Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames assay:

The test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the Salmonella typhimurium and E. coli strains in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
Experimental data from various test chemicals
Justification for type of information:
Data for the target chemical is summarized based on data from various test chemicals
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Including Prival modification / 3
Principles of method if other than guideline:
WoE for the target CAS is summarized based on data from various test chemicals
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine for Salmonella strains and Tryptophan for E. coli strains
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Remarks:
2
Details on mammalian cell type (if applicable):
No data
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium, other: TA98, TA100
Remarks:
3 / 5
Details on mammalian cell type (if applicable):
No data
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Remarks:
4
Details on mammalian cell type (if applicable):
No data
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Remarks:
4
Details on mammalian cell type (if applicable):
No data
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
2. Aroclor 1254 induced S9 metabolic activation system

3/5. Type and composition of metabolic activation system: Liver S9 homogenate was prepared from Syrian golden hamsters that had been injected with Aroclor 1254 at 500 mg/kg body weight. The components of the S9 mix were 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 4 mM NADP, 100 mM sodium phosphate (pH 7.4), and the appropriate S9 homogenate at a concentration of 0.1 m L/mL of mix
- source of S9 : Liver S9 homogenate was prepared from Syrian golden hamsters
- method of preparation of S9 mix : The postmitochondrial (microsomal) enzyme fractions were prepar ed as described by Ames et al.
- concentration or volume of S9 mix and S9 in the final culture medium : Uninduced hamster liver S9 (30% v/v), cofactors (FMN, NADH, glucose-6-phosphate dehydrogenase, and glucose-6-phosphate)
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): No data

4. The S9 liver microsomal fraction was obtained from the livers of 8 - 12 weeks old male Wistar rats
Test concentrations with justification for top dose:
2. 0.0 (NC), 0.005, 0.016, 0.050, 0.158, 0.501 mg/plate
3. 0, 10, 33, 100, 333, 1000 µg/plate
4. 0, 33, 100, 333, 1000, 2500 or 5000 µg/plate
5. 0, 10, 33, 100, 333, 1000, 3333 or 10000 µg/plate
Vehicle / solvent:
2. - Vehicle(s)/solvent(s) used: RO water
- Justification for choice of solvent/vehicle: The test chemical was solulble in RO water

3. - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO

4. - Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: The test chemical was soluble in water

5. - Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: The test chemical was soluble in water
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
RO water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-Nitro-o-phenylenediamine (TA 1537, TA 98, without S9); 2-Aminoanthracene (TA 1535, TA 1537, TA 98, TA 100 and TA 102, with S9)
Remarks:
2
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
congo red
Remarks:
3
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine (TA1537, TA98, -S9); 2-aminoanthracene (TA1535, TA1537, TA98, TA100, WP2 uvrA + S9)
Remarks:
4
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
congo red
Remarks:
5
Details on test system and experimental conditions:
2. METHOD OF APPLICATION: in agar (plate incorporation- Trial I); preincubation (Trial II)

DURATION
- Preincubation period: Trial I: Not applicable Trial II: 60 min
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data

SPINDLE INHIBITOR (cytogenetic assays): No data

STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: Each concentration, including the negative, vehicle and positive controls was tested in triplicate in two independent experiments performed

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Not applicable

NUMBER OF CELLS EVALUATED: No data

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): No data

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data
- Any supplementary information relevant to cytotoxicity: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No data

- OTHER: No data

3/5. METHOD OF APPLICATION: Preincubation

DURATION
- Preincubation period: 30 mins without shaking. Nitrogen was blown over the preincubation tube to keep the atmosphere reduced
- Exposure duration: 48 h
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): No data available

SELECTION AGENT (mutation assays): No data available
SPINDLE INHIBITOR (cytogenetic assays): No data available
STAIN (for cytogenetic assays): No data available
NUMBER OF REPLICATIONS: Triplicate
NUMBER OF CELLS EVALUATED: No data available

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Cytotoxicity was checked in preliminary dose range finding study

OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available
- Other: No data available

OTHER: No data available

4. NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): No data
- Test substance added : in agar (plate incorporation) (experiment 1); preincubation (experiment 2)

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition
- Any supplementary information relevant to cytotoxicity: No data
Rationale for test conditions:
No data
Evaluation criteria:
2. 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 TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding vehicle/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 control and vehicle control such an increase is not considered biologically relevant.

