Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation assay (Ames test: OECD TG 471): Negative

In vitro cytogenic assay (chromosomal aberrations) (OECD TG 473): Negative

Link to relevant study records

Referenceopen allclose all

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:
The experimental phase of this study was performed between 10 January 2014 and 28 March 2014.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with GLP and OECD guideline, available as unpublished report, no restrictions, fully adequate for assessment.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
other: US Food and Drug Administration, Center for Food Safety& Applied Nutrition, Redbook 2000 Toxicological Principles for the safety of Food Ingredients. IV.C.1.a. Bacterial Reverse Mutation Test, July 2000
GLP compliance:
yes (incl. QA statement)
Remarks:
TNO Triskelion, Utrechtseweg 48, 3704 HE Zeist, The Netherlands
Type of assay:
bacterial reverse mutation assay
Target gene:
Salmonella typhimurium: histidine
Escherichia coli: tryptophan
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Frozen stocks of each strain were checked for histidine requirement and for sensitivity to ampicillin, crystal violet and UV radiation.
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Frozen stocks of each strain were checked for tryptophan requirement and for sensitivity to ampicillin, crystal violet and UV radiation.
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 mix
Test concentrations with justification for top dose:
- First test: 5 concentrations, 62 to 5000 μg/plate
- Repeated first test: 6 concentrations, 4.7 to 150 μg/plate (Salmonella); 5 concentrations,313 to 5000 μg/plate (E. Coli)
- Second test: 5 concentrations, 11 to 900 μg/plate (Salmonella); 5 concentrations, 62 to 5000 μg/plate (E. Coli)
- Repeated second test: 5 concentrations, 1.9 to 150 μg/plate (Salmonella); 5 concentrations, 62 to 5000 μg/plate (E. Coli)
Vehicle / solvent:
- DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Strain 1537, with metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
Strain TA 1535, TA 98, TA 100 and WP 2 uvrA, with metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-ethyl-N-nitrosourea
Remarks:
Strain WP 2 uvrA, without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Strain TA 98, without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Strain TA1537, without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Strain TA 1535 and TA 100, without metabolic activation
Details on test system and experimental conditions:
METHODS
- Plate-incorporation method used in the first, repeated first and repeated second test: Molten top agar, bacteria, the test substance (vehicle or positive control) and when applicable S9-mix were thoroughly mixed and the mix was immediately poured onto minimal glucose agar plates. All determinations were made in triplicate. The plates were incubated for 48-72 hours at ca. 37°C. Subsequently, the his+ and trp+ revertants were counted.
- Treat and plate method, used in the second test: Bacteria, the test substance (vehicle or positive control) and when applicable S9-mix were mixed and incubated for ca. 3 hours at ca. 37°C while shaking. Thereafter, the mixtures were centrifuged and bacteria, after washing, were added to molten top agar, and the mixtures were poured onto minimal glucose agar plates. All determinations were made in triplicate. The plates were incubated for 48-72 hours at ca. 37°C. Subsequently, the his+ and trp+ revertants were counted.

