Isopentyl phenylacetate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames Assay:

The test chemical was evaluated using Salmonella Typhimurium strains TA 98, TA 100. The maximum dose of test chemical was 50 µg/plate.S9 mix treated with PCB EC-100- induced rat liver 9900g supernatant was used as the metabolic activation system. The test chemical showed lethal effects on both strains when tested at the maximum dose 50 µg/plate without S9 activation system, so it was tested at lower dose of 10 µg/plate in the presence and absence of S9 activation system. The test chemical did not show any mutagenic effects when tested at the low concentration. Hence, the test chemical can be considered to non-mutagenic to Salmonella Typhimurium strains TA 98, TA 100 at 10 µg/plate in the presence and absence of S9 activation system.

In vitro chromosomal Aberration study

Based on the available studies and applying the weight of evidence approach, it can be concluded that the given test chemical did not induce chromosomal aberrations in Chinese hamster fibroblast cell line and hence it is not likely to classify as a gene mutant in vitro.

In vitro gene mutation study in mammalian cells

Based on the available studies and applying the weight of evidence approach, it can be concluded that the given test chemical did not induce mutation in mammalian cell line in the presence and absence of metabolic activation and hence it is not likely to classify as a gene mutant in vitro.

 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from secondary literature

Qualifier:

according to guideline

Guideline:

other: as mentioned below

Principles of method if other than guideline:

To evaluate the genotoxicity of the test chemical in Salmonella typhimurium strain TA98 and TA100 by Ames reverse mutation assay

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium, other: TA98 and TA100

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:

PCB KC-500-induced rat liver 9000g S9 supernatant

Test concentrations with justification for top dose:

10 or 50 µg/plate

Vehicle / solvent:

Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

furylfuramide

Remarks:

Positive control substance used With S9 mixture Benzo[a]pyrene- TA98and TA100, Without S9 mixture Furylframide- TA98and TA100

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar(plate incorporation)
DETERMINATION OF CYTOTOXICITY-Yes, cytotoxicity was determined by relative total growth.

Rationale for test conditions:

No data

Evaluation criteria:

Reversal colonies per plate were observed.

Statistics:

No data available

Species / strain:

S. typhimurium, other: TA 98 and TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA98 and TA100

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

50 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

Ames test:
- Signs of toxicity: The test chemical showed lethal effects on both strains when tested at the maximum dose 50 µg/plate without S9 activation system, so it was tested at lower dose of 10 µg/plate in the presence and absence of S9 activation system.

Remarks on result:

other: No mutagenic potential

Results from Salmonella typhimurium With and without S9 Mix from PCB treated rat

 

Compound	Amount (µg/plate)	Reversal colonies per plate observed.
		TA100		TA98
		-S9	+S9	-S9	+S9
test chemical	10	160	180	68	40
	50	22*	120	7*	37
Benzo[a]pyrene	5	-	604	-	237
Furylframide	0.2	1335	-	768	-
Control	100**	165	125	63	36

 

Each compound was tested in the presence and absence of S9 from PCB KC-500-induced rat liver 9000g supernatant

*Lethal effect

**µl per plate

Conclusions:

The test chemical showed lethal effects on both strains when tested at the maximum dose 50 µg/plate without S9 activation system but did not show any mutagenic effects when tested at the low concentration. Hence, the test chemical can be considered to non-mutagenic to Salmonella Typhimurium strains TA 98, TA 100 at 10 µg/plate in the presence and absence of S9 activation system.
.

Executive summary:

The test chemical was evaluated using Salmonella Typhimurium strains TA 98, TA 100. The maximum dose of test chemical was 50 µg/plate.S9 mix treated with PCB EC-100- induced rat liver 9900g supernatant was used as the metabolic activation system. The test chemical showed lethal effects on both strains when tested at the maximum dose 50 µg/plate without S9 activation system, so it was tested at lower dose of 10 µg/plate in the presence and absence of S9 activation system. The test chemical did not show any mutagenic effects when tested at the low concentration. Hence, the test chemical can be considered to non-mutagenic to Salmonella Typhimurium strains TA 98, TA 100 at 10 µg/plate in the presence and absence of S9 activation system.

