Producto P0310

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Key study: Bacterial reverse mutation assay: OECD guideline 471 and EU method B.13/14. GLP study.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Key study: Chromosomal aberrations: OECD guideline 473 and EU method B.10. GLP study.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. Therefore, the test item is considered to be non-clastogenic in this chromosome aberration test with and without S9 mix, when tested up to the highest required concentration in Experiment IA (with and without S9 mix), IB (with S9 mix), and Experiment II (with S9 mix) or to the highest scorable concentration in Experiment II (without S9 mix).

Key study: Gene mutation assay in mammalian cells: OECD guideline 476 and EU method B.17. GLP study.

In conclusion it can be stated that 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. Therefore, the test item is considered to be non-mutagenic in this mouse lymphoma assay.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

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

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Note: Salmonella typhimurium and Scherichia coli

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 Lg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 Lg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 1535, TA 100; without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine, 4-NOPD

Remarks:

TA 1537, TA 98; without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

WP2 uvrA; without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 2-AA

Remarks:

TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA; with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION:
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 μL Test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control),
500 μL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 μL Bacteria suspension (cf. test system, pre-culture of the strains),
2000 μL Overlay agar
In the pre-incubation assay 100 μL test solution, 500 μL S9 mix / S9 mix substitution buffer and 100 μL bacterial suspension were mixed in a test tube and shaken at 37 ºC for 60 minutes. After pre-incubation 2.0 mL overlay agar (45 ºC) was added to each tube. The mixture was poured on selective agar plates. After solidification the plates were incubated upside down for at least 48 hours at 37 ºC in the dark.

NUMBER OF REPLICATIONS: Triplicates

DETERMINATION OF CYTOTOXICITY
- Method: To evaluate the toxicity of the test item a pre-experiment was performed with strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA. Eight concentrations were tested for toxicity and mutation induction with three plates each. The experimental conditions in this pre-experiment were the same as described below for the experiment I (plate incorporation test). Toxicity of the test item results in 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

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The plates incubated with the test item showed normal background growth up to 5000 Lg/plate with and without S9 mix in both experiments.
No toxic effects, evident as a reduction in the number of revertants, occurred in 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 item 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.

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of the test item to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 Lg/plate; Experiment II: 33; 100; 333; 1000; 2500; and 5000 Lg/plate. The plates incubated with the test item showed normal background growth up to 5000 Lg/plate with and without metabolic activation in both independent experiments. No toxic effects, evident as a reduction in the number of revertants, occurred in 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 item 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, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

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

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Experiment IA: exposure period 4 hours: without metabolic activation: 156.3, 312.5, 625.0, 1250.0, 2500.0 and 5000.0 μg/ml
Experiment IA: exposure period 4 hours: with metabolic activation: 156.3, 312.5, 625.0, 1250.0, 2500.0 and 5000.0 μg/ml
Experiment IB: exposure period 4 hours: with metabolic activation: 2500.0, 3000.0, 3500.0, 4000.0, 4500.0 and 5000.0 μg/ml

Experiment II: exposure period 18 hours: without metabolic activation: 156.3, 312.5, 625.0, 1250.0, 2500.0 and 5000.0 μg/ml
Experiment II: exposure period 28 hours: without metabolic activation: 625.0, 1250.0, 2500.0 and 5000.0 μg/ml
Experiment II: exposure period 4 hours: with metabolic activation: 156.3, 312.5, 625.0, 1250.0, 2500.0 and 5000.0 μg/ml

Vehicle / solvent:

Water

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Exposure period 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. For the treatment with metabolic activation 50 μL S9 mix per mL medium were used. Concurrent solvent and positive controls were performed. After 4 hrs the cultures were washed twice with "Saline G" and then the cells were cultured in complete medium for the remaining culture time.

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. All cultures were incubated at 37 ºC in a humidified atmosphere with 1.5 % CO2

DURATION
- Exposure duration: 4, 18 or 28 hours

SPINDLE INHIBITOR (cytogenetic assays):
Colcemid was added (0.2 μg/mL culture medium) to the cultures 15.5 hrs and 25.5 hrs, respectively after the start of the treatment. The cells on the slides were treated 2.5 hrs later, in the chambers with hypotonic solution (0.4 % KCl) for 20 min at 37 ºC. After incubation in the hypotonic solution the cells were fixed with a mixture of methanol and glacial acetic acid (3:1 parts, respectively).

STAIN (for cytogenetic assays): Giemsa


NUMBER OF REPLICATIONS: Duplicates

NUMBER OF CELLS EVALUATED: 100 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides, except for the positive control in Experiment II, at the 28 hrs preparation interval without metabolic activation, where only 50 metaphase plates were scored.

DETERMINATION OF CYTOTOXICITY
- Method: For evaluation of cytotoxicity indicated by reduced cell numbers two additional cultures per test item and solvent control group, not treated with colcemid, were set up in parallel. These cultures were stained after 18 hrs and 28 hrs, respectively, in order to determine microscopically the cell number within 10 defined fields per coded slide. The cell number of the treatment groups is given in percentage compared to the respective solvent control.

