Ethylene carbonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ethylene carbonate was not mutagenic in the key in vitro mutagenicity study, in vitro chromosome aberration test and the mammalian cell gene mutation assay.

Three key studies were selected: an AMES Salmonella/Microsome Plate test of Huntsman (1983), performed according to OECD guideline 471, a chromosome aberration study of Evonik (2000), performed according to OECD guideline 473 and a cell mutation assay at the Thymidine Kinase Locus in Mouse Lymphoma Cells (RCC Cytotest Cell Research GmbH, 2000), performed according OECD guideline 476.

Other reliable studies include a number of AMES tests, an in vitro mammalian cell gene mutation test (CHO/HGPRT Assay), performed according to OECD guideline 476 of Huntsman (2010) which tested mutagenicity with and without metabolic activation and in the presence of positive and negative (and/or vehicle control) and a rat hepatocyte primary culture / DNA repair assay, performed according to OECD guideline 482. These studies support the results identified in the key studies.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

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.

Target gene:

tryptophan locus of Escherichia coli strain WP2 uvrA
histidine locus of Salmonella strains

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.

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.

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.

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP study conducted according to OECD Guideline 473 and EU method B10 with minor deviation: updating final concentration and exposure duration of the positive control. However, this deviation had no detrimental impact on the outcome of the study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

update of final concentration and exposure duration of the positive control

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

update of final concentration and exposure duration of the positive control

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: exposure period of 4 hours: the culture medium of exponentially growing cell cultures was replaced with serum-free medium (for treatment with S9 mix) or complete medium (for treatment without S9 mix) with 10% FCS (v/v), containing the test item. Exposure period 18 and 28 hours: the culture medium of exponentially growing cell cultures was replaced with complete medium (with 10% FCS) containing different concentrations of the test item without S9 mix. The medium was not changed until preparation of the cells.
- Properly maintained: yes (all cultures were incubated at 37°C in a humidified atmosphere with 4.5% CO2 (95.5% air)
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction from male rats

Test concentrations with justification for top dose:

Pre-test on toxicity: solvent control, 6.95, 13.91, 27.81, 55.63, 111.3, 222.5, 445.0, 890.0 µg/mL
Experiment I and II: 27.8, 55.6, 111.3, 222.5, 445.0, 890.0 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionized water
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative nontoxicity to the cell cultures.

Untreated negative controls:

yes

Remarks:

culture medium

Negative solvent / vehicle controls:

yes

Remarks:

deionized water

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation; at 600 µg/mL = 4.8 mM (continuous exposure), 1000 µg/mL = 8.0 mM (4 h exposure), dissolved in nutrient medium

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation; at 0.7 µg/mL = 2.5 µM (4h exposure; 18 h preparation interval); 1.0 µg/mL = 3.5 µM (4 h exposure; 28 h preparation interval); in nutrient medium

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: not applicable
- Exposure duration: 4 hours, 18 and 28 hours
- Expression time (cells in growth medium): continuous exposure
- Selection time (if incubation with a selection agent): 2 hours
- Fixation time (start of exposure up to fixation or harvest of cells): no data

SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): colcemid (0.2 µg/mL culture medium)
STAIN (for cytogenetic assays): After incubation in hypotonic solution the cells were fixed with 3 + 1 methanol + glacial acetic acid. Per experiment both slides per group were prepared. After preparation the cells were stained with Giemsa. Additionally, two cultures per test item and solvent control treatment group, not treated with Colcemid, were set up in parallel. These cultures were stained in order to determine microscopically the cell number within 10 defined fields per slide. The toxicity of the test item is given as reduction of % cells as compared to the solvent control.

NUMBER OF REPLICATIONS: In each experimental group two parallel cultures were set up.

NUMBER OF CELLS EVALUATED: Per culture 100 metaphase plates were scored for structural chromosome aberrations

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: the number of polyploid cells was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype)
- Determination of endoreplication: no data
- Other: no data

OTHER: no data

Evaluation criteria:

A test item is classified as non-clastogenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups are in the range of the historical control data (0.0 - 4.0% aberrant cells exclusive gaps)
and/or
- no significant increase of the number of structural chromosome aberrations is observed.

A test item is classified as clastogenic if:
- the number of induced structural chromosome aberrations are not in the range of our historical control data (0.0 - 4.0 % aberrant cells exclusive gaps)
and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

Statistics:

Statistical significance was confirmed by means of the Fischer's exact test (p< 0.05). However, both biological and statistiscal significance should be considered together. If the criteria of evaluation are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

In this study, neither in absence nor in the presence of S9 mix, relevant toxic effects indicated by reduced mitotic indices or reduced cell numbers were observed

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
Precipitation of the test item in culture medium was not observed, neither in the pre-test nor in the main experiments. no relevant influence of the test item on the osmolarity (solvent control 284 mOsm versus 294 mOsm at 890 µg/mL) or the pH-value (both pH 7.2) was determined.