Reference 2

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:

Weight of evidence approach based on various test chemicals

Justification for type of information:

Weight of evidence approach based on 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:

other: Weight of evidence approach based on various test chemicals

Principles of method if other than guideline:

Weight of evidence approach based on various test chemicals

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung (CHL/IU)

Remarks:

Study 8

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

Species / strain / cell type:

Chinese hamster lung (CHL/IU)

Remarks:

Study 9

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: the lung of new born female at the cancer research institute,Tokyo
- Suitability of cells: No data available
- Normal cell cycle time (negative control): No data available
For cell lines:
- Absence of Mycoplasma contamination: No data available
- Number of passages if applicable: 4 passages
- Methods for maintenance in cell culture: No data available
- Cell cycle length, doubling time or proliferation index : 15hours
- Modal number of chromosomes: 25
- Periodically checked for karyotype stability: [yes/no]
- Periodically ‘cleansed’ of spontaneous mutants: [yes/no]
MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if
applicable:Minimum essential medium, supplememted by 10% calf serum
- Type and identity of media: Minimum
Essential Medium (MEM; GIBCO) supplemented by 10% calf serum
- Properly maintained: yes by 4 day passages
- 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

Species / strain / cell type:

Chinese hamster lung (CHL/IU)

Remarks:

Study 10

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: the lung of new born female at the cancer research institute,Tokyo
- Suitability of cells: No data available
- Normal cell cycle time (negative control): No data available
For cell lines:
- Absence of Mycoplasma contamination: No data available
- Number of passages if applicable: 4 passages
- Methods for maintenance in cell culture: No data available
- Cell cycle length, doubling time or proliferation index : 15hours
- Modal number of chromosomes: 25
- Periodically checked for karyotype stability: [yes/no]
- Periodically ‘cleansed’ of spontaneous mutants: [yes/no]
MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if
applicable:Minimum essential medium, supplememted by 10% calf serum
- Type and identity of media: Minimum
Essential Medium (MEM; GIBCO) supplemented by 10% calf serum
- Properly maintained: yes by 4 day passages
- 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

Cytokinesis block (if used):

no data available

Metabolic activation:

with and without

Metabolic activation system:

8. A liver S9 fraction prepared from phenobarbital- and 5,6-benzoflavone-pretreated male Sprague-Dawley rats
9,10. without S9 activation system

Test concentrations with justification for top dose:

8. 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
9. Max dose 2 mg/ml, The maximum dose of each sample was selected by a preliminary test in which the dose needed for 50% cell-growth inhibition.
10. Max dose 1 mg/ml, The maximum dose of each sample was selected by a preliminary test in which the dose needed for 50% cell-growth inhibition.

Vehicle / solvent:

8. DMSO, the test chemical was soluble in solvent
9. DMSO, the test chemical was soluble in solvent
10. DMSO, the test chemical was soluble in solvent

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

Study 8

Untreated negative controls:

yes

Remarks:

Untreated cells served as control

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Remarks:

Study 9

Untreated negative controls:

yes

Remarks:

Untreated cells served as control

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Remarks:

study 10

Details on test system and experimental conditions:

8.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

NUMBER OF CELLS EVALUATED: 100 well spread metaphase cells

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes, 100 well spread metaphases were observed
9. FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): Colcemid was added to culture 2hour before the harvesting time.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): Giemsa
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): 100 cells were spread and stained
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): The incidence of polyploid cells as well as of cells with structural chromosomal aberrations
such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate.
10. FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): Colcemid was added to culture 2hour before the harvesting time.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): Giemsa
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): 100 cells were spread and stained
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): The incidence of polyploid cells as well as of cells with structural chromosomal aberrations
such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate.

Evaluation criteria:

8. 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.
9.The results were considered to be negative if the incidence was less than 4 .9%, equivocal if it was between 5 .0 and 9.9%, and positive if it was more than 10 .0%. When no reasonable dose-response relationships were found, additional experiments were carried out at similar dose levels . For a quantitative evaluation of the clastogenic potential of the positive samples, the D20 was calculated, which is the dose (mg/ml) at which structural aberrations (including gaps) were detected in 20% of the metaphases observed .
10.

