(2-methoxyethyl)benzene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames assay:

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

 

In vitro mammalian chromosome aberration study:

The test chemical did not induce chromosome aberrations in the mammalian cell line in the presence and absence of S9 metabolic activation system and hence it is not mutagenic in the chromosome aberration study performed.

 

In vitro gene mutation study in mammalian cells:

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

Remarks:

read across data

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

Data is from peer reviewed publication.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Gene mutation toxicity study was performed to evaluate the mutagenic nature of the test chemical.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium, other: TA97, TA98, TA100, TA1535, TA1537

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:

The S-9 (9,000 x g supernatant) fractions of Aroclor 1254-induced, male Sprague-Dawley rat and male Syrian hamster livers

Test concentrations with justification for top dose:

0, 10, 33, 100, 333, 666, 1000, 1666, 3333 and 6666 µg/plate

Vehicle / solvent:

- 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 (TA98 and TA1538; Without S9); 2-aminoanthracene (With S9; all strains)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 mins
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: No data

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

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

OTHER: Plates were machine counted unless precipitate was present which interfered with the count, or the color of the test chemical on the plate reduced the contrast between the colonies and the agar.

Rationale for test conditions:

No data

Evaluation criteria:

The plates were observed for a dose dependent increase in the number of Histidine- independent (his+) colonies.

Evaluations were made at both the individual trial and chemical levels.

Individual trials were judged mutagenic (+), weakly mutagenic (+ W), questionable (?), or nonmutagenic (-), depending on the magnitude of the increase in his+ revertants, and the shape of the dose response. A trial was considered questionable (?) if the dose-response was judged insufficiently high to support a call of “+ W”, if only a single dose was elevated over the control, or if a weak increase was not dose-related. The distinctions between a questionable response and a nonmutagenic or weakly mutagenic response, and between a weak mutagenic response and mutagenic response are highly subjective. It was not necessary for a response to reach two-fold over background for a trial to be judged mutagenic.

A chemical was judged mutagenic (+) or weakly mutagenic (+W) if it produced a reproducible, dose-related response over the solvent control, under a single metabolic activation condition, in replicate trials. A chemical was judged questionable (?) if the results of individual trials were not reproducible, if increases in his+ revertants did not meet the criteria for a “+W” response, or if only single doses produced increases in his+ revertants in repeat trials. Chemicals were judged nonmutagenic (-) if they did not meet the criteria for a mutagenic or questionable response.

Statistics:

No data

Species / strain:

S. typhimurium, other: TA97, TA98, TA100, TA1535, TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: All chemicals were run initially in a toxicity assay to determine the appropriate dose range for the mutagenicity assay. The toxicity assay was performed using TA100. Toxic concentrations were defined as those that produced a decrease in the number of his+ colonies, or a clearing in the density of the background lawn, or both.

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

Remarks on result:

other: No mutagenic potential

Table: Results for the test chemical

Dose (µg/plate)	TA100
	-S9		10% HLI		30% HLI		10% RLI		30% RLI
	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM
0	114	7.3	141	11.0	150	11.8	133	10.1	165	3.8
10	126	4.7
33	126	9.5	137	7.6			133	10.0
100	136	7.5	120	5.4	170	4.7	151	6.4	132	7.8
333	110	1.2	117	5.5	169	1.9	158	3.4	150	1.7
666	89	3.6
1000			108	4.6	162	5.3	144	10.0	154	7.4
1666
3333			82	20.7	117	5.0	83	18.0	124	1.2
6666					74	4.9			63	1.2
Positive control	319	7.4	1223	32.8	736	21.3	906	15.4	536	14.4

 

Dose (µg/plate)	TA1535
	-S9		10% HLI		30% HLI		10% RLI		30% RLI
	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM
0	19	1.8	12	3.1	9	1.2	9	3.3	18	0.7
10	19	0.7
33	17	3.3	5	0.6			8	1.2
100	24	0.6	11	0.9	10	1.9	13	2.8	14	1.7
333	18	2.4	9	1.2	9	0.3	11	2.9	13	0.3
666	9	0.3
1000			6	1.2	8	0.3	6	1.5	9	0.9
1666
3333			7	1.5	9	0.6	5	1.5	6	1.5
6666					4	1.5			4	0.7
Positive control	189	2.6	160	14.8	633	57.1	119	19.3	176	14.2

