Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test:

The test chemical is not mutagenic in the bacterium Salmonella typhimurium strains in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as gene mutant in vitro.

In vitro chromosome aberration assay:

The test chemical did not induce chromosome aberrations in the mammalian cell line in the presence and absence of S9 metabolic activation system and hence it is not mutagenic in the chromosome aberration study performed.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
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 the various test chemicals
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
other: Refer below principle
Principles of method if other than guideline:
Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Remarks:
LT2 strains / 1
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
other: TA98, TA100, TA97 and TA1535
Remarks:
2
Details on mammalian cell type (if applicable):
Not specified
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
other: TA97, TA98, TA100, TA1535, TA1537
Remarks:
3
Details on mammalian cell type (if applicable):
Not specified
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
Liver fraction (S-9) from Aroclor 1254 or methylcholanthrene induced male Sprague Dawley rats
Test concentrations with justification for top dose:
1. 3 µmole/plate
2. No data
3. 0, 10, 33, 100, 333, 666, 1000, 1666, 3333 or 6666 µg/plate
Vehicle / solvent:
1. - Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: The test chemical was dissolved in ethanol as a solvent only

2. No data

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

Untreated negative controls:
yes
Remarks:
The control plates were checked for spontaneous revertants
Negative solvent / vehicle controls:
not specified
Remarks:
The details of ethanol being used as solvent control is not available
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosoguanidin (without metabolic activation) and 2-aminoanthracene (with activation)
Remarks:
1
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
not specified
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:
9-aminoacridine
sodium azide
other: 4-nitro-o-phenylenediamine (TA98 and TA1538, -S9); 2-aminoanthracene (All strains, +S9)
Remarks:
3
Details on test system and experimental conditions:
1. METHOD OF APPLICATION: Spot test (in agar)

DURATION
- Preincubation period: No data available
- Exposure duration: No data available
- Expression time (cells in growth medium): No data available
- 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: No data available

NUMBER OF CELLS EVALUATED: No data available

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Yes, viable count was determined

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

OTHER: No data available

2. METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: No data
- Exposure duration: No data
- Expression time (cells in growth medium): No data
- 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: Duplicate

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

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

OTHER: No data

3. METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 mins
- 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: No data

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

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

OTHER: Plates were machine counted unless precipitate was present which interfered with the count, or the color of the test chemical on the plate reduced the contrast between the colonies and the agar.
Rationale for test conditions:
No data
Evaluation criteria:
1. The plates were observed for the presence of revertant colonies

2. The plates were observed for a dose dependent increase in the number of revertants/plate. The combination of a questionable (?) and negative (-) response was considered negative (-); the combination of a weakly positive (+w) and negative response was considered questionable (?).

3. The plates were observed for a dose dependent increase in the number of Histidine- independent (his+) colonies.

Evaluations were made at both the individual trial and chemical levels.

Individual trials were judged mutagenic (+), weakly mutagenic (+ W), questionable (?), or nonmutagenic (-), depending on the magnitude of the increase in his+ revertants, and the shape of the dose response. A trial was considered questionable (?) if the dose-response was judged insufficiently high to support a call of “+ W”, if only a single dose was elevated over the control, or if a weak increase was not dose-related. The distinctions between a questionable response and a nonmutagenic or weakly mutagenic response, and between a weak mutagenic response and mutagenic response are highly subjective. It was not necessary for a response to reach two-fold over background for a trial to be judged mutagenic.

A chemical was judged mutagenic (+) or weakly mutagenic (+W) if it produced a reproducible, dose-related response over the solvent control, under a single metabolic activation condition, in replicate trials. A chemical was judged questionable (?) if the results of individual trials were not reproducible, if increases in his+ revertants did not meet the criteria for a “+W” response, or if only single doses produced increases in his+ revertants in repeat trials. Chemicals were judged nonmutagenic (-) if they did not meet the criteria for a mutagenic or questionable response.
Statistics:
No data
Species / strain:
S. typhimurium TA 1535
Remarks:
LT2 strains
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: TA98, TA100, TA97 and TA1535
Remarks:
2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
other: TA97, TA98, TA100, TA1535, TA1537
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
Positive controls validity:
valid
Additional information on results:
1.
The following controls were also made:
1. The viable count was determined
2. The number of spontaneous revertants was measured
3. The presence of the rfa-mutation was checked by crystal violet inhibition
4. The presence of the plasmid pKM 101 in strains TA 98 and TA 100 was checked by resistance to ampicillin
5. The response to the positive controls N-methyl-N'-nitro-N-nitrosoguanidin (not requiring metabolic activation) and 2-aminoanthracene (requiring activation) was checked

