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Genetic toxicity in vitro

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Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: other: chromosome breakage (structural chromosomal aberrations) and misdistribution of chromosomes
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
(2014)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
not applicable
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
- Type and identity of media: Dulbecco's Modified Eagle Medium/Ham's F12 supplemented with 200 mM GlutaMAX TM, penicillin/streptomycin 100 U/mL/100 µg/mL, PHA 3 µg/mL, 10 % fetal bovine serum, 10 mM HEPES, and heparin 125 U.S.P.-U/mL.
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from the liver of Phenobarbital/beta-naphthoflavone induced rats.
Test concentrations with justification for top dose:
Exp. I, pulse exposure: 0.8, 1.5, 3.0, 6.0, 12.0, 24.1, 48.2, 96.4, 192.8, 385.5, 771, 1542 µg/mL (without and with S9 mix), repeat without S9 mix
Exp. II, continuous exposure: 3.0, 6.0, 12.0, 24.1, 48.2, 96.4, 192.8, 385.5, 771, 1542 µg/mL (solely without S9 mix)

The following concentrations were selected for reading: pulse exposure, without S9 mix: 96.4, 192.8, and 385.5 µg/mL; pulse exposure, with S9 mix: 48.2, 96.4, and 192.8 µg/mL; continuous exposure, without S9 mix: 48.2, 96.4, and 192.8 µg/mL.
Vehicle / solvent:
EGDE (Ethylene glycol dimethylether)
Untreated negative controls:
yes
Remarks:
culture medium
Negative solvent / vehicle controls:
yes
Remarks:
EGDE
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Demecolcin
Remarks:
without metabolic activation: Mitomycin C (1 µg/mL) and Demecolcin (150 ng/mL, for continuous exposure); with metabolic activation: cyclophosphamide (17.5 µg/mL)
Details on test system and experimental conditions:
Blood samples were drawn from healthy non-smoking donors not receiving medication. The lymphocytes of the respective donors have been shown to respond well to stimulation of proliferation with phytohemeagglutinine (PHA) and to positive control substances. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes. The lymphocytes were stimulated for proliferation by the addition of the mitogen PHA to the culture medium for a period of 48 hours.

Pulse exposure
About 48 hrs after seeding 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test item concentration. The culture medium was replaced with serum-free medium containing the test item. For the treatment with metabolic activation 50 μL S9 mix per mL culture medium was added. After 4 hrs the cells were gently centrifugated, separated from the supernatant, washed and resuspended in complete culture medium for a 16-hour recovery period. After this period Cytochalasin B (4 μg/mL) was added and the cells were cultured another approximately 20 hours until preparation.

Continuous exposure (without S9 mix)
About 48 hrs after seeding 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test item concentration. The culture medium was replaced with complete medium (with 10 % FBS) containing the test item. After 20 hours the cells were gently centrifugated, separated from the supernatant, washed and resuspended in complete culture medium. Cytochalasin B (4 μg/mL) was added and the cells were cultured another approximately 20 hours until preparation.

STAIN (for cytogenetic assays): Giemsa

NUMBER OF CELLS EVALUATED: At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides.

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity is characterized by the percentages of reduction in the CBPI (Cytokinesis-block proliferation index) in comparison with the controls (% cytostasis) by counting 500 cells per culture in duplicate.

DOSE SELECTION
With regard to the molecular weight of the test item, 1542.0 µg/mL (approx. 10 mM) were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 0.8 to 1542.0 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test for toxicity, phase separation of the test item was observed at the end of treatment at 771.0 µg/mL and above in the absence and at 1542.0 µg/mL in the presence of S9 mix.
Clear toxic effects were observed after 4 hours treatment with 771.0 µg/mL and above in the absence of S9 mix and with 385.5 µg/mL and above in the presence of S9 mix.
Dose selection of Experiment II was based on test item toxicity and the occurrence of phase separation. There 1542.0 µg/mL were chosen as top treatment concentration for continuous exposure in the absence of S9 mix.
Evaluation criteria:
A test item is considered to be negative if:
- None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
- There is no concentration-related increase
- The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data