 

Dose (µg/plate)	TA1537
	-S9		30% HLI		30% RLI
	Mean	SEM	Mean	SEM	Mean	SEM
0	10	1.8	14	1.9	11	1.5
10	9	2.3
33	11	3.1
100	9	3.2	10	0.9	9	2.3
333	11	0.9	10	1.0	10	1.5
666	6	1.5
1000			6	0.6	9	1.2
1666
3333			7	1.3	8	1.2
6666			4	0.0	5	1.5
Positive control	216	47.0	68	5.6	44	1.8

 

Dose (µg/plate)	TA97
	-S9		10% HLI		30% HLI		10% RLI		30% RLI
	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM
0	151	6.9	194	8.2	156	11.3	200	7.4	208	3.5
10	162	6.1
33	153	9.0	203	10.0	166	5.7	211	6.8	209	3.7
100	159	2.2	1++	10.0	179	3.6	221	9.0	199	2.3
333	166	4.9	211	2.0	173	3.8	206	7.9	209	5.4
666	118	6.2
1000			181	3.1	174	5.3	165	8.4	192	11.0
1666			167	5.6	140	8.7	166	14.5	139	17.4
3333
6666
Positive control	497	18.9	601	21.1	403	26.7	465	20.6	444	6.1

 

 

Dose (µg/plate)	TA98
	-S9		10% HLI		30% HLI		10% RLI		30% RLI
	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM
0	14	3.0	33	1.8	25	2.1	24	1.0	32	1.9
10	22	1.7
33	15	3.2	34	3.0			29	5.6
100	16	2.0	28	3.8	30	3.8	24	1.5	32	2.7
333	13	1.7	27	3.5	24	2.9	26	2.4	29	4.7
666	8	2.0
1000			23	2.4	26	3.1	20	2.6	26	1.8
1666
3333			18	1.8	18	3.8	14	3.1	23	2.6
6666					15	1.9			20	3.2
Positive control	908	22.3	1161	61.8	425	22.2	725	6.1	147	18.8

 

Conclusions:

The test chemical did not induce gene mutation in Salmonella typhimurium strains TA97, TA98, TA100, TA1535, TA1537 in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

Executive summary:

The mutagenicity of the analogue substance, Methyl benzoate (CAS Number: 98-85-1, EC Number: 202-707-1) has been tested in Salmonella typhimurium tester strains (TA1535, TA1537, TA98, TA100 and TA102). The test was performed both in the presence and absence of liver microsomal activation (S9 mix). Based on the solubility and precipitation test results eight different concentrations viz., 0.0 (NC), 0.002, 0.005, 0.0158, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate were selected for pre-experiment. Based on the pre-experiment results, the test item was tested with the following concentrations 0.0 (NC), 0.0158, 0.050, 0.158, 0.501, 1.582 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9). The plates were observed for a dose dependent increase in the number of Histidine- independent (his+) colonies. The test chemical did not induce gene mutation in Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 in the presence and absence of S9 metabolic activation system. The positive controls produced expected increases in the number of revertant colonies, thus confirming the validity of the assay. Hence, the chemical is not likely to classify as a gene mutant in vitro.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Remarks:

Read across data

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

Data is from authoritative source.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Principles of method if other than guideline:

The test chemical was tested in cultured Chinese hamster ovary (CHO) cells for induction of chromosomal aberrations both in the presence and absence of Aroclor 1254-induced male Sprague Dawley rat liver S9 and cofactor mix.