2. 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: All chemicals were run initially in a toxicity assay to determine the appropriate dose range for the mutagenicity assay. The toxicity assay was performed using TA100. Toxic concentrations were defined as those that produced a decrease in the number of his+ colonies, or a clearing in the density of the background lawn, or both.

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data
Remarks on result:
other: No mutagenic potential
Conclusions:
The test chemical is not mutagenic in the bacterium Salmonella typhimurium strains in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as gene mutant in vitro.
Executive summary:

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

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The material was dissolved in ethanol and applied at a concentration of 3 µmole/plate in the spot test performed to Salmonella typhimurium LT-2 strains TA 98, TA 100, TA 1535, and TA 1537 with and without S9 metabolic activation system. The test chemical did not induce reversion of mutant strains and hence is not mutagenic in the bacterium Salmonella typhimurium LT-2 strains TA 98, TA 100, TA 1535, and TA 1537 with and without S9 metabolic activation system and hence the chemical is not likely to classify as gene mutant in vitro.

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed as per the preincubation modification of the Salmonella/mammalian microsome mutagenicity (Ames) test. The chemicals were tested in a preincubation procedure in strains TA98 and TA100 without metabolic activation and with activation provided by Aroclor induced rat and hamster liver homogenates (S9). If a positive response was seen in one of these two strains, the strain/metabolic activation combination producing that response was repeated, and no further testing was performed. If no positive responses were seen, the chemical was tested in strains TA97 and TA1535. The plates were observed for a dose dependent increase in the number of revertants/plate. The combination of a questionable (?) and negative (-) response was considered negative (-); the combination of a weakly positive (+w) and negative response was considered questionable (?). The test chemical did not induce a dose dependent increase in the number of revertants in Salmonella typhimurium TA98, TA100, TA97 and TA1535 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium strains TA97, TA98, TA100, TA1535, TA1537 in the presence and absence of S9 metabolic activation system. The chemical was dissolved in DMSO and used at dose levels 0, 10, 33, 100, 333, 666, 1000, 1666, 3333 or 6666 µg/plate by the preincubation method. The doses were selected on the basis of preliminary dose range finding study and concurrent solvent and positive controls were included in the study. The plates were observed for a dose dependent increase in the number of Histidine- independent (his+) colonies. The test chemical did not induce gene mutation in Salmonella typhimurium strains TA97, TA98, TA100, TA1535, TA1537 in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

Based on the observations made, the test chemical is not mutagenic in the bacterium Salmonella typhimurium strains in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as gene mutant in vitro.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
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 the various test chemicals
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Principles of method if other than guideline:
In vitro mammalian chromosome aberration test was performed to evaluate the mutagenic nature of the test chemical
GLP compliance:
not specified
Type of assay:
other: In vitro mammalian chromosome aberration
Target gene:
No data
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
1
Details on mammalian cell type (if applicable):
- Type and identity of media: Mc- Coy’s 5a medium with 10% fetal calf serum, L-glutamine, and antibiotics
- Properly maintained: No data available
- Periodically checked for Mycoplasma contamination: No data available
- Periodically checked for karyotype stability: No data available
- Periodically "cleansed" against high spontaneous background: No data available
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
other: Chinese hamster lung fibroblast cell line (CHL/IU)
Remarks:
2
Details on mammalian cell type (if applicable):
- Type and identity of media: Eagle's minimum essential medium supplemented with 10% heat-inactivated calf or fetal bovine serum
- Properly maintained: No data available
- Periodically checked for Mycoplasma contamination: No data available
- Periodically checked for karyotype stability: No data available
- Periodically "cleansed" against high spontaneous background: No data available
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
The S9 mix consisted of 15 pl/ml liver homogenate (from male Sprague-Dawley rats, induced with Aroclor 1254), 2.4 mg/ ml NADP, and 4.5 mg/ml isocitric acid in serum-free medium.
Test concentrations with justification for top dose:
1. Without S9: 160-1600 µg/ ml
With S9: 500-5000 µg/ ml