The results were considered to be negative if the incidence was less than 4 .9%, equivocal if it was between 5 .0 and 9.9%, and positive if it was more than 10 .0%. When no reasonable dose-response relationships were found, additional experiments were carried out at similar dose levels . For a quantitative evaluation of the clastogenic potential of the positive samples, the D20 was calculated, which is the dose (mg/ml) at which structural aberrations (including gaps) were detected in 20% of the metaphases observed .

Statistics:

9. TR values were calculated
10. TR values were calculated

Species / strain:

Chinese hamster lung (CHL/IU)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

not specified

Remarks:

Study 9,10

Additional information on results:

8.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.
9.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 maximum dose of each sample was selected by a preliminary test in which the dose needed for 50% cell-growth inhibition was estimated using a cell densitometer
COMPARISON WITH HISTORICAL CONTROL DATA: No data ADDITIONAL INFORMATION ON CYTOTOXICITY: No data
10.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 maximum dose of each sample was selected by a preliminary test in which the dose needed for 50% cell-growth inhibition was estimated using a cell densitometer
COMPARISON WITH HISTORICAL CONTROL DATA: No data ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

Remarks on result:

other: not mutagenic

Conclusions:

Thus, based on the available studies and applying the weight of evidence approach, it can be concluded that the given test chemical did not induce chromosomal aberrations in Chinese hamster fibroblast cell line and hence it is not likely to classify as a gene mutant in vitro.

Executive summary:

In different studies, the given test chemical has been investigated for the mutagenic nature. 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 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.

This is supported by the results of another study where the effects of the test chemical were studied to understand the incidence of chromosomal aberration in a Chinese hamster fibroblast cell line, CHL. The cell line was originally established from the lung of a new born female at the Cancer Research Institute, Tokyo, and was maintained by 4-day passages in Minimum Essential Medium (MEM; GIBCO) supplemented by 100% calf serum . The modal chromosome number was 25 and the doubling time was approximately 15 hr. The cells were exposed to each sample at three different doses for 24 and 48 hr. In the present studies, no metabolic activation systems were applied. The maximum dose of each sample was selected by a preliminary test in which the dose needed for 50% cell-growth inhibition was estimated using a cell densitometer (Monocellater, Olympus Co., Ltd).The maximum dose for the test chemical based on preliminary study was 2 mg/ml .Chromosome preparations were made as follows: Colcemid (final concentration 0.2 μg/ml) was added to the culture 2 hr before cell harvesting. The cells were then trypsinized and suspended in a hypotonic KCl solution (0 .075 M) for 13 min at room temperature. After centrifugation the cells were fixed with acetic acid-methanol (1:3, v/v) and spread on clean glass slides. After air-drying, the slides were stained with Giemsa solution (1 .5%, at pH 6.8) for 12-15 min. A hundred well-spread metaphases were observed under the microscope (x 600 with a no cover objective lens). The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. Untreated cells and solvent-treated cells served as negative controls, in which the incidence of aberrations was usually less than 3 .0%. The results were considered to be negative if the incidence was less than 4 .9%, equivocal if it was between 5 .0 and 9.9%, and positive if it was more than 10 .0%. When no reasonable dose-response relationships were found, additional experiments were carried out at similar dose levels. For a quantitative evaluation of the clastogenic potential of the positive samples, the D20 was calculated, which is the dose (mg/ml) at which structural aberrations (including gaps) were detected in 20% of the metaphases observed. In addition, the TR value was calculated. The results indicated that there was no significant incidence of polyploid conditions in the exposed cultures. Based on the obtained outcomes it is expected that the test chemical is not likely to induce the chromosomal aberrations.

