Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test:

The test chemical did not induce gene mutation in Salmonella typhimurium strains 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.

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.

In vitro mammalian cell gene mutation assay:

In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to the test chemical in the concentration of 0, 0.5, 1, 2.5 or 5 mM and S9-induced metabolic activation for 3 hours. The results showed that there was no evidence of cytotoxicity when CHO cells were treated with the test chemical. Independently of treatment concentration, the results showed no evidence of gene toxicity when cells were exposed to the test chemical. Therefore, it is considered that the test chemical in the concentrations of 0, 0.5, 1, 2.5 or 5mM does not cause genetic mutation(s) in the presence of metabolic activation.

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 data from various test chemicals
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
WoE derived based on the experimental data from various test chemicals
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA 1537, TA 98,TA 97 and TA 100
Remarks:
1
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:
2
Details on mammalian cell type (if applicable):
Not applicable
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 applicable
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
rat and hamster liver derived S-9 mix
Test concentrations with justification for top dose:
1. 0, 10,100,1000 and 10000ug/plate.
2. 0.03, 0.3, 3 and 30 µmol/plate
3. 0, 10, 33, 100, 333, 666, 1000, 1666, 3333 or 6666 µg/plate
Vehicle / solvent:
1./3. - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO

2. - Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: The test chemical was dissolev in ethanol
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; Without S9); 2-aminoanthracene (With S9; all strains)
Remarks:
1/3
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
other: 2-aminoanthracene
Remarks:
2
Details on test system and experimental conditions:
1. METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours
- 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: All assays were repeated no less than one week after completion of the initial test.

NUMBER OF CELLS EVALUATED: No data available

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: 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.

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.

2. 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).

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. Mutagenic response: a dose-related, reproducible increase in the number of revertants over background, even if the increase was less than twofold.
Nonmutagenic response: when no increase in the number of revertants was elicited
Questionable response: when there was an absence of a clear cut dose related increase in revertants, when the dose related increase in revertants was not reproducible or when the response was of insufficient magnitude to support a determination of mutagenicity.

2.
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

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. 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, other: TA97, TA98, TA100, TA1535, TA1537
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: YA1535, TA1537, TA98 and TA100
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
Additional information on results:
1. 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 did not induce gene mutation in Salmonella typhimurium strains 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.
Executive summary:

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

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. The test was conducted both in the presence and absence of metabolic activation using rat and hamster liver derived S-9 mix, over a range of doses. The test chemical was dissolved in DMSO and used at dose level from 0 to 10000 ug/plate. COncurret solvent and positive control chemicals were also included in the study. Based on the results of this study, the test chemical was not considered to be mutagenic under the conditions of this test.

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 does not classify as 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 did not induce gene mutation in Salmonella typhimurium strains 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.

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:
WoE derived based on the experimental 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:
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:
mammalian cell line, 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:
mammalian cell line, 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:
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:
03-07-2014 - 25-03-2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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:
Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical in the presence of S9 metabolic activation system
GLP compliance:
no
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, 2.5 or 5 mM
Vehicle / solvent:
Vehicle(s)/solvent(s) used: DMSO
Justification for choice of solvent/ vehicle: The test chemical was easily dissolved in DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
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:
Chinese Hamster Ovary Cells (CHO) were observed for gene mutation caused by the test compound
Statistics:
No data
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
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: Completed without S9 metabolic activation. A range of test concentrations (0, 0.01, 0.05, 0.1, 0.5,1, 5, 10 or 20mM) 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 assayMTTand 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 furthertesting concentrations were adapted to have a maximum test concentration of 5 mM.Since the test chemical was dissolved in DMSO, higher concentrations of the test chemical than the concentration mentioned above would result in a toxic effect of DMSO. The test chemical could only be dissolved in 100% DMSO.

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

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

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

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Other observations when applicable:No data
Remarks on result:
other: No mutagenic potential
Conclusions:
The test chemical in the concentration of ≥ 0.05 mM may cause genetic mutation(s) when CHO cells are exposed to the test chemical in the presence of metabolic activation.
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, 2.5 or 5 mM and S9-induced metabolic activation for 3 hours. The results showed that there was no evidence of cytotoxicity when CHO cells were treated with the test chemical. Independently of treatment concentration, the results showed no evidence of gene toxicity when cells were exposed to the test chemical. Therefore, it is considered that the test chemical in the concentrations of 0, 0.5, 1, 2.5 or 5mM does not cause genetic mutation(s) in the presence of metabolic activation.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

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

Ames test:

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. The test was conducted both in the presence and absence of metabolic activation using rat and hamster liver derived S-9 mix, over a range of doses. The test chemical was dissolved in DMSO and used at dose level from 0 to 10000 ug/plate. Concurret solvent and positive control chemicals were also included in the study. Based on the results of this study, the test chemical was not considered to be mutagenic under the conditions of this test.

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 does not classify as 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.

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 500-5000µg/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.

In vitro mammalian cell gene mutation assay:

In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to the test chemical in the concentration of 0, 0.5, 1, 2.5 or 5 mM and with and without S9-induced metabolic activation for 3 hours. The results showed that there was no evidence of cytotoxicity when CHO cells were treated with the test chemical. Independently of treatment concentration, the results showed no evidence of gene toxicity when cells were exposed to the test chemical. Therefore, it is considered that the test chemical in the concentrations of 0, 0.5, 1, 2.5 or 5mM does not cause genetic mutation(s) in the presence and absence of metabolic activation.

In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to the test chemical in the concentration of 0, 0.5, 1, 2.5 or 5 mM and S9-induced metabolic activation for 3 hours. The results showed that there was no evidence of cytotoxicity when CHO cells were treated with the test chemical. Independently of treatment concentration, the results showed no evidence of gene toxicity when cells were exposed to the test chemical. Therefore, it is considered that the test chemical in the concentrations of 0, 0.5, 1, 2.5 or 5mM does not cause genetic mutation(s) in the presence of metabolic activation.

Based on the data available for the test chemicals and applying the 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 for the test chemicals and applying the 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.