Methyl anthranilate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames Assay:

The test chemical is expected to not induce a reproducible, dose-related increase in his+ revertants over the corresponding solvent in the S. typhimurium tester strains TA102, TA1535, TA1537, TA 100 and TA 98 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

 

Chromosomal Abberation:

Based on all the available data and from the observations and results, it was concluded that the test chemical did not induce any clastogenicity and did not cause chromosomal aberrations when tested under optimum conditions.

In Vitro Mammalian Cell Mutagenicity Assay:

Based on all the available data and from the observations and results, it was concluded that the test chemical did not induce any cell gene mutations when tested under optimum conditions in in vitro mammalian cell gene mutation assay.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from publication

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Determination of Gene mutation toxicity study of the test chemical.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

The S-9 mixes were prepared immediately prior to use and contained 10% RLI and 10% HLI respectively.
RLI = induced male Sprague Dawley rat liver S9
HLI = induced male Syrian hamster liver S9

- source of S9 : Male Sprague-Dawley rats and male Syrian hamsters

- method of preparation of S9 mix : Aroclor 1254 (200 mg/ml in corn oil) was administered ip at 500 mg/kg 5 days prior to decapitation (EGG, SRI) or cervical dislocation (CWR). The animals were deprived of food 12-24 hr immediately preceding death; otherwise food and water were provided ad libitum. The livers were removed aseptically, washed in ice-cold 0.15 M KCl, and minced and homogenized (3 ml of 0.15 M KC1 per gm of wet tissue) in a Potter-Elvehjem apparatus with a Teflon pestle. CWR initially used a Waring blender, but switched to a Potter-Elvehjem apparatus. The S-9 fraction was obtained by centrifugation of the liver homogenate for 10 min at 9,000 g at 4°C. The S-9 fraction was dispensed into freezing ampules and stored in a -70°C freezer, or in liquid nitrogen.

- concentration or volume of S9 mix and S9 in the final culture medium : 0.5 ml

- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): No data

Test concentrations with justification for top dose:

0.000, 33.000, 100.000, 333.000, 1000.000 and 1800.000 µg/plate

Vehicle / solvent:

Genetox: - 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

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 4-nitro-o-phenylenediamine for TA98, 2-aminoanthracene for all strains

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 mins
- Exposure duration: 2 days
- Expression time (cells in growth medium): 2 days
- 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: At least five doses of the chemical were tested in triplicate. Experiments were repeated at least 1 wk following the initial trial

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:
Preincubation method: The test chemical was assayed for mutagenicity in the preincubation assay. To each of 13 X 100-mm test tubes maintained at 37°C were added in the following order: 0.5 ml of S-9 mix or 0.1 M PO4 buffer (pH 7.4), 0.05 ml of the overnight culture, and 0.05 ml of solvent or chemical dilution. The mixture was mixed and allowed to incubate without shaking at 37°C for 20 min, at which time 2.0 ml (CWR, SRI) of molten (45°C) top agar supplemented with 0.5 mM L-histidine and 0.5 mM D-biotin were added. The contents of the tubes were mixed and poured onto 25 ml of minimal glucose bottom agar [Vogel and Bonner, 19561 in 15 X 100-mm plastic petri dishes. When the top agar had solidified, the plates were inverted and incubated at 37°C for 48 hr. Concurrent solvent and positive controls were tested with and without the metabolic activation systems. At least five dose levels of the chemicals were tested, with three plates per dose level. AU assays were repeated (as described above) no less than 1 wk after completion of the initial test.

Preliminary Dose-Setting Experiment:
The test chemical was initially tested with strain TA100 in the presence and the absence of the metabolic activation systems, over a wide dose range with an upper limit of 10 mg/plate, or less when solubility problems were encountered. Toxicity was evidenced by one or more of the following phenomena: appearance of his pin point
colonies, reduced numbers of revertant colonies per plate, or thinning or absence of the bacterial lawn. Nontoxic chemicals were tested in the initial experiment up to the 10 mg/plate dose level, or to a level determined by their solubility. Toxic chemicals were tested up to a high dose which exhibited some degree of toxicity. As a rule, at least one toxic dose was incorporated into the first mutagenicity test; the repeat test occasionally had the doses adjusted so that an apparent toxic dose was not reached.