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

- Name of test material (as cited in study report): Ethylbenzene
- Molecular formula (if other than submission substance): C8H10
- Molecular weight (if other than submission substance): 106.16 g/mol
- Substance type: Organic
- Physical state: liquid
- Purity: >99%
- Impurities (identity and concentrations): NA

Target gene:

No data

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Details on mammalian cell line
- Type and identity of media:McCoy's 5A medium

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced male Sprague Dawley rat liver S9 and cofactor mix

Test concentrations with justification for top dose:

Dose Range :
75, 100, 125, 150 microgram/L with and without S9

Vehicle / solvent:

- 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:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
DURATION
- Preincubation period:
Without S9: 8.5 hrs
With S9: 2 hrs
- Exposure duration: 10.5 hrs
- Expression time (cells in growth medium): 10.5 hrs

SELECTION AGENT (mutation assays): Geimsa
SPINDLE INHIBITOR (cytogenetic assays): Colcemid

NUMBER OF CELLS EVALUATED:
100 first-division metaphase cells

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

OTHER:
In the chromosomal aberration test without S9, cells were incubated in McCoy's 5A medium with the study chemical for 8.5 hours; colcemid was added, and incubation was continued for 2 hours. The cells were then harvested by mitotic shake-off, fixed, and stained with Giemsa. For the chromosomal aberration test with S9, cells were treated with the study chemical and S9 for 2 hours, after which the treatment medium was removed and the cells were incubated for 8.5 hours in fresh medium, with colcemid present for the final 2 hours. Cells were harvested in the same manner as for the treatment without S9.

Rationale for test conditions:

No data

Evaluation criteria:

Cells were selected for scoring on the basis of good morphology and completeness of karyotype (21 ± 2 chromosomes). All slides were scored blind, and those from a single test were read by the same person. 100 first-division metaphase cells were scored at each dose for the chromosomal aberration test. Classes of aberrations included simple (breaks and terminal deletions), complex (rearrangements and translocations), and other (pulverized cells, despiralized chromosomes, and cells containing 10 or more aberrations).

Statistics:

Chromosomal aberration data were presented as percentage of cells with aberrations. To arrive at a statistical call for a trial, analyses were conducted on both the dose response curve and individual dose points. For a single trial, a statistically significant (P ≤ 0.05) difference from one dose point and a significant trend (P ≤ 0.015) were considered weak evidence for a positive response; significant differences for two or more dose indicated the trail was positive.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

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)

Metabolic activation:

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

Remarks on result:

other: No mutagenic potential

Table: induction of Chromosomal Aberrations in Chinese Hamster Ovary Cells by Ethylbenzene^a.

	-S9					+S9
	Dose (µg/ml)	Total cells scored	No. of Abs	Abs/cell	Cells with Abs (%)		Dose (µg/ml)	Total cells scored	No. of Abs	Abs/cell	Cells with Abs (%)
Harvest time: 10.5 hours						Harvest time: 10.5 hours
Summary: Negative						Summary: Negative
Dimethylsulfoxide^b		100	3	0.03	3	Dimethylsulfoxide		100	3	0.03	3
Mytomicin-C^c	1	50	16	0.32	22	Cyclophosphamide^c	50	50	23	0.46	36
Ethylbenzene	75	100	1	0.01	1	Ethylbenzene	75	100	4	0.04	4
	100	100	3	0.03	3		100	100	1	0.01	1
	125	100	5	0.05	5		125	100	1	0.01	1
	150	0					150	0
P=0.150^d						P=0.917

^aStudy was performed at Litton Bionetics, Inc. The detailed protocol is presented in Galloway et al. (1987). Abs=aberrations

^bSolvent control

^cPositive control

^dSignificance of percent cells with aberrations tested by the linear regression trend test versus log of the dose

Conclusions:

The test substance Ethylbenzene was found to be non mutagenic at any of the concentrations in Chinese Hamster Ovary (CHO) cells, as there were no significant changes observed in structural and numerical chromosome aberrations with or without metabolic activator.

