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 mutation in the Salmonella typhimurium strains both in the presence and absence of S9 metabolic activation system and hence is not likely to be mutagenic under the conditions of this study.

In vitro mammalian chromosome aberration study:

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 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)
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:
2. Histidine locus in the genome of Salmonella typhimurium and tryptophan locus of Escherichia coli
3. Histidine
4. Histidine
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Remarks:
2
Details on mammalian cell type (if applicable):
In addition to a mutation in either the histidine or tryptophan operons, the tester strains contain additional mutations that enhance their sensitivity to some mutagenic compounds. Mutation of either the uvrA gene (Escherichia coli) or the uvrB gene (Salmonella typhimurium) results in a deficient DNA excision repair system, which greatly enhances the sensitivity of these strains to some mutagens. Since the uvrB deletion extends through the bio gene, Salmonella typhimurium tester strains containing this deletion also require the vitamin biotin for growth.

Salmonella typhimurium tester strains also contain the rfa wall mutation, which results in the loss of one of the enzymes responsible for the synthesis of part of the lipopolysaccharide barrier that forms the surface of the bacterial cell wall. The resulting cell wall deficiency increases permeability to certain classes of chemicals such as those containing large ring systems (i.e., benzo[a]pyrene) that would otherwise be excluded by a normal intact cell wall.

Tester strains TA98 and TAlOO also contain the pKMlOl plasmid, which further increases the sensitivity of these strains to some mutagens. The suggested mechanism by which this plasmid increases sensitivity to mutagens is by modification of an existing bacterial DNA repair polymerase complex involved with the mismatch-repair process.

Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strains TAlOO, TA1535, and WP2uvrA are reverted from auxotrophy to prototrophy by base substitution mutagens.
Additional strain / cell type characteristics:
DNA polymerase A deficient
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Remarks:
3
Details on mammalian cell type (if applicable):
Not applicable
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):
Not applicable
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
S9 Homogenate (Aroclor) in S9Mix
Test concentrations with justification for top dose:
2. Salmonella tester strains (with S9 mix): 33.3, 100, 333, 1000, 3330, and 5000 ug per plate
Salmonella tester strains (without S9 mix): 3.33, 10.0, 33.3, 100, 333, 1000, 3330, and 5000 ug per plate
Escherichia coli tester strain (with and without S9 mix): 33.3, 100, 333, 1000, 3330, and 5000 ug per plate

Cytotoxicity was observed in the dose range finding study, and the highest dose level of test article used in the subsequent mutagenicity assay was a dose which gave a reduction of revertants per plate and/or a thinning or disappearance of the bacterial background lawn

3. 0, 33, 100, 333, 1000, 3333 or 10000 ug/plate

4. 0.03, 0.3, 3 and 30 μmol/plate
Vehicle / solvent:
2. The test article was .observed to form a transparent, colorless solution at a concentration of 100 mg per mL in dimethylsulfoxide (DMSO). DMSO was selected as the vehicle. At 100 mg per mL, which was the most concentrated stock dilution prepared for the mutagenicity assay, the test article was observed to form a transparent, non-viscous, colorless solution. The test article remained a solution in all succeeding dilutions prepared for the mutagenicity assay.

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

4. - Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: The test chemical was dissolved in ethanol
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: 2-aminoanthracene - TA100, TA1535, TA1537, WP2uvrA with S9 Mix; ICR-191 - TA1537 without S9 Mix
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, 2-aminoanthracene
Remarks:
3
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
other: 2- aminoanthracene
Remarks:
4
Details on test system and experimental conditions:
2. Tester strains were exposed to the test article via the plate incorporation methodology originally described by Ames et al. (1975) and Maron and Ames (1983). This methodology has been shown to detect a wide range of classes of chemical mutagens. In the plate incorporation methodology, test article, tester strain, and S9 mix (when appropriate) were combined in molten agar, which was overlaid onto a minimal agar plate. Following incubation, revertant colonies were counted. All doses of test article, vehicle controls and positive controls were plated in triplicate.

3. METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 48 hrs
- 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: : All assays were repeated in duplicate one week after completion of the initial test. At least five dose levels of the chemicals were tested, with three plates per dose level.

NUMBER OF CELLS EVALUATED: :No data available

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

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

OTHER: At least one toxic dose was incorporated into the first mutagenicity test, the repeat test(s) occasionally had the doses adjusted so that an apparent toxic dose was not reached.

3. 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: No data available

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

OTHER: Initially, cultures were grown in Difco nutrient broth. Since this medium is suspected to have a weak mutagenic activity, it was substituted for Oxoid nutrient broth No. 2 in later experiments. Revertants were scored on glucosenminimal salts medium supplemented with 0.05 μmol histidine and 0.05 μmol biotin. Plates used for viable counts contained 10 μmol histidine (and 0.05 μmol biotin).