RANGE-FINDING/SCREENING STUDIES: In the range finding pre-test on toxicity cell numbers 24 h after start of treatment were scored as indicator for cytotoxicity. Concentrations between 6.95 and 890.0 µg/mL were applied. In the pre-test toxic effects were not observed up to the highest concentration. Therefore, 890 µg/mL (± 10 mM) were chosen as top concentrations in experiment I

COMPARISON WITH HISTORICAL CONTROL DATA: In the absence and in the presence of S9 mix, the aberration rates of the cells after treatment with the test item (exp I: 0.5% - 2.5 %; exp II: 0.0 - 3.0%) were within the range of the historical control data: 0.0% - 4.0%

ADDITIONAL INFORMATION ON CYTOTOXICITY: no additional information

Results:

A single significant increase (p < 0.05) was observed in experiment I, in the presence of S9 mix after treatment with 890 µg/mL. Although this increase of 2.0% aberrant cells exclusive gaps was statistically significant compared to the low response (0.0% aberrant cell, exclusive gaps) in the solvent control, the increase is clearly within the historical control data range (0% - 4.0% aberrant cells, exclusive gaps). Therefore, the statistical significance has to be regarded as being biologically irrelevant. In addition, two seemingly dose related increases at interval 18h in experiment I in the presence of S9 mix and in experiment II in the absence of S9 mix clearly not exceeding the historical control data range were regarded as statistical fluctuations without any biological relevance.

In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item as compared to the rates of the solvent controls.

Conclusions:

In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test item Ethylene carbonate did not induce structural chromosome aberrations in V79 Chinese hamster cells.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08. May 2000 - 07. Aug 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Autosomal Thymidine Kinase (TK) Locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 (RPMI 1640-HAT)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 -Mix

Test concentrations with justification for top dose:

55.6; 111.3; 222.5; 445.0; 890.0 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: solubility 214g/L

Untreated negative controls:

yes

Remarks:

cell culture medium

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation: methylmethane sulfonate dissolved in nutrient medium; final concentration 13 µg/ml = 0.12 mM; with metabolic activation: 3-methylcholanthrene dissolved in dimethylsulfoxide; final concentration 3 µg/ml

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium (suspension)
DURATION
- Preincubation period: 1d
- Exposure duration: 4h and 24h
- Expression time (cells in growth medium):72h
-Selection time:
SELECTION AGENT (mutation assays):TFT
NUMBER OF REPLICATIONS:2
NUMBER OF CELLS EVALUATED:approx. 4000/well in 96 well format for cytotoxicity and 2.5 cells/well in 96 plate format for cloning efficiency
DETERMINATION OF CYTOTOXICITY
- Method: viability for cytotoxicity and cloning efficiency for mutant frequency
Controls:

Evaluation criteria:

A test item is classified as positive if it induces either a reproducible concentration-related increase in the mutant frequency or a reproducible positive response for at least one of the test points.
A test item producing neither a reproducible concentration-related increase in the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A significant response is described as follows: The test item is classified as mutagen if it reproducibly induces with at least one of the concentrations a mutation frequency that is two times higher than the mean spontaneous mutation frequency in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase in the mutation frequency. Such evaluation may be considered independently of an enhacement factor for induced mutants.
In the evaluation of the test results the historical variability of the mutation rates in negative and solvent controls and the mutation rates of all negative and solvent controls of this study are taken into consideration.
Results of test groups are rejected if the relative total growth, the relative suspension growth and/or cloning efficiency 1 is less than 10 % of the solvent control or the cloning efficiency 2 after the expression period is less than 20 %.