DETERMINATION OF CYTOTOXICITY
- Toxicity was defined as a reduction (by at least 50%) in the number of revertant colonies and/or a clearing of the background lawn of bacterial growth as compared to the negative (vehicle) control and/or the occurrence of pinpoint colonies.
Evaluation criteria:
- The study was considered valid if the mean colony counts of the vehicle control values of the strains are within acceptable ranges, if the results of the positive controls meet the criteria for a positive response, if no more than 5 % of the plates are lost through contamination or other unforeseen events and if at least three doses are non-toxic.
- A test substance was considered to be positive in the bacterial gene mutation test if the mean number of revertant colonies on the test plates was increased in a dose-related manner or if a two-fold or greater increase was observed compared to the negative control plates. A clear positive response does not need to be verified. Marginally or weakly positive results should be verified by additional testing.
- A test substance was considered to be negative in the bacterial gene mutation test if it produces neither a dose-related increase in the mean number of revertant colonies nor a reproducible minimal two-fold increase in the mean number of revertants at any of the test concentrations.
- Both numerical significance and biological relevance are considered together in the evaluation.
Statistics:
No statistical analysis is performed.
Key result
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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION ON GENOTOXICITY:
- Four bacterial reverse mutation tests were performed, of which two were acceptable. In the first test, less than three non-toxic concentrations were tested, therefore the test was considered not acceptable. The test was repeated. The repeated first test was valid as three non-toxic concentrations were tested and the positive and negative controls showed the expected results. The second test was performed according to the treat and plate method. Unexpectedly the onset of cytotoxicity was within the three hours incubation. As a result, the treat and plate method did not allow exposure to higher concentrations. Therefore the test was considered not acceptable and the test was repeated according to the plate incorporation method.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In all tests, a dose related precipitation of the test substance in the final treatment mix (top agar) was observed. Precipitation was observed in all mixtures above 185 μg/plate. No precipitation was observed on the agar plates.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- In the first test, the test substance was considered toxic both in the absence and presence of S9-mix to all strains. Toxicity was observed at and above 185 μg/plate for strains TA 1535, TA 98 and TA 100, at and above 62 μg/plate for strain TA 1537 both in the absence and presence of S9-mix. For strain WP2 uvrA at 5000 μg/plate in the absence of S9-mix, and at and above 2500 μg/plate in the presence of S9-mix toxicity was observed.
- In the repeated first test, the test substance was considered toxic both in the absence and presence of S9-mix to all strains. Toxicity was observed at 150 μg/plate for all Salmonella strains in both the absence and presence S9-mix. For strain WP2 uvrA at 5000 μg/plate in the absence of S9-mix, and at and above 2500 μg/plate in the presence of S9-mix toxicity was observed.
- In the second test (treat and plate method), the test substance was considered toxic both in the absence and presence of S9-mix to all strains. Toxicity was observed at and above 33 μg/mL for strains TA 1535 and TA 98 and at and above 11 μg/mL for strains TA 1537 and TA 100 in both the absence and presence of S9-mix. For strain WP2 uvrA at and above 62 μg/mL in the absence of S9-mix, and at and above 185 μg/mL in the presence of S9-mix toxicity was observed.
- In the repeated second test, the test substance was considered toxic both in the absence and presence of S9-mix to all strains. Toxicity was observed at 150 μg/plate for strains TA 1535, TA 98 and TA 100, at and above 50 μg/plate for strain TA 1537 and for strain WP2 uvrA at 5000 μg/plate in the absence of S9-mix, and at and above 1667 μg/plate in the presence of S9-mix.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

The test material was non-mutagenic, in the absence and presence of metabolic activation, under the conditions of this bacterial reverse mutation test.
Executive summary:

In a reverse mutation assay, performed according to a OECD Guideline 471 and GLP, salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test substance in the absence and presence of a liver fraction of Aroclor 1254-induced rats (S9-mix). Four bacterial reverse mutation tests were performed of which two were acceptable. These two acceptable experiments were performed in triplicate using the plate incorporation method. In the first test, Salmonella strains were exposed to concentrations of the test substance ranging from 4.7 to 150 μg/plate and E. coli strain to concentrations ranging from 313 to 5000 μg/plate. In the second test, the Salmonella strains were exposed to concentrations of the test substance ranging from 1.9 to 150 μg/plate and the E. coli strain to concentrations ranging from 62 to 5000 μg/plate. The vehicle control plates gave counts of revertant colonies within the acceptable range. In all strains the positive controls gave the expected increase in the mean numbers of revertant colonies. In the first and second accepted test, the test substance was considered toxic both in the absence and presence of S9-mix to all strains. In the first test, for all Salmonella strains toxicity was observed at 150 μg/plate and for the E. coli strain at 5000 μg/plate in the absence of S9-mix and at and above 2500 μg/plate in the presence of S9-mix. In the second test, for strains TA 1535, TA 98 and TA 100 toxicity was observed at 150 μg/plate, for strain TA 1537 at and above 50 μg/plate, both in the absence and presence of S9-mix. For the E. coli strain at 5000 μg/plate in the absence of S9-mix and at and above 1667 μg/plate in the presence of S9-mix toxicity was observed. Toxicity was evidenced by a decrease in the mean number of revertants and/or a clearing of the background lawn of bacterial growth compared to the negative controls. In both accepted tests, in all strains tested, in both the absence and presence of S9-mix, the test substance did not induce a more than 2-fold and/or dose related increase in the mean number of revertant colonies compared to the background spontaneous reversion rate observed with the negative control. The test material was therefore considered to be non-mutagenic under the conditions of this test.