Rationale for test conditions:

No Data Available

Evaluation criteria:

The plates were observed for a dose related increase in the number of revertants
The criteria used for data evaluation can be summarized as follows:
1) mutagenic response: a dose-related, reproducible increase in the number of revertants over background, even if the increase was less than twofold;
2) non mutagenic response: when no increase in the number of revertants was elicited by the chemical;
3) questionable response: when there was an absence of a clear-cut dose-related increase in revertants; when the dose-related increases in the number of revertants were not reproducible; or when the response was of insufficient magnitude to support a determination of mutagenicity.

Statistics:

No data available

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Remarks:

Water

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

No data available

Remarks on result:

other: No mutagenic potenrial observed

Tables

1.

Dose	NA		TA100 10% HLI		10% RLI
µg/plate	Mean	SEM	Mean	SEM	Mean	SEM
0.000	145	10.2	118	5.6	138	8.6
33.000	132	8.5	126	15.3	143	4.2
100.000	132	5.5	130	1.7	118	9.6
333.000	141	6.1	119	10.0	131	6.6
1000.000	130	5.3	110	5.5	121	9.6
1800.000	t	t	t		t
Positive control	1399	21.3	706	10.5	901	20.2

2.

Dose	NA		TA1535 10% HLI		10% RLI
µg/plate	Mean	SEM	Mean	SEM	Mean	SEM
0.000	35	3.8	12	1.5	14	0.6
33.000	29	0.6	9	1.7	7	2.6
100.000	31	4.2	9	1.2	13	1.2
333.000	29	4.7	13	2.2	11	2.2
1000.000	13s	1.2	9	0.3	11	0.3
1800.000	t		t		t
Positive control	1316	14.5	64	8.7	108	5.5

3.

Dose	NA		TA1537 10% HLI		10% RLI
µg/plate	Mean	SEM	Mean	SEM	Mean	SEM
0.000	7	0.9	5	0.9	5	1.2
33.000	5	1.7	8	1.2	7	1.3
100.000	7	0.6	7	1.5	9	1.5
333.000	8	1.2	8	3.0	8	1.2
1000.000	2	0.9	8	1.5	8	0.9
1800.000	t		6s	0.5	5s	1.0
Positive control	171	3.7	100	7.0	81	6.2

 

4.

Dose	NA		TA98 10% HLI		10% RLI
µg/plate	Mean	SEM	Mean	SEM	Mean	SEM
0.000	18	2.2	24	2.0	29	2.2
33.000	14	1.5	26	0.9	29	0.3
100.000	18	1.0	27	2.0	26	2.1
333.000	17	2.0	28	0.6	27	0.9
1000.000	13	1.2	24	2.1	24	1.5
1800.000	t		14s	0.9	t
Positive control	1267	44.6	949	94.4	670	26.3

 

t= toxic

s= slight toxic

Conclusions:

The test chemical failed to induce mutation in the Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 in the presence and absence of S9 metabolic activation system and hence is not likely to classify for gene mutation in vitro.

Executive summary:

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium strains TA98, TA1537, TA100 and TA1535 in the presence and absence of S9 metabolic activation system. The chemical was dissolved in DMSO as solvent and used at dose levels 0, 33, 100, 333, 1000 and 1800 µg/plate by the preincubation method. The liver S-9 was isolated from Aroclor-induced male Sprague-Dawley rats and Syrian hamsters. Positive controls like sodium azide for TA1535 and TA 100, 4-nitro-o-phenylenediamine for TA98, and 9-aminoacridine for TA97 and TA1537; 2-aminoanthracene were used in the study. The test chemical failed to induce mutation in the Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 in the presence and absence of S9 metabolic activation system and hence is not likely to classify for gene mutation in vitro.