The above results are further supported by a similar study performed to understand the effect of test chemical on the incidence of chromosomal aberration in a Chinese hamster fibroblast cell line, CHL. The cell line was originally established from the lung of a new born female at the Cancer Research Institute, Tokyo, and was maintained by 4-day passages in Minimum Essential Medium (MEM; GIBCO) supplemented by 100% calf serum . The modal chromosome number was 25 and the doubling time was approximately 15 hours. The cells were exposed to each sample at three different doses for 24 and 48 hr. In the present studies, no metabolic activation systems were applied. The maximum dose of each sample was selected by a preliminary test in which the dose needed for 50% cell-growth inhibition was estimated using a cell densitometer (Monocellater, Olympus Co, Ltd).The maximum dose for the test chemical based on preliminary study was 2 mg/ml .Chromosome preparations were made as follows: Colcemid (final concentration 0.2 μg/ml) was added to the culture 2 hr before cell harvesting. The cells were then trypsinized and suspended in a hypotonic KCl solution (0 .075 M) for 13 min at room temperature. After centrifugation the cells were fixed with acetic acid-methanol (1:3, v/v) and spread on clean glass slides. After air-drying, the slides were stained with Giemsa solution (1 .5%, at pH 6.8) for 12-15 min. A hundred well-spread metaphases were observed under the microscope (x 600 with a no cover objective lens). The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. Untreated cells and solvent-treated cells served as negative controls, in which the incidence of aberrations was usually less than 3 .0%. The results were considered to be negative if the incidence was less than 4 .9%, equivocal if it was between 5 .0 and 9.9%, and positive if it was more than 10 .0%. When no reasonable dose-response relationships were found, additional experiments were carried out at similar dose levels. For a quantitative evaluation of the clastogenic potential of the positive samples, the D20 was calculated, which is the dose (mg/ml) at which structural aberrations (including gaps) were detected in 20% of the metaphases observed. In addition, the TR value was calculated. The results indicated that there was no significant incidence of polyploid conditions in the exposed cultures. Based on the obtained outcomes it is expected that the test chemical is not likely to induce the chromosomal aberrations.

Thus, based on the available studies and applying the weight of evidence approach, it can be concluded that the given test chemical did not induce chromosomal aberrations in Chinese hamster fibroblast cell line and hence it is not likely to classify as a gene mutant in vitro.

Reference 3

Endpoint:

in vitro gene mutation 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:

Weight of evidence approach based on various test chemicals

Justification for type of information:

Weight of evidence approach based on various test chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: Weight of evidence approach based on various test chemicals

Principles of method if other than guideline:

Weight of evidence approach based on various test chemicals

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

12. 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.
12. Thymidine kinase (Tk)

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

12

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

Species / strain / cell type:

mouse lymphoma L5178Y cells

Remarks:

12

Details on mammalian cell type (if applicable):

For cell lines:
- Methods for maintenance in cell culture:
The TK+/- -3 .7 .2C heterozygote of the L5178Y mouse lymphoma cell line was maintained in Fischer's medium containing 10% horse serum, antibiotics, glutamine, sodium pyruvate, and Pluronic F68 .

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

12. S9 liver microsomal fraction obtained from Arcolor 1254-induced male Sprague-Dawley rats (Supplier: Molecular Toxicology Inc. via Trinova Biochem GmbH, Giessen, Germany)
13. Aroclor 1254-induced rat liver S9 activation system.

Test concentrations with justification for top dose:

12. 0, 0.5, 1.0, 2.5 or 5.0 mM
13. 1000, 1500 μg/ml

Vehicle / solvent:

12. Vehicle(s)/solvent(s) used: Ethanol
Justification for choice of solvent/ vehicle: Methyl phenylacetate was easily dissolved in ethanol.
13. No data

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

other: N-ethyl-N-nitrosourea (ENU) - without S9

Remarks:

12

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Remarks:

13

Details on test system and experimental conditions:

12. 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.
12. METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 3x10^6 cells
- Test substance added in medium - in agar

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:No data
- Exposure duration/duration of treatment: 4 hour exposure period
- Harvest time after the end of treatment (sampling/recovery times):

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection):
- Selection time (if incubation with a selective agent):No data
- Fixation time (start of exposure up to fixation or harvest of cells):No data
- Method used: agar or microwell plates for the mouse lymphoma assay.
- If a selective agent is used - the cells were washed and incubated at 37° C for 48 hours to allow phenotypic expression before cloning 3x10^6 cells in Noble agar containing the selective agent trifluorothymidine or bromodeoxyuridine.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells:
Colonies were counted after 10-14 days' growth using an automatic colony counter
- Criteria for small (slow growing) and large (fast growing) colonies: No data

Rationale for test conditions:

No data

Evaluation criteria:

12. The plates were scored for total number of colonies
13. Mutant frequency was determined by calculating the ratio of mutant to viable colonies cloned without selective medium .