Statistics:

No statistical evaluation is required.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Following continuous treatment at the maximal concentration tested (890.0 µg/ml)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:No precipitation or toxicity was observed from the lowest up to the highest test concentration at pulse and continuous treatment.
COMPARISON WITH HISTORICAL CONTROL DATA: Historical data available from 1998 - 1999

Remarks on result:

other: all strains/cell types tested

Experiment I			experiment I, culture I				experiment I, culture II
			relative	relative	mutant		relative	relative	mutant
	conc. µg	S9	cloning	total	colonies/	induction	cloning	total	colonies/	induction
	per ml	mix	efficiency 1	growth	10^6cells	factor	efficiency 1	growth	10^6cells	factor
Column	1	2	3	4	5	6	7	8	9	10
Neg. contr. with medium		-	100.0	100.0	115		100.0	100.0	94
Neg. contr. with water		-	100.0	100.0	92	1.0	100.0	100.0	82	1.0
Pos. control with MMS	13.0	-	71.4	60.9	390	3.4	81.7	45.7	494	6.0
Test item	27.8	-	118.2	culture was not continued^#			95.5	culture was not continued^#
Test item	55.6	-	122.0	119.0	151	1.7	94.1	69.1	157	1.9
Test item	111.3	-	109.4	83.3	167	1.8	101.6	78.4	98	1.2
Test item	222.5	-	111.1	97.4	139	1.5	100.0	85.0	114	1.4
Test item	445.0	-	107.7	101.3	141	1.5	98.5	83.0	100	1.2
Test item	890.0	-	104.5	94.5	131	1.4	98.5	91.0	106	1.3
Neg. contr. with medium		+	100.0	100.0	137		100.0	100.0	73
Neg. contr. with water		+	100.0	100.0	129	1.0	100.0	100.0	103	1.0
Neg. contr. with DMSO		+	100.0	100.0	93	1.0	100.0	100.0	103	1.0
Pos. control with 3-MC	3.0	+	71.9	48.3	342	3.7	94.5	47.8	303	2.9
Test item	27.8	+	103.1	culture was not continued^#			130.8	culture was not continued^#
Test item	55.6	+	98.5	138.9	102	0.8	110.7	133.1	116	1.1
Test item	111.3	+	91.5	198.0	57	0.4	109.1	59.3	193	1.9
Test item	222.5	+	95.6	193.6	61	0.5	114.0	79.8	154	1.5
Test item	445.0	+	103.1	129.6	106	0.8	98.6	61.5	184	1.8
Test item	890.0	+	98.5	140.6	106	0.8	104.4	111.1	117	1.1

Experiment II			experiment II, culture I				experiment II, culture II
			relative	relative	mutant		relative	relative	mutant
	conc. µg	S9	cloning	total	colonies/	induction	cloning	total	colonies/	induction
	per ml	mix	efficiency 1	growth	10^6cells	factor	efficiency 1	growth	10^6cells	factor
Column	1	2	3	4	5	6	7	8	9	10
Neg. contr. with medium		-	100.0	100.0	114		100.0	100.0	75
Neg. contr. with water		-	100.0	100.0	87	1.0	100.0	100.0	88	1.0
Pos. control with MMS	13.0	-	61.9	66.8	436	5.0	51.3	29.7	508	5.8
Test item	27.8	-	87.9	culture was not continued^#			121.4	culture was not continued^#
Test item	55.6	-	86.4	84.5	77	0.9	140.5	68.3	127	1.4
Test item	111.3	-	80.8	112.0	66	0.8	147.2	99.7	76	0.9
Test item	222.5	-	89.4	78.8	74	0.8	121.4	68.5	118	1.3
Test item	445.0	-	87.9	71.9	104	1.2	143.8	100.4	99	1.1
Test item	890.0	-	84.9	28.4	82	0.9	126.3	29.3	92	1.1

Conclusions:

During the mutagenicity test described and under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation. Ethylene Carbonate is considered to be non-mutagenic in this mouse lymphoma assay.

Executive summary:

The study to investigate the potential of Ethylene Carbonate to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y followed the procedures indicated by the OECD Guidelines No. 476. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation (S9 mix) and a treatment period of 4 hours. The second experiment was solely performed in the absence of metabolic activation with a treatment period of 24 hours. In a pre-experiment to select a concentration range no precipitation or toxicity was observed from the lowest up to the highest test concentration at pulse and continuous treatment. Therefore, the following concentrations of Ethylene Carbonate in water were applied in the main experiments: 55.6 µg/ml; 111.3 µg/ml; 222.5 µg/ml; 445.0 µg/ml; and 890.0 µg/ml (~ 10 mM). No toxic effects occurred in the first experiment with and without metabolic activation. Following continuous treatment relevant toxic effects were observed in both cultures at the maximal concentration. No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced total mutant colonies and an increase of the relative quantity of small versus large colonies. In conclusion it can be stated that under the experimental conditions reported Ethylene Carbonate did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation. Ethylene Carbonate is considered to be non-mutagenic in this mouse lymphoma assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No reliable studies were available for the in vivo mutagenicity endpoint.  

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

According to the available data and the criteria of the CLP Regulation, ethylene carbonate should not be classified for genetic toxicity.