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:

experimental data of read across substances

Justification for type of information:

Weight of evidence approach based on the available data of the read-across 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: As mentioned below

Principles of method if other than guideline:

WoE report is based on Chromosomal abberation study for read across chemicals:

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

No data

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

5

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:

Chinese hamster Ovary (CHO)

Remarks:

6

Details on mammalian cell type (if applicable):

- Type and identity of media: CHO cells were maintained in McCoy’s 5A medium (modified) sup
plemented with L-glutamine (2 mM), antibiotics, and 10% fetal bovine serum (FBS).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: No data

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

other: Chinese hamster lung-derived fibroblasts (CHL)

Remarks:

7

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells:
The cell line was originally established from the lung of a newborn female at the Cancer Research Institute, Tokyo (Koyama, Utakoji & Ono, 1970),

For cell lines:
- Absence of Mycoplasma contamination:
No data
- Number of passages if applicable:
4-day passages
- Methods for maintenance in cell culture:
The cell line was maintained in Minimum Essential Medium (MEM; GIBCO) supplemented by 10% calf serum.
- Cell cycle length, doubling time or proliferation index :
The doubling time was approximately 15 hr.
- Modal number of chromosomes:
The modal chromosome number is 25

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

5. No data
6. No data
7. Colcemid (final conc 0.2 microgm/ml) was added to the culture 2 hr before cell harvesting.

Metabolic activation:

with and without

Metabolic activation system:

5. 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.
6. A liver fraction (S9) prepared from Aroclor 1254-induced male Sprague Dawley rats was used to provide exogenous metabolic activation
7. no metabolic activation systems were applied

Test concentrations with justification for top dose:

5. Without S9: 160-1600 µg/ ml
With S9: 500-5000 µg/ ml
6. Without S9:
Trial 1: 0, 0.16, 0.5, 1.6, 5.0 and 16.0 μg/ml
Trial 2: 0, 5, 10 and 20 μg/ml
With S9:
Trial 1: 0, 0.16, 0.5, 1.6, 5.0 and 16.0 μg/ml
Trail 2: 0, 10, 20, 30 and 40 μg/ml
7. 0, 0.25 mg/ml. Top dose was epxected to produce a 50% inhibition of cell growth based on data from a pre-experiment.

Vehicle / solvent:

5. Water, dimethyl sulfoxide (DMSO), ethanol, or Acetone (in the order of preference)
6. - Vehicle(s)/solvent(s) used: DMSO
7. Vehicle(s)/solvent(s) used: 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:

5

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:

6

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Remarks:

7

Details on test system and experimental conditions:

5. 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

6METHOD OF APPLICATION: in medium
Cells at the start of experiment: 1.2 x 10E6 cells
DURATION
- Preincubation period: Not applicable
- Exposure duration:Without S9: 2 hrs; With S9: 2 hrs
- Expression time (cells in growth medium): Without S9: 10 hrs; With S9: 11 hrs
- Harvest time after the end of treatment (sampling/recovery times):
Without S9: 12hr for trial 1 and 2
With S9: 11hr for trail 1 and 12 hr for trial 2
STAIN (for cytogenetic assays): 6% Giemsa stain
NUMBER OF CELLS EVALUATED: One hundred cells were scored for each dose. Only metaphase
cells in which the chromosome number was between 19 and 23 were scored.

7.DURATION
- Exposure duration: 24 & 48 hours
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
NUMBER OF CELLS EVALUATED: 100
DETERMINATION OF CYTOTOXICITY
- Method: 50% cell growth inhibition
OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Rationale for test conditions:

No data

Evaluation criteria:

5. 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).

6. The chromosome number was recorded for each cell and chromosome or chromatid type aberratio ns were classified into three categories: simple (breaks, fragments, double minutes), complex (interchanges, rearrangements), and other (pulverized, more than ten aberrations /cell).