2. Without S9 (24 hrs): 0, 0.15, 0.3, 0.6 or 0.9 mg/mL
Without S9 (48 hrs): 0, 0.3, 0.6, 0.9 or 1.2 mg/mL
Without S9 (6-18 hrs): 0, 0.3, 0.6 or 1.2 mg/mL
With S9 (6-18 hrs): 0, 0.6, 1.2, 1.8 or 2.4 mg/mL
Vehicle / solvent:
1. Water, dimethyl sulfoxide (DMSO), ethanol, or Acetone (in the order of preference)
2. - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Triethylenemelamine
Remarks:
1
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Remarks:
2
Details on test system and experimental conditions:
1. METHOD OF APPLICATION: in medium

DURATION
- Preincubation period:
- Exposure duration: With S9: 2 hrs
Without S9: Apprx. 8.5-9 hrs
- Expression time (cells in growth medium): 8.5-9 hrs
- Selection time (if incubation with a selection agent): after 18-26 hrs
- Fixation time (start of exposure up to fixation or harvest of cells): 8-12 hr after the beginning of treatment.

SELECTION AGENT (mutation assays): Geimsa stain
SPINDLE INHIBITOR (cytogenetic assays): No data available
STAIN (for cytogenetic assays): No data available

NUMBER OF REPLICATIONS: No data available

NUMBER OF CELLS EVALUATED: 100 cells

DETERMINATION OF CYTOTOXICITY
- Method: No data available

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

OTHER: No data available

2. METHOD OF APPLICATION: in medium

DURATION
- Preincubation period:
- Exposure duration: With S9: 6 hrs
Without S9: 24 or 48 hrs
- Expression time (cells in growth medium): 18 hrs
- Selection time (if incubation with a selection agent): after 18-26 hrs
- Fixation time (start of exposure up to fixation or harvest of cells): 8-12 hr after the beginning of treatment.

SELECTION AGENT (mutation assays): Geimsa stain
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): No data available

NUMBER OF REPLICATIONS: No data available

NUMBER OF CELLS EVALUATED: 100 well spread metaphase cells

DETERMINATION OF CYTOTOXICITY
- Method: No data available

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes, 100 well spread metaphases were observed
- Determination of endoreplication: No data available
- Other: No data available

OTHER: No data available
Rationale for test conditions:
No data
Evaluation criteria:
1. Chromosomal aberrations were noted; Cells were selected for scoring on the basis of good morphology and completeness of karyotype (21 ± 2 chromosomes).

Classes of aberrations included simple (breaks and terminal deletions), complex (rearrangements and translocations), and other (pulverized chromosomes). Gaps and endo-reduplications were recorded but were not included in the totals. Aberrations were not scored in polyploidy cells but metaphases with 19-23 chromosomes were used (the modal number being 21).

2. Structural chromosomal aberrations were classified into 6 groups: chromatid and chromosome gap, chromatid break, chromatid exchange, fragmentation, chromosome break and chromosome exchange (mainly dicentrics and ring chromosomes).

A gap was defined as an achromatic lesion equal to or more than the width of a chromatid that was not accompanied by a dislocation of the portion of the chromatid(s) distal to the lesion, and gaps were taken into account in the evaluation.

A treatment was considered positive when the frequency of structurally aberrant cells or polyploidy was 10% or more; marginal when it was 5% to less than 10%; and negative when it was less than 5%. The test results were confirmed on a case-by-case basis. An overall positive evaluation was made when structural aberrations or polyploidy was shown for one or more treatments, regardless of the presence of an exogenous metabolic activation system.
Statistics:
1. For chromosome aberrations, linear regression analysis of the percentage of cells with aberrations vs the log-dose was used as the test for trend. To examine absolute increases over control levels at each dose, a binomial sampling assumption was used. The P values were adjusted to take into account the multiple dose comparisons. For data analysis, we used the “total” aberration category, and the criterion for a positive response was that the adjusted P value be < 0.05.