Rationale for test conditions:
2. Experimental materials, methods and procedures are based on those described by Ames et al. (1975) and Green and Muriel (1976). The assay design is based on the OECD Guideline 471, updated and adopted 21 July 1997.
Evaluation criteria:
2. The condition of the bacterial background lawn was evaluated both macroscopically and microscopically (using a dissecting microscope) for indications of cytotoxicity and test article precipitate. Evidence of cytotoxicity was scored relative to the vehicle control plate and was recorded along with the revertant counts for all plates at that dose level.
Lawns were scored as normal (N), reduced (R), obscured by precipitate (0), macroscopic precipitate present (P), absent (A), or enhanced (E); contaminated plates (C) were also noted.

Revertant colonies were counted by automated colony counter or by hand.

3. 1) mutagenic response: a dose-related, reproducible increase in the number of revertants over background, even if the increase was less than twofold;
2) nomutagenic response: when no increase in the number of revertants was elicited by the chemical;
3) questionable response: when there was an absence of a clear-cut dose-related increase in revertants; when the dose-related increases in the number of revertants were not reproducible; or when the response was of insufficient magnitude to support a determination of mutagenicity.

4. 1. Increase in the number of spontaneous revertants
2. The presence of the rfa-mutation was checked by crystal violet inhibition;
3. The presence of the plasmid pKM 101 in strains TA 98 and TA 100 was checked by resistance to
ampicillin
Statistics:
No data
Species / strain:
S. typhimurium, other: TA98, TA100, TA1535 and TA1537
Remarks:
2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Remarks:
2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98 and TA 100
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
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98 and TA 100
Remarks:
4
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:
valid
Additional information on results:
2. Dose Range Finding Assay

Doses tested in the mutagenicity assay were selected based on results of the dose rangefinding assay conducted on the test article using tester strains TA100 and WP2uvrA in both the presence and absence of S9 mix with one plate per dose. Ten doses of test article, from 6.67 to 5000 ug per plate were tested.

Cytotoxicity was observed with tester strain TA100 at 333 ug per plate and above in the absence of S9 mix as evidenced by reduced background lawns and a decrease in the number of revertants per plate. No cytotoxicity was observed with tester strain TA100 in the presence of S9 mix or with tester strain WP2uvrA in the presence or absence of S9 mix.

Mutagenicity Assay

In the initial mutagenicity assay, first trial (B1), all data were acceptable, and no positive increases in the mean number of revertants per plate were observed with any of the tester strains in either the presence or absence of S9 mix.

In the confirmatory mutagenicity assay, second trial (C1), contamination was observed on many of the assay plates and several of the plates were observed to have reduced or absent bacterial background lawns. Due to the multiple technical problems observed the data generated were not used in the evaluation of the test article (the results have not been included).

The confirmatory assay was repeated in third trial (D1). In the repeat confirmatory mutagenicity assay, all data were acceptable, and no positive increases in the mean number of revertants per plate were observed with any of the tester strains in either the presence or absence of S9 mix. In this trial, a 2.7-fold increase was observed with tester strain WP2uvrA in the presence of S9 mix, however, this increase was not clearly dose-responsive and did not meet the criteria for a positive evaluation. In order to clarify this response, the test article was retested with tester strain WP2uvrA at the same doses in the presence of S9 mix in fourth trial (D2). Also, due to variability in the vehicle control counts for tester strain TA100 in the absence of S9 mix, the test article was retested with tester strain TAI100 at the same doses in the absence of S9 mix. In the fourth trial, all data were acceptable, and no positive increases in the mean number of revertants per plate were observed with tester strain WP2uvrA in the presence of S9 mix or with tester strain T A100 in the absence of S9 mix.

All criteria for a valid study were met.

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 chemical was tested initially with strain TA100 in the presence and the absence of the metabolic activation systems, over a wide dose range with an upper limit of 10 mgjplate, or less when solubility problems were encountered. Toxicity was evidenced
by one or more of the following phenomena: appearance of his+ pinpoint colonies, reduced numbers of revertant colonies per plate, or thinning or absence of the bacterial lawn. Nontoxic chemicals were tested in the initial experiment up to the 10 mg/plate dose level, or to a level determined by their
solubility. Toxic chemicals were tested up to a high dose which exhibited some degree of toxicity.

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

4. No data
Remarks on result:
other: No mutagenic potential
Conclusions:
The test chemical did not induce mutation in the Salmonella typhimurium strains both in the presence and absence of S9 metabolic actuvation system and hence is not likely to be mutagenic under the conditions of this study.
Executive summary:

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

The results of the Salmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay with a Confirmatory Assay indicate that under the conditions of this study, the test article did not cause a positive increase in the mean number of revertants per plate with any of the tester strains in either the presence or absence of Aroclor™ induced rat liver (S9).