A test item is considered to be positive if:
- At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
- The increase is concentration-related in at least one experimental condition
- The results are outside the range of the laboratory historical solvent control data
Statistics:
Statistical significance will be confirmed by using the Chi-squared test (α < 0.05) using the validated R Script CHI2.Rnw for those values that indicate an increase in the number of cells with micronuclei compared to the concurrent solvent control.
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No biologically relevant increase in the number of cells carrying micronuclei was observed, neither in the absence nor in the presence of S9 mix. The micronucleus rates of the cells after treatment with the test item (0.35 – 1.05 %) surpassed the range of the solvent controls (0.30-0.45 %) as well as the negative controls (0.40- 0.55 %), but were within the range of the laboratory historical control values for organic solvents other than EGDE. The positive controls used (Demecolcin, MMC or CPA) showed distinct increases in cells with micronuclei.
Thus, the test item was considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest evaluable concentration.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH or osmolality: No relevant influence on osmolarity or pH was observed.
- Other confounding effects: Phase separation of the test item was observed at the end of treatment at 771.0 µg/mL and above in the absence and at 1542.0 µg/mL in the presence of S9 mix.

ADDITIONAL INFORMATION ON CYTOTOXICITY
In this study, at both treatment intervals, in the absence at a concentration at 771.0 µg/mL and above as well as in the presence of S9 mix at a concentration of 385.5 µg/mL, a biologically relevant cytotoxicity indicated by not evaluable concentrations could be observed.
Remarks on result:
other: strain/cell type: human lymphocytes
Remarks:
Migrated from field 'Test system'.
Executive summary:

The substance was assessed for its potential to induce micronuclei in human lymphocytes in vitro in accordance to OECD Guideline 487. In two independent experiments the cells were exposed either for 4 hours (pulse exposure) with or without S9 mix, or for 20 hours (continuous exposure) without S9 mix. The highest concentration applied (1542.0 µg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 487.

In this study, at both treatment intervals, a biologically relevant cytotoxicity indicated by not evaluable concentrations could be observed (in the absence of S9 mix at a concentration of 771.0 μg/mL and above, in the presence of S9 mix at a concentration of 385.5 μg/mL). In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying micronuclei was observed. The micronucleus rates of cells after treatment with the test item (0.35-1.05 %) surpassed the range of the solvent controls (0.30-0.45 %) as well as the negative controls (0.40- 0.55 %), but were within the range of the laboratory historical control values for organic solvents other than EGDE.

The positive controls used (Demecolcin, MMC or CPA) showed distinct increases in cells with micronuclei.

Thus, the test item was considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest evaluable concentration.

 

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
(1997)
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT locus
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM containing Hank's salts supplemented with 10 % FBS, neomycin (5 µg/mL) and amphotericin B (1%); for the selection of mutant cells the complete medium was supplemented with 11 µg/mL 6-thioguanine.
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix from the liver of phenobarbital/beta-naphthoflavone induced male Wistar rats.
Test concentrations with justification for top dose:
Exp. I (4 hours exposure): without S9-mix 1.9, 3.8, 7.5, 15.0, 30.0, 60.0, 120, and 240 µg/mL; with S9-mix 1.9, 3.8, 7.5, 15.0, 30.0, 60.0, 120, and 240 µg/mL
Exp. II: 24 hours exposure, without S9-mix 1.9, 3.8, 7.5, 15.0, 30.0, 60.0, 120, and 240 µg/mL; 4 hours exposure, with S9-mix 7.5, 15.0, 30.0, 60.0, 120, 180, and 240 µg/mL

The following concentrations were selected for reading: Exp. I without S9-mix 7.5, 15.0, 30.0, 60.0, and 120 µg/mL and with S9-mix 7.5, 15.0, 30.0, 60.0, and 120 µg/mL; Exp. II without S9-mix (24 hours exposure) 15.0, 30.0, 60.0, 120, and 240 µg/mL, and with S9-mix (4 hours exposure) 30.0, 60.0, 120, 180, and 240 µg/mL
Vehicle / solvent:
EGDE (Ethylene glycol dimethylether)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
EGDE
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Remarks:
without metabolic activation: ethylmethane sulphonate (1.2 mM); with metabolic activation: 7,12-dimethylbenz(a)anthracene (8.6 µM)
Details on test system and experimental conditions:
A pre-test was performed in order to determine the concentration range for the mutagenicity experiments. In this pre-test the colony forming ability of approximately 500 single cells (duplicate cultures per concentration level) after treatment with the test item was observed and compared to the controls. Toxicity of the test item is indicated by a reduction of the cloning efficiency (CE). The pre-experiment was performed in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation. Test item concentrations between 12.1 µg/mL and 1542 µg/mL (equal to a molar concentration of approximately 10 mM) were used. The highest concentration in the pre-experiment was chosen with regard to the molecular weight (154.2 g/mol) of the test item.