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
Study period:
The experimental phase of this study was performed between 16 December 2013 and 6 March 2014.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with GLP and OECD guideline, available as unpublished report, no restrictions, fully adequate for assessment.
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. QA statement)
Remarks:
TNO Triskelion, Utrechtseweg 48, 3704 HE Zeist, The Netherlands
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
Blood samples were obtained by venapuncture from young (23 and 30 years old) healthy, non-smoking females with no known recent exposures to genotoxic chemicals or radiation.
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9-mix
Test concentrations with justification for top dose:
3.6, 7.2, 14, 29, 58, 115, 230, 460, 920, 1842 μg/mL (first assay, 4 hr treatment with and without metabolic activation); selection of highest concentration based on solubility test.
2.0, 4.0, 8.0, 16, 21, 29, 38, 51, 68, 90, 120 (second assay, 24 hr treatment without metabolic activation)
2.0, 4.0, 8.0, 16, 21, 29, 38, 51, 68, 90, 120, 160 (second assay, 4 hr treatment with metabolic activation)
Vehicle / solvent:
- Solvent used: DMSO for test substance, culture medium for positive control substances
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Remarks:
Mitomycin C: in the absence of S9, Cyclophosphamide: in the presence of S9
Details on test system and experimental conditions:
FIRST EXPERIMENT
In both the absence and presence of S9-mix, the treatment times were 4 hours (pulse treatment) and the harvest time of the cells was 24 hours after onset of the treatment. Two hours before the end of the total incubation period, the cultured lymphocytes were arrested in the metaphase stage of their mitosis by the addition of colcemid.

SECOND EXPERIMENT
In the presence of S9-mix a pulse treatment (4hours) was carried as described for the first experiment. In the absence of S9-mix the cultures were treated continuously with the test substance for 24 hours and the harvest time of the cells was 24 hours after onset of the treatment. Two hours before the end of the total incubation period, the cultured lymphocytes were arrested in the metaphase stage of their mitosis by the addition of colcemid.

NUMBER OF REPLICATIONS:
In all instances duplicate cultures were used.

NUMBER OF CELLS EVALUATED:
A number of 1000 stimulated lymphocytes (500 on each slide) were examined in each culture to determine the percentage of cells in mitosis (mitotic index). On the basis of the results of the mitotic index scoring and the observations with respect to the number and quality of the metaphases, three concentrations of the test substance together with the negative (solvent) and positive controls were selected for chromosomal aberration analysis. For each treatment group, 200 well-spread metaphases per concentration (100 metaphases per culture and 50 metaphases per slide), each containing 46 centromeres, were analysed by microscopic examination for chromatid-type aberrations (gaps, breaks, fragments, interchanges), chromosome-type aberrations (gaps, breaks, minutes, rings, dicentrics), according to the criteria recommended by Savage (1975). If heavily damaged cells or cells with numerical aberrations (such as endoreduplicated cells or polyploid cells) were observed, these cells were recorded but not counted and included in the 200 analysed cells. The Vernier readings of all aberrant metaphases were recorded.