Statistics:

No data available

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

12

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

not valid

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

13

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

True negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

12. 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
13. No data

Remarks on result:

other: No mutagenic potential

Conclusions:

Based on the available studies and applying the weight of evidence approach, it can be concluded that the given test chemical did not induce mutation in mammalian cell line in the presence and absence of metabolic activation and hence it is not likely to classify as a gene mutant in vitro.

Executive summary:

In different studies, the given test chemical has been investigated for the mutagenic nature. The studies are as mentioned below:

 

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of the given test chemical when administered to Chinese Hamster Ovary (CHO) cells. A preliminary dose-finding study was conducted prior to the main study. A range of different test concentrations were tested in 96-well plates and analyzed by two commonly used assays, i.e. the colorimetric assay of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and the bicinchoninic acid (BCA) assay to assess cell viability and protein concentration, respectively. From the basis of the results from the MTT and BCA assays, test concentrations of the test chemical was chosen to be included in the gene toxicity test. In the genotoxicity test, chemical was administered to CHO cells for 3 hrs at the dose levels of 0, 0.5, 1.0, 2.5 or 5.0 mM and in the absence or presence of exogenous metabolic activation. CHO cells representing the negative controls were exposed to the vehicle. Positive controls, such as N-ethyl-N-nitrosourea (ENU) experiments without metabolic activation and 7,12-dimethylbenz(a) anthracene in experiments with metabolic activation, were also included in each test. The results showed indication of gene mutations occurring in the positive controls ENU and 7,12-dimethylbenz(a) anthracene while no other treatment gave rise to gene toxicity. Two very diffuse colonies were seen in one well out of four at 2.5 mM in the absence with 4% S9 liver microsomal fraction. These diffuse colonies are not regarded to be relevant since the spots were only mildly colored by crystal violet, thus indicating that it were small clusters of apoptotic cells taking their last breath instead of cells surviving the TG-selection. No cytotoxic effects were observed when CHO cells were exposed to test chemical for 3 hrs. Based on the results of the study, it can be concluded that the given test chemical does not give rise to gene mutations when exposed at ≤ 5.0 mM for 3 hrs or more, and does not induce cytotoxic effects at concentrations of ≤ 5.0mM.

 

In another study, the Mouse Lymphoma cell mutagenesis (MLY) assay was performed by using the given test chemical on TK+/- -3.7.2C heterozygote of the L5178Y mouse lymphoma cell line maintained in Fischer's medium containing 10% horse serum, antibiotics, glutamine, sodium pyruvate, and Pluronic F68 with and without Aroclor 1254-induced rat liver S9 activation system at 1000, 1500 μg/ml. In a typical assay procedure, the thymidine kinase competent heterozygote was exposed to the test chemical in both the presence and absence of an induced rat liver S9 and cofactors (CORE). After a 4 hour exposure period, the cells were washed and incubated at 37° C for 48 hours to allow phenotypic expression before cloning 3x10^6 cells in Noble agar containing the selective agent trifluorothymidine or bromodeoxyuridine. Colonies were counted after 10-14 days growth using an automatic colony counter. Mutant frequency was determined by calculating the ratio of mutant to viable colonies cloned without selective medium. The given test chemical shows negative result in Mouse Lymphoma Forward Mutation Assay on Mouse lymphoma L5178Y tk ± cells with and without S9 metabolic activation. This indicates that the substance is not mutagenic in nature.