Executive summary:

The test chemical Ethylbenzene (CAS no.: 100-41-4, E.C. no.: 202-849-4) was tested for in vitro mammalian chromosome aberration test using Chinese Hamster Ovary (CHO) cells for determining the mutagenicity. Ethylbenzene was administered at dose concentrations 75, 100, 125, 150µg/ml. Dimethyl sulfoxide (DMSO) was selected as a vehicle.The test was performed with and without metabolic activation system using rat liver homogenate. The rat liver homogenate was obtained from Aroclor-1254- induced male Sprague-Dawley rats. The non activated cells were exposed to the test substance for duration of 8.5 hours. Whereas the activated cells were exposed to the test substance for a duration of 2 hours. Mitomycin C was selected as a positive control for non-activated cells and Cyclophosphamide was selected as a positive control for activated cells at a concentration of 1µg/ml and 50 µg/mlrespectively. Cells were selected for scoring on the basis of good morphology and completeness of karyotype (21 ± 2 chromosomes). All slides were scored blind, and those from a single test were read by the same person. 100 first-division metaphase cells were scored at each dose for the chromosomal aberration test. Classes of aberrations included simple (breaks and terminal deletions), complex (rearrangements and translocations), and other (pulverized cells, despiralized chromosomes, and cells containing 10 or more aberrations). The test substance Ethylbenzene was found to be non mutagenic at any of the cocentrations in Chinese hamster Ovary (CHO) cells, as there were no significant changes observed in structural and numerical chromosome abberations with and without metabolic activator. Hence, the test substance Ethylbenzene was found to be non mutagenic in Chinese Hamster Ovary (CHO) cells.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Remarks:

Read across data

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from study report.

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

The purpose of this study was to assess toxic and genotoxic effects of test chemical on Chinese Hamster Ovary (CHO) cells by using several different in vitro-based assays, including genotoxicity tests based on the OECD Guideline No. 476 “In Vitro Mammalian Cell Gene Mutation Test.

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

Cells deficient in hypoxanthine-guanine phosphoribosyl transferase (HPRT) due to the mutation HPRT+/- to HPRT-/- are resistant to cytotoxic effects of 6-thioguanine (TG). HPRT proficient cells are sensitive to TG (which causes inhibition of cellular metabolism and halts further cell division since HPRT enzyme activity is important for DNA synthesis), so mutant cells can proliferate in the presence of TG, while normal cells, containing hypoxanthine-guanine phosphoribosyl transferase cannot.

This in vitro test is an assay for the detection of forward gene mutations at the in hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on the X chromosomes of hypodiploid, modal No. 20, CHO cells. Gene and chromosome mutations are considered as an initial step in the carcinogenic process.
The hypodiploid CHO cells are exposed to the test item with and without exogenous metabolic activation. Following an expression time the descendants of the treated cell population are monitored for the loss of functional HPRT enzyme.

HPRT catalyses the transformation of the purine analogues 6-thioguanine (TG) rendering them cytotoxic to normal cells. Hence, cells with mutations in the HPRT gene cannot phosphoribosylate the analogue and survive treatment with TG.

Therefore, mutated cells are able to proliferate in the presence of TG whereas the non-mutated cells die. However, the mutant phenotype requires a certain period of time before it is completely expressed. The phenotypic expression is achieved by allowing exponential growth of the cells for 7 days.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

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

Additional strain / cell type characteristics:

other: Hypodiploid, modal No. 20

Cytokinesis block (if used):

no data

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction obtained from Arcolor 1254-induced male Sprague-Dawley rats

Test concentrations with justification for top dose:

0, 0.5, 1, 2.5 or 5 mM

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: test chemical was easily dissolved in DMSO.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

N-ethyl-N-nitrosourea (ENU) was the positive control substance in the tests done without S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium with pre-incubation

DURATION
- Preincubation period: 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 (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 14 days
- Fixation time (start of exposure up to fixation or harvest of cells): 7 days (harvest of cells)

SELECTION AGENT (mutation assays): 6-thioguanine (TG)
SPINDLE INHIBITOR (cytogenetic assays): Not applicable
STAIN (for cytogenetic assays): Crystal violet

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

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

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

OTHER EXAMINATIONS: Not applicable

OTHER:no data

Rationale for test conditions:

No data

Evaluation criteria:

The cell line was observed for gene mutation.

Statistics:

No data

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not valid

Additional information on results:

Positive controls valid for study without S9.

