL plating aliquot.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water (sterile-filtered)
- Justification for choice of solvent/vehicle: Water was the vehicle of choice based on the solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in water at a concentration of approximately 50 mg/mL in the solubility test conducted at BioReliance.
- Other: All positive controls were diluted in dimethyl sulfoxide (DMSO) except for sodium azide, which was diluted in sterile water.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with metabolic activation, diluted in DMSO, 1µg/plate (TA98, TA1535), 2µg/plate (TA100, TA1537) and 15 µg/plate (WP2 uvrA)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without metabolic activation, diluted in DMSO, 1µg/plate (TA98)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without metabolic activation, diluted in sterile water, 1µg/plate (TA100, TA1535)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without metabolic activation, diluted in DMSO, 75µg/plate (TA1537)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without metabolic activation, diluted in DMSO, 1000 µg/plate (WP2 uvrA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535 and TA1537 as described by Ames et al. (1975) and Escherichia coli WP2 uvrA as described by Green and Muriel (1976).
Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strain TA1535 is reverted by mutagens that cause basepair substitutions. Tester strain TA100 is reverted by mutagens that cause both frameshift and basepair substitution mutations. Specificity of the reversion mechanism in E. coli is sensitive to basepair substitution mutations, rather than frameshift mutations (Green and Muriel, 1976).
Salmonella tester strains were derived from Dr. Bruce Ames’ cultures; E. coli tester strains were from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland.

DURATION
- Exposure duration: 48 to 72h (37+-2°C)

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: 1.1 to 3.1 x 1E08 cells per plate

DETERMINATION OF CYTOTOXICITY
- Method: The condition of the bacterial background lawn was evaluated for evidence of test substance toxicity by using a dissecting microscope.

OTHER:
- Precipitate was evaluated after the incubation period by visual examination without magnification.
- On the day of use in each assay, all tester strain cultures were checked for the appropriate genetic markers.



Rationale for test conditions:
In the preliminary toxicity assay, neither precipitate nor background lawn toxicity was observed. Based upon these results, the maximum dose tested in the mutagenicity assay was 5000 µg per plate.
Evaluation criteria:
A minimum of three non-toxic dose levels is required to evaluate assay data. A dose level is considered toxic if one or both of the following criteria are met:
- A >50 % reduction in the mean number of revertants per plate as compared to the mean vehicle control value. This reduction must be accompanied by an abrupt dose-dependent drop in the revertant count.
- At least a moderate reduction in the background lawn (moderately reduced, extremely reduced or absence of background lawn)

For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance as specified below:
Strains TA1535 and TA1537:
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0 times the mean vehicle control value.

Strains TA98, TA100 and WP2 uvrA:
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.1-times the mean vheicle control value.

An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response was evaluated as negative if it was neither positive or equivocal.
Statistics:
No statistics performed, only calculations of mean and standard deviation of the number of revertants per plate were reported for each replicate plating.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS:
- Effects of pH: not specified
- Effects of osmolality: not specified
- Evaporation from medium: not specified
- Water solubility: The test substance formed a clear solution in water at a concentration of approximately 50 mg/mL in the solubility test conducted at BioReliance
- Precipitation: no precipitation was observed
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES:
In the preliminary toxicity assay, the dose levels tested were 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 µg per plate. Neither precipitate nor background lawn toxicity was observed. Based upon these results, the maximum dose tested in the mutagenicity assay was 5000 µg per plate.

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

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the mutagenicity assay no toxicity was observed.

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
negative with and without metabolic activation

All criteria for a valid study were met as described in the protocol. The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, Ethylene carbonate did not cause a positive mutagenic response with any of the tester strains in either the presence or absence of Aroclor-induced rat liver S9. The study was concluded to be negative without conducting a confirmatory (independent repeat) assay because the results were clearly negative; hence, no further testing was warranted.