To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.

OTHER EXAMINATIONS:
- Determination of polyploidy: the number of polyploid cells in 500 metaphase plates per culture was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype).

Evaluation criteria:

A test item is classified as non-clastogenic if:
- the number of induced structural chromosome aberrations in all scored dose groups is in the range of the laboratory’s historical control data range (0.0 - 4.0 % aberrant cells, excluding 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 is not in the range of the laboratory’s historical control data range (0.0 - 4.0 % aberrant cells, excluding gaps).
and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

Statistical significance was confirmed by means of the Fisher’s exact test (p < 0.05).

However, both biological and statistical significance should be considered together. If the criteria mentioned above for the test item 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. Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications. The following criterion is
valid:
A test item can be classified as aneugenic if:
- the number of induced numerical aberrations is not in the range of the laboratory’s historical control data range (0.0 – 5.6 % polyploid cells).

Statistics:

Statistical significance was confirmed by means of the Fisher’s exact test (p < 0.05).

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(In Experiment II, in the absence of S9 mix, the concentration showing clear cytotoxicity was not scorable for cytogenetic damage).

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In Experiment IA, in the absence and presence of S9 mix and in Experiments IB and II, in the presence of S9 mix, no clear toxic effects indicated by reduced mitotic indices or reduced cell numbers were observed up to the highest applied test item concentration. In Experiment II, in the absence of S9 mix, the concentration showing clear cytotoxicity was not scorable for cytogenetic damage.

DETERMINATION OF POLYPLOIDY:
In all experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (0.5 - 4.8 %) as compared to the rates of the solvent controls (1.5 - 4.1 %).

In Experiment IA, in the absence of S9 mix, no statistically significant or biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.5 - 3.0 % aberrant cells, excluding gaps) were close to the corresponding value of the solvent control (1.5 % aberrant cells, excluding gaps) and clearly within the range of the laboratory’s historical control data: 0.0 - 4.0 % aberrant cells, excluding gaps. In contrast, in the presence of S9 mix, one single statistically significant increase in the number of aberrant cells, excluding gaps (7.5 %), was observed at the highest applied concentration (5000 μg/mL). The value clearly exceeded the laboratory’s historical control data range (0.0 – 4.0 % aberrant cells, excluding gaps). In order to verify this result, a confirmatory experiment, designated Experiment IB, was performed. In Experiment IB, in the presence of S9 mix, the observation of clastogenicity was not confirmed and, therefore, has to be regarded as biologically irrelevant. In Experiment II, in the absence and presence of S9 mix, no statistically significant or biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.5 - 3.5 % aberrant cells, excluding gaps) were close to the corresponding solvent controls (1.0 - 2.0 % aberrant cells, excluding gaps) and clearly within the range of the laboratory’s historical control data: 0.0 - 4.0 % aberrant cells, excluding gaps.

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. Therefore, the test item is considered to be non-clastogenic in this chromosome aberration test with and without S9 mix, when tested up to the highest required concentration in Experiment IA (with and without S9 mix), IB (with S9 mix), and Experiment II (with S9 mix) or to the highest scorable concentration in Experiment II (without S9 mix).

Executive summary:

The test item dissolved in deionised water, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in three independent experiments. In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations, except for the positive control in Experiment II, in the absence of S9 mix, at preparation interval 28 hrs, where only 50 metaphase plates were scored. The highest applied concentration in the pre-test on toxicity (5000 μg/mL) was chosen with respect to the current OECD Guideline 473. Dose selection for the cytogenetic experiments was performed considering the toxicity data. In Experiment IA, in the absence and presence of S9 mix and in Experiments IB and II, in the presence of S9 mix, no toxic effects indicated by reduced cell numbers and/or mitotic indices were observed up to the highest applied concentrations. In Experiment II, in the presence of S9 mix, no clear cytotoxicity was observed up to the highest scorable concentration. In Experiment IA, in the absence of S9 mix, no clastogenicity was observed up to the highest applied concentration. In contrast, in the presence of S9 mix, a statistically significant increase in the number of aberrant cells, excluding gaps, was observed at the highest applied concentration. In order to verify this result, a confirmatory experiment, designated Experiment IB, was performed. In Experiment IB, in the presence of S9 mix, the observation of clastogenicity was not confirmed and, therefore, has to be regarded as biologically irrelevant. In Experiment II, in the absence and presence of S9 mix, neither a statistically significant or

biologically relevant increase in the number of aberrant cells, excluding gaps, were observed. No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. Appropriate mutagens were used as positive controls. In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. Therefore, the test item is considered to be non-clastogenic in this chromosome aberration test with and without S9 mix, when tested up to the highest required concentration in Experiment IA (with and without S9 mix), IB (with S9 mix), and Experiment II (with S9 mix) or to the highest scorable concentration in Experiment II (without S9 mix).