7. Results were considered negative if incidence of aberrations was less than 4.9%, equivoval if it was between 5 and 9.9% and positive if it was more than 10%

Statistics:

5. 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.

6.The percentage of cells with aberrations was analyzed. Both the dose-response curve and individual dose points were statistically analyzed. A statistically significant (P < 0.003) trend test or a significantly elevated dose point (P < 0.05) was sufficient to indicate a chemical effect.

7. No data

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

5

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

6

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

other: Chinese hamster lung-derieved

Remarks:

7

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

not specified

Additional information on results:

5. 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.
6. RANGE-FINDING/SCREENING STUDIES: The dose levels for ABS studies were chosen based on the toxicity of the test chemical observed in the SCE studies.
7. No data

Remarks on result:

other: No mutagenic potential observed

Conclusions:

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.

Executive summary:

In vitro mammalian chromosome aberration test:

The In-Vitro chromosomal abberation test of the test chemical is as follows:

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

In another Study, In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of test chemical. The study was performed using Chinese hamster ovary cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at different dose levels. The study was performed on two trials with and without metabolic activation. For study with S9 activation dose range of 0, 0.16, 0.50, 1.6, 5.0 and 16.0 μg/plate were used in Trial 1 and dose range of 0, 5, 10 and 20 μg/plate were used in Trial 2 similarly for study without S9 activation dose range of 0, 0.16, 0.50, 1.6, 5.0 and 16.0 μg/plate were used in Trial 1 and dose range of 0, 10, 20, 30 and 40 μg/plate were used in Trial 2.Concurrent solvent and negative control chemicals were also included in the study. Approximately 24 hr prior to cell treatment, 1.2 x 106 cells were seeded per 75 cm2 flask. For assays without metabolic activation, the cells were treated for about 10 hr. Colcemid was added 2-3 hr prior to cell harvest by mitotic shake-off. In the test protocol for assays with metabolic activation cells were harvested approximately 11 hr and 12 hr and for assays without metabolic activation cells were harvested at 12hr. Colcemid was added 2 hr prior to harvest. Slides were stained in 6% Giemsa for 5-10 min. One hundred cells were scored for each dose in early studies and 200 cells per dose in later studies. All slides except high-dose positive controls were coded. Only metaphase cells in which the chromosome number was between 19 and 23 were scored. Based on the observations made, the test chemical did not induce chromosome aberration in Chinese hamster ovary cells 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 a similar study, In vitro mammalian chromosome aberration study was conducted for the given test chemical in Chinese hamster lung-derived fibroblasts (CHL) in the absence of metabolic activation system. The cell line was originally established from the lung of a newborn female at the Cancer Research Institute, Tokyo (Koyama, Utakoji & Ono, 1970), and was maintained by 4-day passages in Minimum Essential Medium (MEM; GIBCO) supplemented by 10% calf serum. The modal chromosome number is 25 and the doubling time was approximately 15 hr. The cells were exposed to chemical at three concentrations up to 0.25 mg/ml for 24 and 48 hr. DMSO was used as solvent. The top dose was expected to produce a 50% inhibition on cell growth based on data from a pre-experiment. Colcemid (final conc 0.2 microgm/ml) was added to the culture 2 hr before cell harvesting. The cells were then trypsinized and suspended in a hypotonic KCI 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; E. Merck) for 12-15 min. A hundred well-spread metaphases were observed under the microscope (x 600 with a nocover 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. Results were considered negative if incidence of aberrations was less than 4.9%, equivoval if it was between 5 and 9.9% and positive if it was more than 10%.The test chemical tested negative for mutachromosomal effects in Chinese hamster lung-derived fibroblasts (CHL).

Thus based on the observations madefrom the above studies, the test chemical did not induce chromosome aberration in Chinese hamster ovary cells 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.

