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 mammalian cell gene mutation assay:

The test chemical did not show any evidence of gene toxicity when CHO cells were exposed to the test chemical in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

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 from handbook or collection of data
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:
S. typhimurium, 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:
S. typhimurium, 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:
S. typhimurium, 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:
S. typhimurium, 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 gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
20-05-2015 to 07-07-2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from study report
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
In vitro Mammalian Cell Gene Mutation assay was performed for the test chemical in the absence of S9 metabolic activation system
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
Cells deficient in hypoxanthine-guanine phosphoribosyl transferase (HPRT) due to the mutation HPRT+/- to HPRT-/- are resistant to cytotoxic effects of 6-thioguanine (TG). HPRT proficient cells are sensitive to TG (which causes inhibition of cellular metabolism and halts further cell division since HPRT enzyme activity is important for DNA synthesis), so mutant cells can proliferate in the presence of TG, while normal cells, containing hypoxanthine-guanine phosphoribosyl transferase cannot.

This in vitro test is an assay for the detection of forward gene mutations at the in hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on the X chromosomes of hypodiploid, modal No. 20, CHO cells. Gene and chromosome mutations are considered as an initial step in the carcinogenic process.

The hypodiploid CHO cells are exposed to the test item with and without exogenous metabolic activation. Following an expression time the descendants of the treated cell population are monitored for the loss of functional HPRT enzyme. HPRT catalyses the transformation of the purine analogues 6-thioguanine (TG) rendering them cytotoxic to normal cells. Hence, cells with mutations in the HPRT gene cannot phosphoribosylate the analogue and survive treatment with TG.

Therefore, mutated cells are able to proliferate in the presence of TG whereas the non-mutated cells die. However, the mutant phenotype requires a certain period of time before it is completely expressed. The phenotypic expression is achieved by allowing exponential growth of the cells for 7 days.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Ham's F-12K (Kaighn's) Medium containing 2 mM L-Glutamine supplemented with 10% Fetal Bovine Serum and 1% Penicillin-Streptomycin (10,000 U/mL).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Not applicable
- Periodically checked for karyotype stability: Not applicable
Additional strain / cell type characteristics:
other: Hypodiploid, modal No. 20
Cytokinesis block (if used):
No data
Metabolic activation:
without
Metabolic activation system:
S9 liver microsomal fraction obtained from Arcolor 1254-induced male Sprague-Dawley rats (Supplier: Molecular Toxicology Inc. via Trinova Biochem GmbH, Giessen, Germany)
Test concentrations with justification for top dose:
0, 0.5, 1.0, 2.5 or 5.0 mM
Vehicle / solvent:
Vehicle(s)/solvent(s) used: Ethanol
Justification for choice of solvent/ vehicle: Methyl phenylacetate was easily dissolved in ethanol.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-ethyl-N-nitrosourea (ENU)
Details on test system and experimental conditions:
METHOD OF APPLICATION: In medium with pre-incubation

Pre-incubation
One week involving 3 days of incubation with Hypoxanthine-aminopterin-thymidine (HAT) in medium as a mutant cleansing stage, followed by overnight incubation with hypoxanthine-thymidine (HT) in medium prior to a 3-4 days incubation in regular cell medium. After seeding and prior to treatment, the mutant-free cells were incubated for an additional of 24 hours.

Exposure duration
3 hours

Expression time
7 days

Selection time
14 days

Fixation time
7 days (harvest of cells)

SELECTION AGENT
(mutation assays):
6-thioguanine (TG)

STAIN (for cytogenetic assays):
Crystal violet

NUMBER OF REPLICATIONS:
A minimum of 2 replicates per dose concentration including negative and positive control.

NUMBER OF CELLS EVALUATED:
5 x 10 E5 cells were plated 7 days after treatment and whatever cells left, after 14 days of incubation with the selection medium, were evaluated.

DETERMINATION OF CYTOTOXICITY

Cytotoxicity test
After being exposed to the test chemical for 3 hours, in the absence or presence of S9, cells were trypsinized and 0.5 x 10 E5 cells per well was seeded in duplicates from two parallel duplicate cultures into 6-well plates in fresh medium. The relative total growth and cytotoxicity was evaluated 24 and 48 hours after seeding.