Based on the available studies and applying the weight of evidence approach, it can be concluded that the given test chemical did not induce mutation in mammalian cell line in the presence and absence of metabolic activation and hence it is not likely to 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

Ames Assay

In different studies, the given test chemical has been investigated for the mutagenic nature. The studies are as mentioned below:

The test chemicals found mutagenic in REC assay were evaluated using Salmonella Typhimurium strains TA 98, TA 100. The maximum dose of test chemical evaluated was 50 µg/plate.S9 mix treated with PCB EC-100- induced rat liver 9900g supernatant was used as the metabolic activation. The test chemical showed lethal effects on both strains when tested at the maximum dose 50 µg/plate without S9 activation system, so it was tested at lower dose of 10 µg/plate in the presence and absence of S9 activation system. The test chemical did not show any mutagenic effects when tested at the low concentration. Hence, the test chemical can be considered to non-mutagenic to Salmonella Typhimurium strains TA 98, TA 100 at 10 µg/plate in the presence and absence of S9 activation system.

 

This is supported by the results of another study where the mutagenicity of total particulate matter (TPM) containing the test material in cigarettes was investigated using an Ames assay protocol that conformed to OECD Guideline 471. For this purpose, prototype cigarettes containing a mixture of ingredients, reference cigarettes without these ingredients, and 2R4F cigarettes (a standard reference cigarette). 0-300 µg/ml (TPM Dose levels) of the test cigarette in which the concentration of the test chemical was around 6.5 ppm. The test chemical was tested with and without S9 metabolic activation in five strains of Salmonella typhimurium strains TA98, TA100, TA102, TA1535, and TA1537. Evaluation of the Ames assay data was carried out in terms of the mutagenic response, No sporadic responses in revertants were recorded. The highest sensitivity and specificity of the mutagenic response were observed using TA98 with metabolic activation. From the comparison of the data obtained for the test and reference cigarettes, it was concluded that the addition of ingredients did not result in a positive mutagenic response in any of the strains under the conditions already described. The test chemical can be considered to be non-mutagenic at a test concentration of 0.0065mg/l under the test conditions. Based on the observations made, test chemical did not induce reversion of gene mutation in the Salmonella typhimrium strains TA98, TA100, TA102, TA1535 and TA1537 with and without S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

The above results are supported by an AMES assay performed according to OECD 471 Guidelines(1997) to determine the mutagenic potential of the test chemical.Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 were used for the study.Doses tested in the mutagenicity assay were selected based on results of the dose range finding 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 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 which in this study was 5000 microgram/plate. S9 Homogenate (Aroclor) in S9 Mix was used as the metabolic activation system.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.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.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).

These results are further supported by a Gene mutation toxicity study performed to determine the mutagenic nature of test chemical using pre-incubation assay. The study was performed using Salmonella typhimurium strain TA97, TA98, TA1535 and TA100 with and without 10% and 30% rat liver and hamster liver S9 metabolic activation system. The study was performed at dose level of 0, 0.3, 1, 3, 10, 33, 66, 100, 333, 1000, 3333 or 10000 µg/plate for TA100 and TA98 and at a dose level of 0, 0.3, 1, 3, 10, 33, 100, 333, 1000, 3333 or 10000 µg/plate for TA1535 and TA97 with DMSO as the solvent. The plates were observed for a dose dependent increase in the number of revertants/plate. Test chemical did not induce a dose dependent increase in the number of revertants per plate and hence exhibited no mutagenic activity under the given test conditions.

Thus, based on the available studies and applying the weight of evidence approach, it can be concluded that the given test chemical did not induce mutations in the Salmonella typhimurium strains tested with and without metabolic activation system hence it is not likely to classify as a gene mutant in vitro.

 

DNA damage and repair assay

In different studies, the given test chemical has been investigated for the mutagenic nature. The studies are as mentioned below:

DNA damage and/or repair study was performed to determine the genetic toxicity for test chemical using Bacillus subtilis strains H17(rec+) and M45 (rec-) by Rec assay. The test chemical was used at the concentration of 20 μl/disk. No Zone of inhibition was observed when 20 μl/disk of the test chemical was incubated with the Bacillus subtilis strains H17 (rec+) and M45 (rec-) in the Rec assay. Hence, the test chemical can be considered to be non-mutagenic.

This is supported by the another similar Rec Assay where Bacillus Subtilis strains H17(rec+) and M45 (rec-) were exposed to test chemical in the concentration of 20 µg/disc by Rec assay. The results showed that there was significant evidence of genotoxicity after treatment. Difference in the length between the inhibition zone with the H17 and M45 strains were observed. Based on the observations made, it was considered that the test chemical showed positive results in Rec assay.