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

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

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - 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:

Thymidine kinase locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Experiment I:
Without S9 mix: 4 h treatment: 156.6, 331.3, 662.5, 1325, 2650 and 5300 μg/ml
With S9 mix: 4 h treatment: 156.6, 331.3, 662.5, 1325, 2650 and 5300 μg/ml

Experiment II:
Without S9 mix: 24 h treatment: 156.6, 331.3, 662.5, 1325, 2650 and 5300 μg/ml
With S9 mix: 4 h treatment: 331.3, 662.5, 1325, 2650, 3975 and 5300 μg/ml

Vehicle / solvent:

Water

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION:
In the mutation experiment 1×10E07 (3x10E06 during 24 h exposure) cells/flask (80 cm2 flasks) suspended in 10 ml RPMI medium with 3 % horse serum (15 % horse serum during 24 h exposure) were exposed to various concentrations of the test item either in the presence or absence of metabolic activation. After 4 h (24 h in the second experiment) the test item was removed by centrifugation (425 x g, 10 min) and the cells were washed twice with "saline G". Subsequently the cells were resuspended in 30 ml complete culture medium and incubated for an expression and growth period of 48 h.

DURATION
- Exposure duration: 4 hours or 24 hours
- Expression time (cells in growth medium): 48 hours

SELECTION AGENT (mutation assays): TFT (Trifluorothymidine)

NUMBER OF REPLICATIONS: Duplicates

NUMBER OF CELLS EVALUATED:
After the expression period the cultures were selected. Cells from each experimental group were seeded into 2 microtiter plates so that each well contained approximately 4×10E03 cells in selective medium with TFT. The viability (cloning efficiency) was determined by seeding about 2 cells per well into microtiter plates (same medium without TFT). The plates were incubated at 37 ± 1.5 ºC in 4.5 % CO2/95.5 % water saturated air for 10 - 15 days. Then the plates were evaluated.

DETERMINATION OF CYTOTOXICITY
- Method: RSG (Relative Suspension Growth) or RTG (Relative Total Growth) values (main experiment) below 50 % are considered toxic. The cell density was determined each day and adjusted to 3×10E05 cells/ml, if necessary. The relative suspension growth (RSG) of the treated cell cultures was calculated by the day 1 fold-increase in cell number multiplied by the day 2 fold-increase in cell number according to the method of Clive and Spector.

Evaluation criteria:

A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 106 cells above the corresponding solvent control.

A relevant increase of the mutation frequency should be dose-dependent.

A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.

However, in the evaluation of the test results the historical variability of the mutation rates in the solvent controls of this study are taken into consideration.
Results of test groups are generally rejected if the relative total growth is less than 10 % of the vehicle control unless the exception criteria specified by the IWGT recommendations are fulfilled.

Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT®11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software. The number of mutant colonies obtained for the groups treated with the test item was 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 relevance and statistical significance were considered together.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(Moderate cytotoxic effects indicated by a relative total growth of less than 50 % of survival in both parallel cultures were solely observed at the maximum concentration of 5300 μg/mL following 4 hours of treatment without metabolic activation).

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Moderate cytotoxic effects indicated by a relative total growth of less than 50 % of survival in both parallel cultures were solely observed at the maximum concentration of 5300 μg/mL following 4 hours of treatment without metabolic activation.

No substantial and reproducible dose dependent increase of the mutation frequency was noted in both experiments with and without metabolic activation. The threshold of 126 above the corresponding solvent control was exceeded at an intermediate concentration of 1325 μg/mL in the second culture of the first experiment without metabolic activation. However, this isolated increase was not reproduced in the parallel culture under identical conditions or in both cultures at any other, even higher, concentration. Furthermore, the increase was not dose dependent as indicated by the lacking statistical significance. Consequently, the isolated increase of the mutation frequency described above was judged as biologically irrelevant outlier.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT®11 statistics software. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.

Conclusions:

In conclusion it can be stated that 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. Therefore, the test item is considered to be non-mutagenic in this mouse lymphoma assay.

Executive summary:

The study was performed to investigate the potential of the test item to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. 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 and a treatment period of 4 h. The second experiment was performed with a treatment period of 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation. The highest applied concentration of the test item was 5300 μg/mL corresponding to approximately 5000 μg/mL of the pure substance. 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 of the test item. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid. In conclusion it can be stated that 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. Therefore, the test item is considered to be non-mutagenic in this mouse lymphoma assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro studies:

Bacterial reverse mutation assay: OECD guideline 471 and EU method B.13/14. GLP study. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Chromosomal aberrations: OECD guideline 473 and EU method B.10. GLP study. In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. Therefore, the test item is considered to be non-clastogenic in this chromosome aberration test with and without S9 mix, when tested up to the highest required concentration in Experiment IA (with and without S9 mix), IB (with S9 mix), and Experiment II (with S9 mix) or to the highest scorable concentration in Experiment II (without S9 mix).

Gene mutation assay in mammalian cells: OECD guideline 476 and EU method B.17. GLP study. In conclusion it can be stated that 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. Therefore, the test item is considered to be non-mutagenic in this mouse lymphoma assay.

Justification for classification or non-classification

Based on the available negative results obtained from in vitro studies, the substance is not classified for genetic toxicity in accordance to CLP Regulation (EC) 1272/2008.