3. The criteria used to evaluate a test were as follows: for a test article to be considered positive, it had to induce at least a doubling (TA98, TA100) in the mean number of revertants per plate of at least on e tester strain. This increase in the mean revertants per plate had to be accompanied by a dose res ponse to increasing concentrations of the test chemical.

4. The plates were observed for the an increase in the number of revertants
Statistics:
No data
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
Remarks:
2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA98, TA100
Remarks:
3
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA1535, TA1537, TA98, TA100
Remarks:
4
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Remarks:
4
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
2. TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No precipitation was noted at a dose upto 5 mg/plate in the pre-experiment
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: To evaluate the toxicity of the test item, a pre-experiment was performed with strains TA 98 and TA 100. Eight concentrations (0.0 (NC), 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5.0 mg/plate) were tested for toxicity and mutation induction with 3 plates each (triplicates). The experimental conditions in this pre-experiment were the same as described below for the Trial-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.

In the pre-experiment, the concentration range of the test item was 0.002 – 5 mg/plate based on the solubility and precipitation test. There was no reduction in colony count as well as in background lawn in treated concentrations 5 (T8) mg/plate – 0.002 (T1) mg/plate) both in absence and in the presence of metabolic activation. Based on the results of pre-experiment following doses were selected for the main study trials: 0.050, 0.158, 0.501, 1.582 and 5 mg/plate, both in the absence (-S9) as well as in the presence of metabolic activation (+S9).

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: No data

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: No data
- Indication whether binucleate or mononucleate where appropriate: No data

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No data
- Negative (solvent/vehicle) historical control data: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Other observations when applicable: No data

3. TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: The doses that were tested in the mutagenicity assay were selected based on the levels of cytotoxici ty observed in a preliminary dose range-finding study using strain TA100. Ten dose levels of the ch emical, one plate per dose, were tested in both the presence and the absence of induced hamster S9. If no toxicity was observed, a total maximum dose of 10 mg of test chemical per plate was used. Ten dose levels of the chemical, one plate per dose, were tested in both the presence and the absence of induced hamster S9. If no toxicity was observed, a total maximum dose of 10 mg of test chemical per plate was used.

COMPARISON WITH HISTORICAL CONTROL DATA: No data
ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

Remarks on result
other: No mutagenic potential

4. TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No data
- Data on osmolality: No data
- Possibility of evaporation from medium: No data
- Water solubility: No data
- Precipitation and time of the determination: No data
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES (if applicable): The pre-experiment was performed with strains TA1535, TA1537, TA98, TA100, 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 article results in a reduction in the number of spontaneous revertants or a clearing of the bacterial ba ckground lawn. Based upon the results of the pre-experiment the concentrations applied in the main experiments were chosen. According to the dose selection criteria the test article was tested at the following concentrations: 33; 100; 333; 1000; 2500; and 5000 µg/plate The results of the pre-expe riment ate included are reported as a part of the main experiment I.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies

Ames test:
- Signs of toxicity : The plates incubated with the test article showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments.
- Individual plate counts : No data
- Mean number of revertant colonies per plate and standard deviation : No data

Remarks on result other: No mutagenic potential
Remarks on result:
other: No mutagenic potential
Conclusions:
The test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the Salmonella typhimurium and E. coli strains in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.
Executive summary:

Data available for the various test chemicals was reviewed to determine the mutagenic nature of the test chemical. The summaries are as mentioned below:

Ames assay was performed to investigate the potential of the test chemical to induce gene mutations in comparison to negative control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using th e Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the negative, positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz., 0.0 (NC), 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5.0 mg/plate were selected for pre-experiment. Based on the pre-experiment results, the test item was tested with the following concentrations 0.0 (NC), 0.005, 0.016, 0.050, 0.158, 0.501 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9). No substantial increase in revertant colony numbers in any of the tester strains were observed following treatment with the test chemical at any dose level in both the confirmatory trials, neither in the presence nor in the 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. The spontaneous reversion rates in the negative, positive controls are within the range of our historical data. The positive controls used for various strains showed a distinct in­crease in induced revertant colonies in both the methods i.e. Plate incorporation method and Pre-incubation method. In conclusion, it is stated that during the described mutagenicity test and under the experimental conditions reported, the test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.  