Eventhough one study claims that the test chemical showed positive results in Rec assay at 0.2 and 2.2 µg/disc but when tested at 20 μl/disk the test chemical showed negative results. Hence, the test chemical can be considered to be non-mutagenic in nature.

In vitro chromosomal Aberration study

In different studies, the given test chemical has been investigated for the mutagenic nature. 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 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.

This is supported by the results of another study where the effects of the test chemical were studied to understand the incidence of chromosomal aberration in a Chinese hamster fibroblast cell line, CHL. The cell line was originally established from the lung of a new born female at the Cancer Research Institute, Tokyo, and was maintained by 4-day passages in Minimum Essential Medium (MEM; GIBCO) supplemented by 100% calf serum . The modal chromosome number was 25 and the doubling time was approximately 15 hr. The cells were exposed to each sample at three different doses for 24 and 48 hr. In the present studies, no metabolic activation systems were applied. The maximum dose of each sample was selected by a preliminary test in which the dose needed for 50% cell-growth inhibition was estimated using a cell densitometer (Monocellater, Olympus Co., Ltd).The maximum dose for the test chemical based on preliminary study was 2 mg/ml .Chromosome preparations were made as follows: Colcemid (final concentration 0.2 μg/ml) was added to the culture 2 hr before cell harvesting. The cells were then trypsinized and suspended in a hypotonic KCl solution (0 .075 M) for 13 min at room temperature. After centrifugation the cells were fixed with acetic acid-methanol (1:3, v/v) and spread on clean glass slides. After air-drying, the slides were stained with Giemsa solution (1 .5%, at pH 6.8) for 12-15 min. A hundred well-spread metaphases were observed under the microscope (x 600 with a no cover objective lens). The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. Untreated cells and solvent-treated cells served as negative controls, in which the incidence of aberrations was usually less than 3 .0%. The results were considered to be negative if the incidence was less than 4 .9%, equivocal if it was between 5 .0 and 9.9%, and positive if it was more than 10 .0%. When no reasonable dose-response relationships were found, additional experiments were carried out at similar dose levels. For a quantitative evaluation of the clastogenic potential of the positive samples, the D20 was calculated, which is the dose (mg/ml) at which structural aberrations (including gaps) were detected in 20% of the metaphases observed. In addition, the TR value was calculated. The results indicated that there was no significant incidence of polyploid conditions in the exposed cultures. Based on the obtained outcomes it is expected that the test chemical is not likely to induce the chromosomal aberrations.

The above results are further supported by a similar study performed to understand the effect of test chemical on the incidence of chromosomal aberration in a Chinese hamster fibroblast cell line, CHL. The cell line was originally established from the lung of a new born female at the Cancer Research Institute, Tokyo, and was maintained by 4-day passages in Minimum Essential Medium (MEM; GIBCO) supplemented by 100% calf serum . The modal chromosome number was 25 and the doubling time was approximately 15 hours. The cells were exposed to each sample at three different doses for 24 and 48 hr. In the present studies, no metabolic activation systems were applied. The maximum dose of each sample was selected by a preliminary test in which the dose needed for 50% cell-growth inhibition was estimated using a cell densitometer (Monocellater, Olympus Co, Ltd).The maximum dose for the test chemical based on preliminary study was 2 mg/ml .Chromosome preparations were made as follows: Colcemid (final concentration 0.2 μg/ml) was added to the culture 2 hr before cell harvesting. The cells were then trypsinized and suspended in a hypotonic KCl solution (0 .075 M) for 13 min at room temperature. After centrifugation the cells were fixed with acetic acid-methanol (1:3, v/v) and spread on clean glass slides. After air-drying, the slides were stained with Giemsa solution (1 .5%, at pH 6.8) for 12-15 min. A hundred well-spread metaphases were observed under the microscope (x 600 with a no cover objective lens). The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. Untreated cells and solvent-treated cells served as negative controls, in which the incidence of aberrations was usually less than 3 .0%. The results were considered to be negative if the incidence was less than 4 .9%, equivocal if it was between 5 .0 and 9.9%, and positive if it was more than 10 .0%. When no reasonable dose-response relationships were found, additional experiments were carried out at similar dose levels. For a quantitative evaluation of the clastogenic potential of the positive samples, the D20 was calculated, which is the dose (mg/ml) at which structural aberrations (including gaps) were detected in 20% of the metaphases observed. In addition, the TR value was calculated. The results indicated that there was no significant incidence of polyploid conditions in the exposed cultures. Based on the obtained outcomes it is expected that the test chemical is not likely to induce the chromosomal aberrations.