2. No data
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
other: Chinese hamster lung fibroblast cell line (CHL/IU)
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
Positive controls validity:
valid
Additional information on results:
1. TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data available
- Effects of osmolality: No data available
- Evaporation from medium: No data available
- Water solubility: No data available
- Precipitation: No data available
- Other confounding effects: No data available

RANGE-FINDING/SCREENING STUDIES: doses were chosen for the aberration test based on a preliminary test of cell survival 24 hr after treatment. Doses were based on observations of cell confluence and mitotic cell availability in the SCE test.

COMPARISON WITH HISTORICAL CONTROL DATA: No data available

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data available

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

RANGE-FINDING/SCREENING STUDIES: The maximum concentration for each chemical, which was determined by preliminary cytotoxicity tests, was the concentration showing more than 50% inhibition of cell growth regardless of solubility.

COMPARISON WITH HISTORICAL CONTROL DATA: No data available

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data available
Remarks on result:
other: No mutagenic potential
Conclusions:
The test chemical did not induce chromosome aberrations in the mammalian cell line in the presence and absence of S9 metabolic activation system and hence it is not mutagenic in the chromosome aberration study performed.
Executive summary:

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

In vitro mammalian chromosome aberration test was performed to evaluate the mutagenic nature of the test chemical. Cloned Chinese hamster ovary cells (CHO-W-B1) were cultured in Mc-Coy’s 5a medium with 10% fetal calf serum, L-glutamine, and antibiotics. Tests were carried out with and without an in vitro metabolic activation system (S9 mix). In tests without metabolic activation, the test chemical was left in culture until colcemid addition, whereas with activation the test chemical was added along with S9 mix for only 2 hr at the beginning of the test period. The doses used for the study were 160-1600µg/mL without S9 and 500-5000µg/mL with S9. The test chemical did not induce chromosome aberrations in the Chinese hamster ovary cell line in the presence and absence of S9 metabolic activation system and hence it is not mutagenic in the chromosome aberration study performed.

In vitro mammalian chromosome aberration test was performed to evaluate the mutagenic nature of the test chemical. Cloned Chinese hamster lung fibroblast cell line (CHL/IU) were cultured in Eagle's minimum essential medium supplemented with 10% heat-inactivated calf or fetal bovine serum. Tests were carried out with and without an in vitro metabolic activation system (S9 mix). In tests without metabolic activation, the test chemical was left in culture until colcemid addition for 24 or 48 hrs, whereas with activation the test chemical was added along with S9 mix for only 6 hr at the beginning of the test period. The doses used for the study were 0, 0.15, 0.3, 0.6 or 0.9 mg/mL without S9 (24 hrs), 0, 0.3, 0.6, 0.9 or 1.2 mg/mL without S9 (48 hrs), 0, 0.3, 0.6 or 1.2 mg/mL without S9 (6-18 hrs) and 0, 0.6, 1.2, 1.8 or 2.4 mg/mL with S9 (6-18 hrs). The test chemical showed a marginal induction of structural aberrations, predominantly chromatid exchanges, but only at the highest dose in 24-h continuous treatment without S9 mix. However, because there was no structural aberration induction with any other treatment and the dose range used, the test chemical was considered to be negative forin vitro mammalian chromosome aberration test.

Based on the observations made, the test chemical did not induce chromosome aberrations in the mammalian cell line in the presence and absence of S9 metabolic activation system and hence it is not mutagenic in the chromosome aberration study performed.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Gene mutation in vitro:

Ames assay:

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The material was dissolved in ethanol and applied at a concentration of 3 µmole/plate in the spot test performed toSalmonella typhimuriumLT-2 strains TA 98, TA 100, TA 1535, and TA 1537 with and without S9 metabolic activation system. The test chemical did not induce reversion of mutant strains and hence is not mutagenic in the bacteriumSalmonella typhimurium LT-2 strains TA 98, TA 100, TA 1535, and TA 1537 with and without S9 metabolic activation system and hence the chemical is not likely to classify as gene mutant in vitro.