The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. Experiment I was pre-maturely terminated due to bacterial contamination of the cells and repeated under identical general experimental conditions except with an increased concentration range (experiment IA). The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

Treatment Protocol without Metabolic Activation: Approximately 1.5 x 10exp6 (single culture) and 5 x 10exp2 cells (in duplicate) were seeded in plastic culture flasks. The cells were grown for 24 hours prior to treatment. After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 µl/mL S9 mix. Concurrent solvent and positive controls were treated in parallel. After 4 hours this medium was replaced with complete medium following two washing steps. In the second experiment the cells were exposed to the test item for 24 hours in complete medium, supplemented with 10 % FBS, in the absence of metabolic activation. The colonies used to determine the cloning efficiency (survival) were fixed and stained approx. 7 days after treatment as described below. Three or four days after treatment 1.5 x 10exp6 cells per experimental point were sub-cultivated in 175 cm² flasks containing 30 mL medium. Following the expression time of 7 days five 80 cm² cell culture flasks were seeded with about 3 - 5 x 10exp5 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability.
The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 for about 8 days. The colonies were stained with 10 % methylene blue in 0.01 % KOH solution.
The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

DETERMINATION OF CYTOTOXICITY: Toxicity of the test item is indicated by a reduction of the cloning efficiency.
Evaluation criteria:
A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.
Statistics:
A linear regression (least squares, calculated using Sum_neu_v2.xltm, version 2.0) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.
- Phase separation: Based on the pre-experiment phase separation occurred at 771.0 µg/mL and above after 4 hours treatment without metabolic activation and at 385.2 mg/mL and above after 4 hours treatment with metabolic activation. Following 24 hours treatment phase separation was noted at 192.8 µg/mL and above.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Base on the pre-experiment strong toxic effects were noted after 4 hour treatment at 12.1 µg/mL and above without metabolic activation and at 24.1 µg/mL and above with metabolic activation. Following 24 hour treatment strong cytotoxicity occurred at 96.4 µg/mL and above without metabolic activation.
Remarks on result:
other: strain/cell type: Chinese hamster lung fibroblasts (V79)
Remarks:
Migrated from field 'Test system'.
Executive summary:

A study according to OECD TG 476 was conducted in order to investigate the potential of the substance to induce gene mutations at the HPRT locus in mammalian cells. For that purpose, V79 cells were exposed to the test item for 4 hours with and without metabolic activation in the first experiment and for 4 hours with and for 24 hours without metabolic activation in the second experiment. The maximum concentration of the pre-experiment (1542 µg/mL) was equal to a molar concentration of about 10 mM. The concentration range of the main experiments was limited by cytotoxicity.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations in mammalian cells.

 

 

 

 

 

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(1997)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine locus
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix from rat liver induced with phenobarbital/beta-naphthoflavone.
Test concentrations with justification for top dose:
Experiment I (plate incorporation), with S9-mix: 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate for TA98 and TA100 (initial pre-experiment), 0.03; 0.1; 0.3; 1; 3; 10; 33; 100; 333 and 1000 µg/plate for TA1535, TA1537, and WP2uvrA; without S9-mix: 0.03; 0.1; 0.3; 1; 3; 10; 33; 100; 333 and 1000 µg/plate for all strains.
Experiment II (pre-incubation method), with and without S9-mix: 0.03; 0.1; 0.3; 1; 3; 10; 33; 100; 333 and 1000 µg/tube for all strains.
Vehicle / solvent:
EGDE (Ethylene glycol dimethylether)
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
EGDE
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Na-azide (TA 1535, TA 100), 4-nitro-1,2-phenylene diamine (TA 1537 and TA 98), methyl methane sulfonate (WP2 uvrA), 2-aminoanthracene (TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA).
Remarks:
The positive controls sodium azide, 4-nitro-1,2-phenylene diamine and methyl methane sulfonate were only used without S9 mix; the positive control 2-aminoanthracene was only used with S9 mix.
Details on test system and experimental conditions:
METHOD OF APPLICATION: Initial test (plate incorporation); as independent repeat a preincubation modification (60 min. at 37°C) was performed.
For each strain and dose level, including the controls, three plates were used.