DETERMINATION OF CYTOTOXICITY
Method: mitotic index
Evaluation criteria:
- The study was considered valid if the positive controls demonstrated a statistically significantly increase in the number of aberrant cells and the solvent controls were within the historical range.
- There were several criteria for determining a positive response, such as a statistically significant dose-related or a reproducible statistically significant increase in the number of metaphases containing one or more aberrations at one or more test points analysed.
- A response was considered to be equivocal if the percentage of aberrant cells was statistically marginal higher than that of the solvent control (0.05- A test substance was considered to be negative if it demonstrated neither a statistically significant dose-related or reproducible statistically significant increase in the number of metaphases containing one or more aberrations, at any of the test points analysed.
- Statistical methods were used as an aid in evaluating the test results but were not the only determining factor for a positive response. Both statistical methods and biological relevance of the results were considered together in the evaluation.
Statistics:
The number of metaphases containing one or more aberrations of the test substance treated groups were compared with those of the concurrent solvent controls using Fisher's exact test (one-sided). The difference was considered statistically significant when the p-value of the Fisher’s exact test was less than 0.05.
Key result
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:
not examined
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- In the first test, in the presence of S9-mix, at the four highest dose levels (1842, 920, 460 and 230 μg/mL), the test substance was severely cytotoxic. The mitotic indices of lower dose levels (115, 58, 29, 14 and 7.2 μg/mL) showed a dose related decrease to 52%, 46%, 55%, 70% and 90%, respectively when compared to the mitotic index of the concurrent solvent control. At the lowest dose level of 3.6 μg/mL apparent variation in the mitotic index of the duplicate cultures was observed. Based on the results, the cultures of three dose levels of the test substance (58, 14 and 7.2 μg/mL) were selected for chromosomal aberration analysis. In the absence of S9-mix, at the five highest dose levels (1842, 920, 460, 230 and 115 μg/mL), the test substance was severely cytotoxic to the cells. The mitotic indices of the intermediate dose levels (58 and 29 μg/mL) showed a dose-related decrease to 47% and 82%, respectively, when compared to the mitotic index of the concurrent negative control (solvent). The lower dose levels did not show a reduction of the mitotic index. Based on the results of the mitotic index analysis, the cultures of three dose levels of the test substance (58, 29 and 7.2 μg/ml) were selected for chromosomal aberration analysis.
- In the second test, in the presence of S9-mix, at the highest dose levels (160 and 120 μg/mL), the test substance was severely cytotoxic to the cells. The mitotic indices of the highest five dose levels of the test substance (90, 68, 51, 38 and 29 μg/mL) showed a dose-related decrease to 41%, 55%, 87%, 77% and 92%, respectively, when compared to the mitotic index of the concurrent negative control (solvent). The lowest dose level selected (21 μg/mL) did not reduce the mitotic index. Based on the results of the mitotic index analysis obtained in pulse treatment group with metabolic activation (S9-mix), the cultures of three dose levels of the test substance (90, 51 and 29 μg/mL) were selected for chromosomal aberration analysis. In the continuous treatment group, in the absence of S9-mix, at the highest dose levels of 120 μg/mL, the test substance was severely cytotoxic to the cells. The mitotic indices of the highest six selected dose levels of the test substance (90, 68, 51, 38, 29 and 21 μg/mL) showed a dose-related decrease to 26%, 21%, 47%, 73%, 85% and 92%, respectively, when compared to the mitotic index of the concurrent negative control (DMSO). At the highest dose level of 90 μg/mL apparent variation in the mitotic index of the duplicate cultures was observed (one of the cultures was severely cytotoxic; only a few metaphase cells were observed). The lowest dose level selected (16 μg/mL) did not reduce the mitotic index. Based on the results of the mitotic index analysis obtained in continuous treatment group without metabolic activation, the cultures of three dose levels of the test substance (51, 38 and 21 μg/mL) were selected for chromosomal aberration analysis.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

The test material was not clastogenic to cultured human lymphocytes under the conditions of this chromosome aberration test.
Executive summary:

In an in vitro mammalian cell chromosome aberration test, performed according to OECD 473 and GLP, the test substance was examined for its potential to induce chromosome aberrations in human lymphocytes in both the absence and presence of a metabolic activation system (S9 -mix). In the first chromosomal aberration test the treatment/harvesting times were 4/24 hours (pulse treatment). A dose-related increase in cytotoxicity was observed. Three dose levels of the test substance (7.2, 14 and 58 μg/mL) in the presence of S9 -mix, and three dose levels of the test substance (7.2, 29 and 58 μg/mL) in the absence of S9 -mix were analysed for chromosomal aberrations. In the second test in the presence of S9-mix, the treatment/harvesting times were 4/24 hours (pulse treatment). A dose-related increase in cytotoxicity was observed and three dose levels (29, 51 and 90 μg/mL) were analysed for chromosomal aberrations. In the absence of S9-mix, the treatment/harvesting times were 24/24 hours (continuous treatment). A dose-related increase in cytotoxicity was observed and three dose levels (21, 38 and 51 μg/mL) were analysed for chromosomal aberrations. In both chromosomal aberration tests, the numbers of cells with structural aberrations observed in the solvent control (DMSO) cultures were within the historical range. Treatment with the positive controls Cyclophosphamide and Mitomycin C resulted in statistically significant increases in the numbers of metaphases containing one or more chromosomal aberrations, when compared to the numbers observed in the cultures treated with the solvent (DMSO). This demonstrates the validity of the study. In both chromosomal aberration tests, the test substance did not induce a statistically significant increase in the number of aberrant cells, at any of the concentrations and treatment periods analysed, when compared to the number of aberrant cells observed in the solvent control cultures. The test material was therefore considered to be non-clastogenic under the conditions of this test.