In another study, gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed as per the preincubation modification of the Salmonella/mammalian microsome mutagenicity (Ames) test. The chemicals were tested in a preincubation procedure in strains TA98 and TA100 without metabolic activation and with activation provided by Aroclor induced rat and hamster liver homogenates (S9). If a positive response was seen in one of these two strains, the strain/metabolic activation combination producing that response was repeated, and no further testing was performed. If no positive responses were seen, the chemical was tested in strains TA97 and TA1535. The plates were observed for a dose dependent increase in the number of revertants/plate. The combination of a questionable (?) and negative (-) response was considered negative (-); the combination of a weakly positive (+w) and negative response was considered questionable (?). The test chemical did not induce a dose dependent increase in the number of revertants in Salmonella typhimurium TA98, TA100, TA97 and TA1535 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Gene mutation toxicity study was also performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium strains TA97, TA98, TA100, TA1535, TA1537 in the presence and absence of S9 metabolic activation system. The chemical was dissolved in DMSO and used at dose levels 0, 10, 33, 100, 333, 666, 1000, 1666, 3333 or 6666 µg/plate by the preincubation method. The doses were selected on the basis of preliminary dose range finding study and concurrent solvent and positive controls were included in the study. The plates were observed for a dose dependent increase in the number of Histidine- independent (his+) colonies. The test chemical did not induce gene mutation in Salmonella typhimurium strains TA97, TA98, TA100, TA1535, TA1537 in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

Based on the observations made, the test chemical is not mutagenic in the bacterium Salmonella typhimurium strains in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as gene mutant in vitro.

In vitro chromosome aberration assay:

In vitro mammalian chromosome aberration test was performed to evaluate the mutagenic nature of the test chemical. Cloned Chinese hamster ovary cells (CHO-W-B1) were cultured in Mc-Coy’s 5a medium with 10% fetal calf serum, L-glutamine, and antibiotics. Tests were carried out with and without an in vitro metabolic activation system (S9 mix). In tests without metabolic activation, the test chemical was left in culture until colcemid addition, whereas with activation the test chemical was added along with S9 mix for only 2 hr at the beginning of the test period. The doses used for the study were 160-1600µg/mL without S9 and 500-5000µg/mL with S9. The test chemical did not induce chromosome aberrations in the Chinese hamster ovary cell line in the presence and absence of S9 metabolic activation system and hence it is not mutagenic in the chromosome aberration study performed.

In another study, In vitro mammalian chromosome aberration test was performed to evaluate the mutagenic nature of the test chemical. Cloned Chinese hamster lung fibroblast cell line (CHL/IU) were cultured in Eagle's minimum essential medium supplemented with 10% heat-inactivated calf or fetal bovine serum. Tests were carried out with and without an in vitro metabolic activation system (S9 mix). In tests without metabolic activation, the test chemical was left in culture until colcemid addition for 24 or 48 hrs, whereas with activation the test chemical was added along with S9 mix for only 6 hr at the beginning of the test period. The doses used for the study were 0, 0.15, 0.3, 0.6 or 0.9 mg/mL without S9 (24 hrs), 0, 0.3, 0.6, 0.9 or 1.2 mg/mL without S9 (48 hrs), 0, 0.3, 0.6 or 1.2 mg/mL without S9 (6-18 hrs) and 0, 0.6, 1.2, 1.8 or 2.4 mg/mL with S9 (6-18 hrs). The test chemical showed a marginal induction of structural aberrations, predominantly chromatid exchanges, but only at the highest dose in 24-h continuous treatment without S9 mix. However, because there was no structural aberration induction with any other treatment and the dose range used, the test chemical was considered to be negative forin vitro mammalian chromosome aberration test.

Based on the observations made, the test chemical did not induce chromosome aberrations in the mammalian cell line in the presence and absence of S9 metabolic activation system and hence it is not mutagenic in the chromosome aberration study performed.

On the basis of available data for the test chemicals and applying the weight of evidence approach, the test chemical is not likely to classify as a gene mutant in vitro. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

On the basis of available data for the test chemicals and applying the weight of evidence approach, the test chemical is not likely to classify as a gene mutant in vitro. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.