Ames mutagenicity test with Prival modifications was also conducted as per the OECD 471 guideline for the test chemical to evaluate its genetoxic effects. In the FMN-modified assay, the test chemical was exposed toSalmonella typhimuriumstrains TA98 and TA100 at dose concentrations of 0, 10, 33, 100, 333, 1000  µg/plate. The doses for the main study were based on preliminary dose range study conducted. The bacteria, uninduced hamster liver S9 (30% v/v), cofactors (FMN, NADH, glucose-6-phosphate dehydrogenase, and glucose-6-phosphate), and test chemical were added, mixed, and incubated at 30 °C for 30 min without shaking. Nitrogen was blown over the preincubation tube to keep the atmosphere reduced. At the end of the incubation period, 2 mL of molten top agar was added to each sample tube and the mixture was poured on a minimal agar plate containing 0.5% glucose rather than the 2% glucose specified by Ames et al.. The plates were then incubated at 37 °C for 48 h. The positive control used for the experiment was Congo red. All plates were counted with an Artek automated colony counter or Mini-count colony counter, which was calibrated prior to use. The test chemical did not induce gene mutation in the Salmonella typhimurium strains TA98 and TA100 both in the presence and absence of hamster S9 activation system and hence the chemical is not likely to be a genotoxic in vitro.

In another study, Bacterial reverse mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed usingSalmonella typhimuriumstrains TA1535, TA1537, TA98, TA100, andEscherichia colistrain WP2 uvrA in the presence and absence of S9 metabolic activation system. The test chemical dissolved in water was used at dose level of 0, 33, 100, 333, 1000, 2500 or 5000 µg/plate. The doses for the main study was based on pre-experiment conducted. Main study was performed as per plate incorporation and pre-incubation assay in two independent experiments with triplicate concentrations used. Concurrent untreated, solvent and positive control plates were also included in the study. The plates incubated with the test article showed normal background growth up to 5000 µg/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 chemical at any dose level, neither in the presence nor in the 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 used as positive controls showed a distinct increase of induced revertant colonies. Based on the details of the study, the test chemical did not induce gene mutation inSalmonella typhimuriumstrains TA1535, TA1537, TA98, TA100, andEscherichia colistrain WP2 uvrA in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In the same study as mentioned above, Ames mutagenicity test with Prival modification was conducted as per OECD 471 guideline for the test chemical to evaluate its genetoxic effects when exposed to Salmonella typhimurium strains TA98 and TA100 with dose concentration of 0, 10, 33, 100, 333, 1000, 3333 or 10000  µg/plate in pre-incubation assay. The doses for the main study were based on preliminary dose range study conducted. The bacteria, uninduced hamster liver S9 (30% v/v), cofactors (FMN, NADH, glucose-6-phosphate dehydrogenase, and glucose-6-phosphate), and test chemical were added, mixed, and incubated at 30 °C for 30 min without shaking. Nitrogen was blown over the preincubation tube to keep the atmosphere reduced. At the end of the incubation period, 2 mL of molten top agar was added to each sample tube and the mixture was poured on a minimal agar plate containing 0.5% glucose rather than the 2% glucose specified by Ames et al.. The plates were then incubated at 37 °C for 48 h. The positive control in all FMN experiments was Congo red. All plates were counted with an Artek automated colony counter or Minicount colony counter, which was calibrated prior to use. The test chemical did not induce gene mutation in the Salmonella typhimurium strains TA98 and TA100 both in the presence and absence of hamster S9 activation system and hence the chemical is not likely to be a genotoxic in vitro.

Based on the data available and applying the weight of evidence approach, the test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the Salmonella typhimurium and E. coli strains in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

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

Additional information

Ames assay:

Data available for the various test chemicals was reviewed to determine the mutagenic nature of the test chemical. The summaries are as mentioned below:

Ames assay was performed to investigate the potential of the test chemical to induce gene mutations in comparison to negative control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using th eSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98, TA 100 and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the negative, positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz., 0.0 (NC), 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5.0 mg/plate were selected for pre-experiment. Based on the pre-experiment results, the test item was tested with the following concentrations 0.0 (NC), 0.005, 0.016, 0.050, 0.158, 0.501 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9). No substantial increase in revertant colony numbers in any of the tester strains were observed following treatment with the test chemical at any dose level in both the confirmatory trials, neither in the presence nor in the 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. The spontaneous reversion rates in the negative, positive controls are within the range of our historical data. The positive controls used for various strains showed a distinct in­crease in induced revertant colonies in both the methods i.e. Plate incorporation method and Pre-incubation method. In conclusion, it is stated that during the described mutagenicity test and under the experimental conditions reported, the test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.  