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

Additional information

Additional information from genetic toxicity in vitro:

In a reverse mutation assay, performed according to a OECD Guideline 471 and GLP, salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test substance in the absence and presence of a liver fraction of Aroclor 1254-induced rats (S9-mix). Four bacterial reverse mutation tests were performed of which two were acceptable. These two acceptable experiments were performed in triplicate using the plate incorporation method. In the first test, Salmonella strains were exposed to concentrations of the test substance ranging from 4.7 to 150 μg/plate and E. coli strain to concentrations ranging from 313 to 5000 μg/plate. In the second test, the Salmonella strains were exposed to concentrations of the test substance ranging from 1.9 to 150 μg/plate and the E. coli strain to concentrations ranging from 62 to 5000 μg/plate. The vehicle control plates gave counts of revertant colonies within the acceptable range. In all strains the positive controls gave the expected increase in the mean numbers of revertant colonies. In the first and second accepted test, the test substance was considered toxic both in the absence and presence of S9-mix to all strains. In the first test, for all Salmonella strains toxicity was observed at 150 μg/plate and for the E. coli strain at 5000 μg/plate in the absence of S9-mix and at and above 2500 μg/plate in the presence of S9-mix. In the second test, for strains TA 1535, TA 98 and TA 100 toxicity was observed at 150 μg/plate, for strain TA 1537 at and above 50 μg/plate, both in the absence and presence of S9-mix. For the E. coli strain at 5000 μg/plate in the absence of S9-mix and at and above 1667 μg/plate in the presence of S9-mix toxicity was observed. Toxicity was evidenced by a decrease in the mean number of revertants and/or a clearing of the background lawn of bacterial growth compared to the negative controls. In both accepted tests, in all strains tested, in both the absence and presence of S9-mix, the test substance did not induce a more than 2-fold and/or dose related increase in the mean number of revertant colonies compared to the background spontaneous reversion rate observed with the negative control. The test material was therefore considered to be non-mutagenic under the conditions of this test.

In an in vitro mammalian cell chromosome aberration test, performed according to OECD 473 and GLP, the test substance was examined for its potential to induce chromosome aberrations in human lymphocytes in both the absence and presence of a metabolic activation system (S9 -mix). In the first chromosomal aberration test the treatment/harvesting times were 4/24 hours (pulse treatment). A dose-related increase in cytotoxicity was observed. Three dose levels of the test substance (7.2, 14 and 58 μg/mL) in the presence of S9 -mix, and three dose levels of the test substance (7.2, 29 and 58 μg/mL) in the absence of S9 -mix were analysed for chromosomal aberrations. In the second test in the presence of S9-mix, the treatment/harvesting times were 4/24 hours (pulse treatment). A dose-related increase in cytotoxicity was observed and three dose levels (29, 51 and 90 μg/mL) were analysed for chromosomal aberrations. In the absence of S9-mix, the treatment/harvesting times were 24/24 hours (continuous treatment). A dose-related increase in cytotoxicity was observed and three dose levels (21, 38 and 51 μg/mL) were analysed for chromosomal aberrations. In both chromosomal aberration tests, the numbers of cells with structural aberrations observed in the solvent control (DMSO) cultures were within the historical range. Treatment with the positive controls Cyclophosphamide and Mitomycin C resulted in statistically significant increases in the numbers of metaphases containing one or more chromosomal aberrations, when compared to the numbers observed in the cultures treated with the solvent (DMSO). This demonstrates the validity of the study. In both chromosomal aberration tests, the test substance did not induce a statistically significant increase in the number of aberrant cells, at any of the concentrations and treatment periods analysed, when compared to the number of aberrant cells observed in the solvent control cultures. The test material was therefore considered to be non-clastogenic under the conditions of this test.


Justification for selection of genetic toxicity endpoint
The two studies available are reliable and are adequate for covering this endpoint.

Justification for classification or non-classification

Based on the available information in the dossier, FRET 10-0367 does not need to be classified for genotoxicity when considering the criteria outlined in Annex I of 1272/2008/EC and Annex VI of 67/548/EEC.