The test chemical was examined for its ability to cause mutagenic changes when tested in five strains of the bacteria Salmonella typhimurium, specifically, TA 1535, TA 1537, TA97, TA 98 and TA 100 through the preincubation assay method. Preliminary dose range finding study was performed initially to set the doses for the main study. The test was conducted both in the presence and absence of metabolic activation using male rat and hamster liver derived S-9 mix at dose levels of 0, 33, 100, 333, 1000, 3333 or 10000 ug/plate. The test was repeated and atleast three plates were used at each dose level. The test chemical did not induce mutation in the Salmonella typhimurium strain TA98, TA100, TA1535 or TA1537 both in the presence and absence of S9 metabolic activation system and hence is not likely to be mutagenic under the conditions of this study.

In another study, the test chemical was investigated for its ability to induce mutagenic activity when tested in an in vitro reverse mutagenicity test using four strains of the bacteria Salmonella typhimurium, specifically TA 98, TA 100, TA 1535 and TA 1537. Spot test was performed for the chemical at dose levels of 0.03, 0.3, 3 and 30 μmol/plate. The study was conducted both in the presence and absence of metabolic activation using S9 mix from Aroclor 1254 or methylcholanthrene induced rats. The test chemical is not mutagenic in the bacterium Salmonella typhimurium LT-2 strains TA 98, TA 100, TA1535 and TA37 with and without S9 metabolic activation system and hence is not likely to classify as gene mutant in vitro.

Based on the data available for the various test chemicals and applying the weight of evidence approach, the test chemical did not induce mutation in the Salmonella typhimurium strains both in the presence and absence of S9 metabolic actuvation system and hence is not likely to be mutagenic under the conditions of this study.

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 for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
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:
other: In vitro mammalian chromosome aberration
Target gene:
No data
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
6
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:
mammalian cell line, other: Chinese hamster lung fibroblast cell line (CHL/IU)
Remarks:
7
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:
6. Without S9: 160-1600 µg/ ml
With S9: 500-5000 µg/ ml

7. 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:
6. Water, dimethyl sulfoxide (DMSO), ethanol, or Acetone (in the order of preference)
7. - 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:
6
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:
7
Details on test system and experimental conditions:
6. 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

7. 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:
6. 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).

7. 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:
6. 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.

7. No data
Species / strain:
Chinese hamster Ovary (CHO)
Remarks:
6
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:
mammalian cell line, other: Chinese hamster lung fibroblast cell line (CHL/IU)
Remarks:
7
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:
6. 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

7. 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 500-5000 µg/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 for in 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:

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

Ames assay:

The results of the Salmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay with a Confirmatory Assay indicate that under the conditions of this study, the test article did not cause a positive increase in the mean number of revertants per plate with any of the tester strains in either the presence or absence of Aroclor™ induced rat liver (S9).

The test chemical was examined for its ability to cause mutagenic changes when tested in five strains of the bacteria Salmonella typhimurium, specifically, TA 1535, TA 1537, TA97, TA 98 and TA 100 through the preincubation assay method. Preliminary dose range finding study was performed initially to set the doses for the main study. The test was conducted both in the presence and absence of metabolic activation using male rat and hamster liver derived S-9 mix at dose levels of 0, 33, 100, 333, 1000, 3333 or 10000 ug/plate. The test was repeated and atleast three plates were used at each dose level. The test chemical did not induce mutation in the Salmonella typhimurium strain TA98, TA100, TA1535 or TA1537 both in the presence and absence of S9 metabolic activation system and hence is not likely to be mutagenic under the conditions of this study.

In another study, the test chemical was investigated for its ability to induce mutagenic activity when tested in an in vitro reverse mutagenicity test using four strains of the bacteria Salmonella typhimurium, specifically TA 98, TA 100, TA 1535 and TA 1537. Spot test was performed for the chemical at dose levels of 0.03, 0.3, 3 and 30 μmol/plate. The study was conducted both in the presence and absence of metabolic activation using S9 mix from Aroclor 1254 or methylcholanthrene induced rats. The test chemical is not mutagenic in the bacterium Salmonella typhimurium LT-2 strains TA 98, TA 100, TA1535 and TA37 with and without S9 metabolic activation system and hence is not likely to classify as gene mutant in vitro.

Based on the data available for the various test chemicals, the test chemical did not induce mutation in the Salmonella typhimurium strains both in the presence and absence of S9 metabolic activation system and hence is not likely to be mutagenic under the conditions of this study.

In vitro mammalian chromosome aberration study:

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 500-5000 µg/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.

Based on the data available and applying weight of evidence approach, the test chemical does not exhibit gene mutation 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

Based on the data available and applying weight of evidence approach, the test chemical does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.