DETERMINATION OF CYTOTOXICITY
To evaluate the toxicity of the test item a pre-experiment was performed with strains TA 98 and TA 100 with and without metabolic activation. Eight concentrations were tested for toxicity and mutation induction with each 3 plates.
- Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item occurred up to and including the highest investigated dose.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The plates incubated with the test item showed reduced background growth in all strains used. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all of the test groups with and without metabolic activation.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Executive summary:

A bacterial reverse mutation (Ames) test according to OECD TG 471 was conducted to investigate the potential of the test substance to induce gene mutations. The assay was performed in two independent experiments, the initial plate incorporation and the subsequent preincubation test, both with and without S9 mix and the following tester strains: Salmonella typhimurium TA 1535, TA 100, TA 1537, and TA 98, and Escherichia coli WP2 uvrA. The doses in the main experiments (up to and including 1000 µg/plate) were chosen based on a pre-experiment with doses up to 5000 µg/plate.

No precipitation of the test item occurred up to and including the highest investigated dose. The plates incubated with the test item showed reduced background growth in all strains used. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all of the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, the test substance was considered to be non-mutagenic without and with S9 mix based on this Ames-test.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A bacterial reverse mutation (Ames) test according to OECD TG 471 was performed in order to investigate the potential of the test substance to induce gene mutations. The assay used two independent experiments, the initial plate incorporation and the subsequent preincubation test, both with and without S9 mix and with the following tester strains: Salmonella typhimurium TA 1535, TA 100, TA 1537, and TA 98, and Escherichia coli WP2 uvrA. The doses in the main experiments (up to and including 1000 µg/plate) were chosen based on a pre-experiment with doses up to 5000 µg/plate.

No precipitation of the test item occurred up to and including the highest investigated dose. The plates incubated with the test item showed reduced background growth in all strains used. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all of the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, the test substance was considered to be non-mutagenic without and with S9 mix based on this Ames-test.

Further available is a study according to OECD TG 476 that investigates the potential of the substance to induce gene mutations at the HPRT locus in mammalian cells. For that purpose, V79 cells were exposed to the test item for 4 hours with and without metabolic activation in the first experiment and for 4 hours with and for 24 hours without metabolic activation in the second experiment. The maximum concentration of the pre-experiment (1542 µg/mL) was equal to a molar concentration of about 10 mM. The concentration range of the main experiments was limited by cytotoxicity.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

In conclusion, the test item did not induce gene mutations in mammalian cells under the experimental conditions reported.

The substance was also assessed for its potential to induce micronuclei in human lymphocytes in vitro in accordance to OECD Guideline 487. In two independent experiments the cells were exposed either for 4 hours (pulse exposure) with or without S9 mix, or for 20 hours (continuous exposure) without S9 mix. The highest applied concentration in this study (1542.0 µg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 487.

In this study, at both treatment intervals, in the absence of S9 mix at a concentration of 771.0 µg/mL and above as well as in the presence of S9 mix at a concentration of 385.5 µg/mL, a biologically relevant cytotoxicity indicated by not evaluable concentrations could be observed.

In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying micronuclei was observed. The micronucleus rates of cells after treatment with the test item (0.35-1.05 %) surpassed the range of the solvent controls (0.30-0.45 %) as well as the negative controls (0.40- 0.55 %), but were within the range of the laboratory historical control values for organic solvents other than EGDE.

The positive controls used (Demecolcin, MMC or CPA) showed distinct increases in cells with micronuclei.

Thus, the test item was considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest evaluable concentration.

Overall, a potential for genetic toxicity is not concluded for the substance.

Justification for classification or non-classification

According to Regulation (EC) No 1272/2008, Annex I, no classification is warranted for genetic toxicity.