Rationale for test conditions:
No data
Evaluation criteria:
The plates were scored for total number of colonies
Statistics:
No data available
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
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:
Preliminary dose-finding/toxicity test
Completed without S9 metabolic activation. A range of test concentrations (0, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0 or 5.0 mM) was applied 24 hours after seeding to single cultures in fresh medium in 96-well plates. The cell population (control and treated cells) were assessed 24 and 48 hours after treatment using the colorimetric assay MTT and the BCA assay to assess cell viability and total protein concentration, respectively. From the basis of these results, the test concentrations of the chemical was chosen to be included in the gene toxicity test. Since cytotoxicity was evident at the tested concentration in this preliminary dose-finding test further testing concentrations were adapted to have a maximum test concentration of 0.5 mM. Since the test chemical was dissolved in ethanol, higher concentrations of the test chemical than the concentration mentioned above would result in a toxic effect of ethanol. The test chemical could only be dissolved in 99.5% ethanol.

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data
Remarks on result:
other: No mutagenic potential
Conclusions:
The test chemical in the concentration of 0, 0.5, 1.0, 2.5 or 5.0 mM did not show any evidence of gene toxicity when CHO cells were exposed to the test chemical in the absence of S9 metabolic activation system and hence dose not classify for gene mutation in vitro.
Executive summary:

In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to the test chemical in the concentration of 0, 0.5, 1.0, 2.5 or 5.0 mM and without metabolic activation for 3 hours. The results showed that there was no evidence of cytotoxicity after treatment. Independently of tested concentration, the results showed no evidence of gene toxicity. Therefore, it is considered that the test chemical in the concentration of 0, 0.5, 1.0, 2.5 or 5.0 mM does not cause genetic mutation(s) when CHO cells are exposed to the test chemical in the absence of metabolic activation and hence does not classify for gene mutation in vitro.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
20-05-2015 to 07-07-2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from study report
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
In vitro Mammalian Cell Gene Mutation assay was performed for the test chemical in the presence of S9 metabolic activation system
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
Cells deficient in hypoxanthine-guanine phosphoribosyl transferase (HPRT) due to the mutation HPRT+/- to HPRT-/- are resistant to cytotoxic effects of 6-thioguanine (TG). HPRT proficient cells are sensitive to TG (which causes inhibition of cellular metabolism and halts further cell division since HPRT enzyme activity is important for DNA synthesis), so mutant cells can proliferate in the presence of TG, while normal cells, containing hypoxanthine-guanine phosphoribosyl transferase cannot.

This in vitro test is an assay for the detection of forward gene mutations at the in hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on the X chromosomes of hypodiploid, modal No. 20, CHO cells. Gene and chromosome mutations are considered as an initial step in the carcinogenic process.
The hypodiploid CHO cells are exposed to the test item with and without exogenous metabolic activation. Following an expression time the descendants of the treated cell population are monitored for the loss of functional HPRT enzyme.
HPRT catalyses the transformation of the purine analogues 6-thioguanine (TG) rendering them cytotoxic to normal cells. Hence, cells with mutations in the HPRT gene cannot phosphoribosylate the analogue and survive treatment with TG.

Therefore, mutated cells are able to proliferate in the presence of TG whereas the non-mutated cells die. However, the mutant phenotype requires a certain period of time before it is completely expressed. The phenotypic expression is achieved by allowing exponential growth of the cells for 7 days.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Cell line used: Chinese Hamster Ovary (CHO) cells
- Type and identity of media: Ham's F-12K (Kaighn's) Medium containing 2 mM L-Glutamine supplemented with 10% Fetal Bovine Serum and 1% Penicillin-Streptomycin (10,000 U/mL).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Not applicable
- Periodically checked for karyotype stability: Not applicable
Additional strain / cell type characteristics:
other: Hypodiploid, modal No. 20
Cytokinesis block (if used):
No data
Metabolic activation:
with
Metabolic activation system:
S9 liver microsomal fraction obtained from Arcolor 1254-induced male Sprague-Dawley rats (Supplier: Molecular Toxicology Inc. via Trinova Biochem GmbH, Giessen, Germany)
Test concentrations with justification for top dose:
0, 0.5, 1.0, 2.5 or 5.0 mM
Vehicle / solvent:
Vehicle(s)/solvent(s) used: Ethanol
Justification for choice of solvent/ vehicle: Methyl phenylacetate was easily dissolved in ethanol.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION:
In medium with pre-incubation

DURATION
Pre-incubation
One week involving 3 days of incubation with Hypoxanthine-aminopterin-thymidine (HAT) in medium as a mutant cleansing stage, followed by overnight incubation with hypoxanthine-thymidine (HT) in medium prior to a 3-4 days incubation in regular cell medium. After seeding and prior to treatment, the mutant-free cells were incubated for an additional of 24 hours.