Thus, based on the available studies and applying the weight of evidence approach, it can be concluded that the given test chemical did not induce chromosomal aberrations in Chinese hamster fibroblast cell line and hence it is not likely to classify as a gene mutant in vitro.

In vitro gene mutation study in mammalian cells

In different studies, the given test chemical has been investigated for the mutagenic nature. The studies are as mentioned below:

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of the given test chemical when administered to Chinese Hamster Ovary (CHO) cells. A preliminary dose-finding study was conducted prior to the main study. A range of different test concentrations were tested in 96-well plates and analyzed by two commonly used assays, i.e. the colorimetric assay of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and the bicinchoninic acid (BCA) assay to assess cell viability and protein concentration, respectively. From the basis of the results from the MTT and BCA assays, test concentrations of the test chemical was chosen to be included in the gene toxicity test. In the genotoxicity test, chemical was administered to CHO cells for 3 hrs at the dose levels of 0, 0.5, 1.0, 2.5 or 5.0 mM and in the absence or presence of exogenous metabolic activation. CHO cells representing the negative controls were exposed to the vehicle. Positive controls, such as N-ethyl-N-nitrosourea (ENU) experiments without metabolic activation and 7,12-dimethylbenz(a) anthracene in experiments with metabolic activation, were also included in each test. The results showed indication of gene mutations occurring in the positive controls ENU and 7,12-dimethylbenz(a) anthracene while no other treatment gave rise to gene toxicity. Two very diffuse colonies were seen in one well out of four at 2.5 mM in the absence with 4% S9 liver microsomal fraction. These diffuse colonies are not regarded to be relevant since the spots were only mildly colored by crystal violet, thus indicating that it were small clusters of apoptotic cells taking their last breath instead of cells surviving the TG-selection. No cytotoxic effects were observed when CHO cells were exposed to test chemical for 3 hrs. Based on the results of the study, it can be concluded that the given test chemical does not give rise to gene mutations when exposed at ≤ 5.0 mM for 3 hrs or more, and does not induce cytotoxic effects at concentrations of ≤ 5.0mM.

In another study, the Mouse Lymphoma cell mutagenesis (MLY) assay was performed by using the given test chemical on TK+/- -3.7.2C heterozygote of the L5178Y mouse lymphoma cell line maintained in Fischer's medium containing 10% horse serum, antibiotics, glutamine, sodium pyruvate, and Pluronic F68 with and without Aroclor 1254-induced rat liver S9 activation system at 1000, 1500 μg/ml. In a typical assay procedure, the thymidine kinase competent heterozygote was exposed to the test chemical in both the presence and absence of an induced rat liver S9 and cofactors (CORE). After a 4 hour exposure period, the cells were washed and incubated at 37° C for 48 hours to allow phenotypic expression before cloning 3x10^6 cells in Noble agar containing the selective agent trifluorothymidine or bromodeoxyuridine. Colonies were counted after 10-14 days growth using an automatic colony counter. Mutant frequency was determined by calculating the ratio of mutant to viable colonies cloned without selective medium. The given test chemical shows negative result in Mouse Lymphoma Forward Mutation Assay on Mouse lymphoma L5178Y tk ± cells with and without S9 metabolic activation. This indicates that the substance is not mutagenic in nature.

 

Thus, based on the available studies and applying the weight of evidence approach, it can be concluded that the given test chemical did not induce mutation in mammalian cell line in the presence and absence of metabolic activation and hence it is not likely to classify as a gene mutant in vitro.

Justification for classification or non-classification

Based on the available results, the test chemical is not likely to be mutagenic in nature. It can be further classified under the category "Not Classified" as per CLP Regulation.