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:

comparable to guideline study

Remarks:

experimental data of read across substances

Justification for type of information:

Data for the target chemical is summarized based on the structurally similar read across chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Principles of method if other than guideline:

To determine the mutagenic potential of test chemical from WoE report number 8 and 9.

GLP compliance:

not specified

Type of assay:

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

Target gene:

8.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.
9.Thymidine kinase

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Ham's F-12K (Kaighn's) Medium containing 2 mM L-Glutamine supplemented with 10% Fetal Bovine Serum and 1% Penicillin-Streptomycin (10,000 U/mL).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Not applicable
- Periodically checked for karyotype stability: Not applicable

Additional strain / cell type characteristics:

other: Hypodiploid, modal No. 20

Remarks:

8

Species / strain / cell type:

mouse lymphoma L5178Y cells

Remarks:

TK+/- 3.7.C

Details on mammalian cell type (if applicable):

- Type and identity of media: The cells were grown in Fischer’s medium for leukemic cells of mice supplemented with 10% horse serum and 0.02% pluronic F-68.
- Properly maintained: No data available
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: No data available
- Periodically "cleansed" against high spontaneous background: No data available

Additional strain / cell type characteristics:

not specified

Remarks:

9

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

8.S9 liver microsomal fraction obtained from Arcolor 1254-induced male Sprague-Dawley rats (Supplier: Molecular Toxicology Inc. via Trinova Biochem GmbH, Giessen, Germany)
9.Liver S9 fraction was prepared from Aroclor 1254-induced male Sprague- Dawley rats.

Test concentrations with justification for top dose:

8. 0, 0.5, 1.0, 2.5 or 5.0 mM
9. 5-52 µg/mL

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

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

Remarks:

8

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

methylmethanesulfonate

other: 3-methylcholanthrene at 1.86 × 10-5 M (or dimethylbenz[a]- anthracene at 0.5-4 µg/mL) for the test with metabolic activation.

Remarks:

9

Details on test system and experimental conditions:

8.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.
9.METHOD OF APPLICATION: in medium;
- Cell density at seeding (if applicable): 6000000 cells

DURATION
- Preincubation period: No data available
- Exposure duration: 4 h
- Expression time (cells in growth medium):48 h
- 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): 1×106 cells/plate for mutant selection
SPINDLE INHIBITOR (cytogenetic assays): No data available
STAIN (for cytogenetic assays): No data available

NUMBER OF REPLICATIONS: Duplicate

NUMBER OF CELLS EVALUATED: 1 X 106 cells/plate for mutant selection and 200
cells/plate for viable count determinations

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: The rate of cell growth was determined for each of the treated cultures

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

OTHER: No data available

Rationale for test conditions:

No data

Evaluation criteria:

8.The plates were scored for total number of colonies
9.Results were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase. Doubling of the mutant frequency was previously reported as representing a positive effect. Only doses yielding total growth values of 10% were used in the analysis of induced mutant frequency. Doses yielding less than 10% total growth were used in determining dose response.

Statistics:

No data available

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

8

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:

TK+/- 3.7.C

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Remarks:

9

Additional information on results:

8.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
9.No data

Remarks on result:

other: No mutagenic potential observed.

Conclusions:

The test chemical in the concentration of 0, 0.5, 1.0, 2.5 or 5.0 mM did not show any evidence of gene toxicity when CHO cells were exposed to the test chemical in the absence of S9 metabolic activation system and hence dose not classify for gene mutation in vitro.

Executive summary:

In different studies, the given test chemical has been investigated for mammalian cell mutation to a greater or lesser extent. The studies are summarized as 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.

The gene mutation study was conducted according to L5178Y TK+/-Mouse Lymphoma Mutagenicity Assay to determine the mutagenic nature of test chemical.