Exposure duration
3 hours

Expression time
7 days

Selection time
14 days

Fixation time
7 days (harvest of cells)

SELECTION AGENT (mutation assays):
6-thioguanine (TG)

SPINDLE INHIBITOR (cytogenetic assays):
Not applicable

STAIN (for cytogenetic assays):
Crystal violet

NUMBER OF REPLICATIONS:
A minimum of 2 replicates per dose concentration including negative and positive control.

NUMBER OF CELLS EVALUATED:
5 x 10 E5 cells were plated 7 days after treatment and whatever cells left, after 14 days of incubation with the selection medium, were evaluated.

DETERMINATION OF CYTOTOXICITY
Cytotoxicity test
After being exposed to the test chemical for 3 hours, in the absence or presence of S9, cells were trypsinized and 0.5 x 10 E5 cells per well was seeded in duplicates from two parallel duplicate cultures into 6-well plates in fresh medium. The relative total growth and cytotoxicity was evaluated 24 and 48 hours after seeding.

Rationale for test conditions:
No data
Evaluation criteria:
The plates were scored for total number of colonies
Statistics:
No data available
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
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: Preliminary dose-finding/toxicity test
Completed with S9 metabolic activation. A range of test concentrations (0, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0 or 5.0 mM) was applied 24 hours after seeding to single cultures in fresh medium in 96-well plates. The cell population (control and treated cells) were assessed 24 and 48 hours after treatment using the colorimetric assay MTTand the BCA assay to assess cell viability and total protein concentration, respectively. From the basis of these results, the test concentrations of the chemical was chosen to be included in the gene toxicity test. Since cytotoxicity was evident at the tested concentration in this preliminary dose-finding test further testing concentrations were adapted to have a maximum test concentration of 5.0 mM. Since the test chemical was dissolved in ethanol, higher concentrations of the test chemical than the concentration mentioned above would result in a toxic effect of ethanol. The test chemical could only be dissolved in 99.5% ethanol.

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data
Remarks on result:
other: No mutagenic potential
Conclusions:
The test chemical in the concentration of 0, 0.5, 1.0, 2.5 or 5.0 mM did not show any evidence of gene toxicity when CHO cells were exposed to the test chemical in the presence of S9 metabolic activation system and hence does not classify as a gene mutant in vitro.
Executive summary:

In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to the test chemical in the concentration of 0, 0.5, 1.0, 2.5 or 5.0 mM and S9-induced metabolic activation for 3 hours. The results showed that there was no evidence of cytotoxicity after treatment. Independently of tested concentration, the results showed no evidence of gene toxicity. Therefore, it is considered that the test chemical in the concentration of 0, 0.5, 1.0, 2.5 or 5.0 mM does not cause genetic mutation(s) when CHO cells are exposed to the test chemical in the presence of metabolic activation and hence dose not classify as a gene mutant in vitro.

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 mammalian cell gene mutation assay:

In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to the test chemicals in the concentration of 0, 0.5, 1.0, 2.5 or 5.0 mM . The test substance were exposed with and without metabolic activation for 3 hours. The results showed that there was no evidence of cytotoxicity after treatment. Independently of tested concentration, the results showed no evidence of gene toxicity. Therefore, it is considered that the test chemical in the concentration of 0, 0.5, 1.0, 2.5 or 5.0 mM does not cause genetic mutation(s) when CHO cells are exposed to the test chemical in the presence and absence of S9 metabolic activation system. Hence the substance cannot be classified as gene mutant in vitro as per the criteria mentioned in CLP regulation.

Based on the observations made, the test chemical in the concentration of 0, 0.5, 1.0, 2.5 or 5.0 mM did not show any evidence of gene toxicity when CHO cells were exposed to the test chemical in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

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.