Ames mutagenicity test with Prival modifications was also conducted as per the OECD 471 guideline for the test chemical to evaluate its genetoxic effects. In the FMN-modified assay, the test chemical was exposed toSalmonella typhimuriumstrains TA98 and TA100 at dose concentrations of 0, 10, 33, 100, 333, 1000  µg/plate. The doses for the main study were based on preliminary dose range study conducted. The bacteria, uninduced hamster liver S9 (30% v/v), cofactors (FMN, NADH, glucose-6-phosphate dehydrogenase, and glucose-6-phosphate), and test chemical were added, mixed, and incubated at 30 °C for 30 min without shaking. Nitrogen was blown over the preincubation tube to keep the atmosphere reduced. At the end of the incubation period, 2 mL of molten top agar was added to each sample tube and the mixture was poured on a minimal agar plate containing 0.5% glucose rather than the 2% glucose specified by Ames et al.. The plates were then incubated at 37 °C for 48 h. The positive control used for the experiment was Congo red. All plates were counted with an Artek automated colony counter or Mini-count colony counter, which was calibrated prior to use. The test chemical did not induce gene mutation in the Salmonella typhimurium strains TA98 and TA100 both in the presence and absence of hamster S9 activation system and hence the chemical is not likely to be a genotoxic in vitro.

In another study, Bacterial reverse mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed usingSalmonella typhimuriumstrains TA1535, TA1537, TA98, TA100, andEscherichia colistrain WP2 uvrA in the presence and absence of S9 metabolic activation system. The test chemical dissolved in water was used at dose level of 0, 33, 100, 333, 1000, 2500 or 5000 µg/plate. The doses for the main study was based on pre-experiment conducted. Main study was performed as per plate incorporation and pre-incubation assay in two independent experiments with triplicate concentrations used. Concurrent untreated, solvent and positive control plates were also included in the study. The plates incubated with the test article showed normal background growth up to 5000 µg/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 chemical at any dose level, neither in the presence nor in the 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 used as positive controls showed a distinct increase of induced revertant colonies. Based on the details of the study, the test chemical did not induce gene mutation inSalmonella typhimuriumstrains TA1535, TA1537, TA98, TA100, andEscherichia colistrain WP2 uvrA in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In the same study as mentioned above, Ames mutagenicity test with Prival modification as per OECD 471 guideline was conducted for the test chemical to evaluate its genetoxic effects when exposed to Salmonella typhimurium strains TA98 and TA100 with dose concentration of 0, 10, 33, 100, 333, 1000, 3333 or 10000  µg/plate in pre-incubation assay. The doses for the main study were based on preliminary dose range study conducted. The bacteria, uninduced hamster liver S9 (30% v/v), cofactors (FMN, NADH, glucose-6-phosphate dehydrogenase, and glucose-6-phosphate), and test chemical were added, mixed, and incubated at 30 °C for 30 min without shaking. Nitrogen was blown over the preincubation tube to keep the atmosphere reduced. At the end of the incubation period, 2 mL of molten top agar was added to each sample tube and the mixture was poured on a minimal agar plate containing 0.5% glucose rather than the 2% glucose specified by Ames et al.. The plates were then incubated at 37 °C for 48 h. The positive control in all FMN experiments was Congo red. All plates were counted with an Artek automated colony counter or Minicount colony counter, which was calibrated prior to use. The test chemical did not induce gene mutation in the Salmonella typhimurium strains TA98 and TA100 both in the presence and absence of hamster S9 activation system and hence the chemical is not likely to be a genotoxic in vitro.

Based on the data available and applying the weight of evidence approach, the test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the Salmonella typhimurium and E. coli strains in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

Based on the data available and applying the weight of evidence approach, the test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the Salmonella typhimurium and E. coli strains in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.