The Cells at a concentration of 6 X 10⁵/mL (6 X10⁶cells total) were exposed for 4 h to a range of concentrations from 5 -52 µg/mL of the test chemical. The cells were then washed, resuspended in growth medium, and incubated at 37°C for 48 h. The rate of cell growth was determined for each of the treated cultures and compared to the rate of growth of the solvent controls.Results were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase .The test chemical did not induce a doubling of the mutant frequency both in the presence and absence of S9 activation system and hence is not likely to be gene mutant in vitro. The gene mutation study was conducted according to L5178Y TK+/-Mouse Lymphoma Mutagenicity Assay to determine the mutagenic nature of test chemical.

Therefore based on above summarized studies it can be concluded that the test chemical in not mutagenic to mammalian cell line and thus the test chemical can be classified as 'Not Mutagenic' as per CLP regulation.

Reference 4

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Data is from peer reviewed publication

Justification for type of information:

Data is from peer reviewed publication

Qualifier:

according to guideline

Guideline:

other: As mention below

Principles of method if other than guideline:

The genotoxicity of test chemical, was examined by a DNA repair test with rat hepatocytes.

GLP compliance:

not specified

Type of assay:

DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro

Target gene:

No data

Species / strain / cell type:

hepatocytes: ACI rat hepatocytes

Details on mammalian cell type (if applicable):

- Type and identity of media: Williams' Medium E
- Properly maintained: No data
- Periodically checked for Mycoplasma contamination: No data
- Periodically checked for karyotype stability: No data
- Periodically “cleansed” against high spontaneous background: No data

Additional strain / cell type characteristics:

not specified

Metabolic activation:

not specified

Metabolic activation system:

No data

Test concentrations with justification for top dose:

10-3, 10-4, 10-5, 10-6 M

Vehicle / solvent:

DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: N-2-fluorenylacetamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: No data
- Exposure duration: 20 hrs
- Expression time (cells in growth medium): 20 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: Duplicate

NUMBER OF CELLS EVALUATED: 50 cells/coverslip

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: The test was performed in accordance with the method of Williams et al. Hepatocytes were isolated from the livers of male ACI rats weighing 200-250 g. The isolated hepatocytes were allowed to attach for 2 h on plastic coverslips in primary culture using Williams' Medium E. The cultures were then washed and exposed to the test chemical and [Me- 3H]thymidine (10 µCi/ml; 49 Ci/mmole) for 20 h. It was logarithmically diluted before addition to the cultures. At the end of incubation, the cultures were washed, and the coverslips were mounted on glass slides. The slides were dipped in Sakura NR-M2 photographic emulsion and exposed for 14 days. Autoradiographic grains were counted on a television screen (Olympus, type S) with a microscopic attachment.

The data were expressed as the average net counts/nucleus for 3 coverslips + the standard deviation (50 cells/coverslip). Two experiments were performed for the test compound for the assays with rat hepatocytes.

Evaluation criteria:

The test chemical was considered positive when the mean net nuclear grain count was more than 5 grains above background and statistically greater than that of controls (unpaired t-test; P < 0.01).

Statistics:

Mean ± standard deviation

Species / strain:

hepatocytes: ACI rat hepatocytes

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

No data available

Remarks on result:

other:

Chemical	Dose M	UDS grains/nucleus	% od UDS positive cells	DNA repair
Test chemical	10^-3	0.5 ± 1.5	0	-
	10^-4	0.3 ± 1.0	0
	10^-5	-0.4 ± 1.1	0
	10^-6	-0.3 ± 0.9	0

Conclusions:

The given test material is negative in the rat hepatocyte/DNA repair test.

Executive summary:

The hepatocyte/DNA repair test which measures unscheduled DNA synthesis (UDS) is known to be sensitive to various classes of DNA-reactive carcinogens and is regarded as a reliable short-term test for the detection of chemical carcinogens.The genotoxicity of test substance, was examined by a DNA repair test with rat hepatocytes.The test was performed basically in accordance with the method of Williams et al. The test material was dissolved in DMSO and the positive control used was N-2-fluorenylacetamide. The hepatocytes were exposed to the test chemical for 20 hrs. At the end of incubation, the cultures were washed, and the coverslips were mounted on glass slides. The slides were dipped in Sakura NR-M2 photographic emulsion and exposed for 14 days. Autoradiographic grains were counted on a television screen (Olympus, type S) with a microscopic attachment. The results of the hepatocyte/DNA repair test suggests that the test chemical is negative for genotoxicity in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Gene mutation in vitro:

Ames Assay:

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

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium strains TA98, TA1537, TA100 and TA1535 in the presence and absence of S9 metabolic activation system. The chemical was dissolved in DMSO as solvent and used at dose levels 0, 33, 100, 333, 1000 and 1800 µg/plate by the preincubation method. The liver S-9 was isolated from Aroclor-induced male Sprague-Dawley rats and Syrian hamsters. Positive controls like sodium azide for TA1535 and TA 100, 4-nitro-o-phenylenediamine for TA98, and 9-aminoacridine for TA97 and TA1537; 2-aminoanthracene were used in the study. The test chemical failed to induce mutation in the Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 in the presence and absence of S9 metabolic activation system and hence is not likely to classify for gene mutation in vitro.

Gene mutation in vitro study was performed for the test chemical using S typhimurium strain TA98 and TA100 in the presence and absence of S9 metabollic activation system. Rat-liver microsome (S9) was prepared from Sprague-Dawley rats treated with Aroclor 1254 by the method of Ames (Ames et al., 1975). The test chemical was dissolved in and diluted with sterilized dimethyl sulfoxide (DMSO). A dose level of 0.05 to 500 µg/plate was used during the study. The mutagenicity test was conducted in the Salmonella/microsome mutagenicity assay on plates according to the method of Ames (Ames et al., 1975), with the S. typhimurium histidine (-) mutants, TA98 and TA100. Positive control substances like Afl B1, B[a]P, 2-NF and 4NQ0 showed considerable mutagenic potency as positive standard mutagens. The test chemical induced a number of revertants that was over half the number of spontaneous revertants of TA98 or TA100 at the indicated dose ranges, either with or without S9 mix. These results show that the test chemical has negative mutagenic potency against S. typhimurium TA98 and TA100 strains indicating that the test chemical is not mutagenic in nature.

Gene mutation toxicity study was performed to evaluate the mutagenic nature of the test chemical . Preincubation assay was performed at dose levels of 3 -1000 µg/plate inSalmonella typhimuriumstrains TA102, TA1535, TA1537 with and without S9 metabolic activation system. S9 was obtained from rat liver homogenate. Histidine was used as a target gene in the study. Increase in the number of revertants was noted. In the pre-incubation assay, cytotoxicity was observed at 333 and 1000 µg/plate (+/-S9) in all the strains, except in strain TA102 at 333µg/plate +S9. Thus the test chemical is not likely to classify as a gene mutant in vitro.

Chromosomal Abberation:

In vitro mammalian chromosome aberration test:

The In-Vitro chromosomal abberation test of the test chemical is as follows:

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

In another Study, In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of test chemical. The study was performed using Chinese hamster ovary cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at different dose levels. The study was performed on two trials with and without metabolic activation. For study with S9 activation dose range of 0, 0.16, 0.50, 1.6, 5.0 and 16.0 μg/plate were used in Trial 1 and dose range of 0, 5, 10 and 20 μg/plate were used in Trial 2 similarly for study without S9 activation dose range of 0, 0.16, 0.50, 1.6, 5.0 and 16.0 μg/plate were used in Trial 1 and dose range of 0, 10, 20, 30 and 40 μg/plate were used in Trial 2.Concurrent solvent and negative control chemicals were also included in the study. Approximately 24 hr prior to cell treatment, 1.2 x 106 cells were seeded per 75 cm2 flask. For assays without metabolic activation, the cells were treated for about 10 hr. Colcemid was added 2-3 hr prior to cell harvest by mitotic shake-off. In the test protocol for assays with metabolic activation cells were harvested approximately 11 hr and 12 hr and for assays without metabolic activation cells were harvested at 12hr. Colcemid was added 2 hr prior to harvest. Slides were stained in 6% Giemsa for 5-10 min. One hundred cells were scored for each dose in early studies and 200 cells per dose in later studies. All slides except high-dose positive controls were coded. Only metaphase cells in which the chromosome number was between 19 and 23 were scored. Based on the observations made, the test chemical did not induce chromosome aberration in Chinese hamster ovary cells 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 a similar study, In vitro mammalian chromosome aberration study was conducted for the given test chemical in Chinese hamster lung-derived fibroblasts (CHL) in the absence of metabolic activation system. The cell line was originally established from the lung of a newborn female at the Cancer Research Institute, Tokyo (Koyama, Utakoji & Ono, 1970), and was maintained by 4-day passages in Minimum Essential Medium (MEM; GIBCO) supplemented by 10% calf serum. The modal chromosome number is 25 and the doubling time was approximately 15 hr. The cells were exposed to chemical at three concentrations up to 0.25 mg/ml for 24 and 48 hr. DMSO was used as solvent. The top dose was expected to produce a 50% inhibition on cell growth based on data from a pre-experiment. Colcemid (final conc 0.2 microgm/ml) was added to the culture 2 hr before cell harvesting. The cells were then trypsinized and suspended in a hypotonic KCI 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; E. Merck) for 12-15 min. A hundred well-spread metaphases were observed under the microscope (x 600 with a nocover 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. Results were considered negative if incidence of aberrations was less than 4.9%, equivoval if it was between 5 and 9.9% and positive if it was more than 10%.The test chemical tested negative for mutachromosomal effects in Chinese hamster lung-derived fibroblasts (CHL).

Thus based on the observations madefrom the above studies, the test chemical did not induce chromosome aberration in Chinese hamster ovary cells 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 gene mutation study in mammalian cells:

In different studies, the given test chemical has been investigated for mammalian cell mutation to a greater or lesser extent. The studies are summarized as 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.

The gene mutation study was conducted according to L5178Y TK+/-Mouse Lymphoma Mutagenicity Assay to determine the mutagenic nature of test chemical.

The Cells at a concentration of 6 X 10⁵/mL (6 X10⁶cells total) were exposed for 4 h to a range of concentrations from 5 -52 µg/mL of the test chemical. The cells were then washed, resuspended in growth medium, and incubated at 37°C for 48 h. The rate of cell growth was determined for each of the treated cultures and compared to the rate of growth of the solvent controls.Results were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase .The test chemical did not induce a doubling of the mutant frequency both in the presence and absence of S9 activation system and hence is not likely to be gene mutant in vitro. The gene mutation study was conducted according to L5178Y TK+/-Mouse Lymphoma Mutagenicity Assay to determine the mutagenic nature of test chemical.

Therefore based on above summarized studies it can be concluded that the test chemical in not mutagenic to mammalian cell line and thus the test chemical can be classified as 'Not Mutagenic' as per CLP regulation.

In vitro DNA damage and/or repair study:

The hepatocyte/DNA repair test which measures unscheduled DNA synthesis (UDS) is known to be sensitive to various classes of DNA-reactive carcinogens and is regarded as a reliable short-term test for the detection of chemical carcinogens.The genotoxicity of test substance, was examined by a DNA repair test with rat hepatocytes.The test was performed basically in accordance with the method of Williams et al. The test material was dissolved in DMSO and the positive control used was N-2-fluorenylacetamide. The hepatocytes were exposed to the test chemical for 20 hrs. At the end of incubation, the cultures were washed, and the coverslips were mounted on glass slides. The slides were dipped in Sakura NR-M2 photographic emulsion and exposed for 14 days. Autoradiographic grains were counted on a television screen (Olympus, type S) with a microscopic attachment. The results of the hepatocyte/DNA repair test suggests that the test chemical is negative for genotoxicity in vitro.

Justification for classification or non-classification

Based on the data available, 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.