Remarks on result:

other: No mutagenic potential

Table 1. Effect of 1-phenylethanol exposure on gene toxicity in CHO cells. After being exposed to the test chemical for 3 hrs, cells was washed with sterile PBS and then incubated for 7 days at 37°C, 5% CO₂. After 7 days, cells were re-seeded in new 6-well plates in the absence or presence of 10mM TG as a selection agent and returned to the incubator for 14 days at 37°C, 5% CO₂. On day 15, all 6-well plates were stained with crystal violet and the number of colonies were counted manually. The results are presented as the total number of colonies found in the number of independent wells analyzed (e.g. 0 colonies in 4 wells will give 0/4) (n = 2 samples from 2 independent cultures).

 

	With S9		Without S9
	with TG	without TG	with TG	without TG
Neg. control	0/4	153/4	0/4	184/4
Pos. control	0/4	188/4	21/4	156/4
0.5 mM	0/4	167/4	0/4	184/4
1.0 mM	0/4	147/4	0/4	181/4
2.5 mM	0/4	163/4	0/4	195/4
5.0 mM	1^a/4	173/4	0/4	193/4

 

^a)One very diffuse colony were found in one single well.

 

 

Table 2.Mutation frequency in CHO cells after 3 hrs of exposure to 1-phenylethanol in the absence or presence of 4% S9 liver microsomal fraction. N/A, no colonies present in the samples selected with TG, i.e. no mutation frequency could be determined.

 

	With S9	Without S9
Neg. control	N/A	N/A
Pos. control	N/A	4.53x10^-4
0.5 mM	N/A	N/A
1.0 mM	N/A	N/A
2.5 mM	N/A	N/A
5.0 mM	N/A^a	N/A

 

^a)Since only one diffuse colony were found in one single well (see Table 1A), this diffuse colony was not regarded as reliable and a true colony since the cells seemed to be apoptotic.

Table 3. CYTOTOXICITY & GENE TOXICITY

 

	Flask 1	Flask 2
Number of cells in cell culture flask (cells/ml)	4.30 x 10^6	4.62 x 10^6
Total number of cells in cell culture flask	19.35 x 10^6	20.79 x 10^6
Number of cells to be seeded per well (for cytotoxicity)	2.0 x 10^5	2.0 x 10^5
Dilution(s) made to create a cell suspension with 2.0 x 10^5cells per 2 ml for cytotoxicity	698ml cell suspension + 14.30 ml cell medium	649ml cell suspension + 14.35 ml cell medium
Number of cells to be seeded per well (for gene toxicity)	5.0 x 10^4	5.0 x 10^4
Dilution(s) made to create a cell suspension with 5.0 x 10^4cells per 2 ml for gene toxicity	174ml cell suspension + 14.83 ml cell medium	162ml cell suspension + 14.84 ml cell medium

 

Table 4. ADDITIONAL GENE TOXICITY TEST(S)

 

	Flask 1	Flask 2
Number of cells in cell culture flask (cells/ml)	6.03 x 10^6	6.29 x 10^6
Total number of cells in cell culture flask	27.14 x 10^6	28.31 x 10^6
Number of cells to be seeded per well (for gene toxicity)	5.0 x 10^4	5.0 x 10^4
Dilution(s) made to create a cell suspension with 5.0 x 10^4cells per 2 ml for gene toxicity	125ml cell suspension + 14.88 ml cell medium	119ml cell suspension + 14.88 ml cell medium

Table 5. RE-SEEDING AFTER EXPOSURE

Total number of cells (x 10⁶) per well after 3 hrs of 1-phenylethanol exposure without or with metabolic activation (S9) and prior to re-seeding. A haemocytometer was used for identifying the total number of cells (x 10⁶) per well:

 

	Flask 1		Flask 2
	Without S9	With S9	Without S9	With S9
Neg. control	0.2331	0.2860	0.3090	0.2660
Pos. control	0.2565	0.2898	0.2590	0.3090
0.5 mM	0.2898	0.2766	0.2860	0.3230
1.0 mM	0.2532	0.2931	0.2700	0.2660
2.5 mM	0.4131	0.2364	0.2366	0.2760
5.0 mM	0.2331	0.2832	0.2190	0.2830

 

 

 

 

Table 6. CYTOTOXICTY AFTER 24 & 48 HOURS

Raw data of cytotoxicity at 24 hours after chemical exposure and re-seeding using haemocytometer for identifying the total number of cells (x 10⁶) per well:

 

Without metabolic activation

	Flask 1		Flask 2
	Sample 1	Sample 2	Sample 1	Sample 2
Neg. control	0.193	0.206	0.113	0.193
Pos. control	0.116	0.103	0.103	0.126
0.5 mM	0.186	0.176	0.113	0.072
1.0 mM	0.186	0.146	0.136	0.166
2.5 mM	0.203	0.153	0.169	0.166
5.0 mM	0.187	0.233	0.150	0.150

 

With metabolic activation

	Flask 1		Flask 2
	Sample 1	Sample 2	Sample 1	Sample 2
Neg. control	0.133	0.096	0.079	0.129
Pos. control	0.156	0.166	0.086	0.096
0.5 mM	0.123	0.139	0.153	0.086
1.0 mM	0.106	0.133	0.129	0.139
2.5 mM	0.183	0.183	0.076	0.093
5.0 mM	0.129	0.150	0.120	0.106

 

Raw data of cytotoxicity at 48 hours after chemical exposure and re-seeding using haemocytometer for identifying the number of cells per well:

 

Without metabolic activation

	Flask 1		Flask 2
	Sample 1	Sample 2	Sample 1	Sample 2
Neg. control	0.663	0.739	0.459	0.506
Pos. control	0.496	0.406	0.333	0.350
0.5 mM	0.583	0.699	0.510	0.453
1.0 mM	0.593	0.463	0.613	0.596
2.5 mM	0.683	0.683	0.183	0.669
5.0 mM	0.666	0.476	0.313	0.699

 

With metabolic activation

	Flask 1		Flask 2
	Sample 1	Sample 2	Sample 1	Sample 2
Neg. control	0.303	0.293	0.346	0.413
Pos. control	0.566	0.519	0.456	0.523
0.5 mM	0.306	0.390	0.293	0.453
1.0 mM	0.406	0.506	0.403	0.600
2.5 mM	0.693	0.649	0.423	0.425
5.0 mM	0.403	0.426	0.373	0.543

 

Table 7. RE-SEEDING AFTER EXPRESSION TIME

Total number of cells (x 10⁶) per well after 3 hrs of 1-phenylethanol exposure without or with metabolic activation (S9) and prior to re-seeding. A haemocytometer was used for identifying the total number of cells (x 10⁶) per well:

 

	Flask 1		Flask 2
	Without S9	With S9	Without S9	With S9
Neg. control	2.750	2.790	2.800	2.270
Pos. control	2.060	2.580	2.170	2.280
0.5 mM	2.770	2.790	2.640	2.980
1.0 mM	2.560	2.580	2.820	2.850
2.5 mM	2.600	2.760	2.690	3.260
5.0 mM	2.750	2.060	2.450	2.660

 

 

Table 8. ADDITIONAL RE-SEEDING AFTER EXPRESSION TIME

 Total number of cells (x 10⁶) per well after 3 hrs of 1-phenylethanol exposure without or with metabolic activation (S9) and prior to re-seeding. A haemocytometer was used for identifying the total number of cells (x 10⁶) per well:

 

	Flask 1		Flask 2
	Without S9	With S9	Without S9	With S9
Neg. control	1.942	1.957	1.702	1.822
Pos. control	1.665	1.344	1.890	2.059
0.5 mM	2.209	1.792	2.040	1.717
1.0 mM	2.139	2.209	2.419	2.130
2.5 mM	2.289	2.220	2.169	2.019
5.0 mM	3.339	1.552	2.229	2.409

 

Table 9. STAINING AND COUNTING OF COLONIES

Number of colonies at 14 days of incubation without or with 6-Thioguanine (TG):

 

Without metabolic activation and without TG

	Flask 1		Flask 2
	Sample 1	Sample 2	Sample 1	Sample 2
Neg. control	44	41	44	55
Pos. control	31	53	36	36
0.5 mM	41	42	50	51
1.0 mM	44	45	46	46
2.5 mM	41	48	50	56
5.0 mM	51	54	46	42

 

With metabolic activation and without TG

	Flask 1		Flask 2
	Sample 1	Sample 2	Sample 1	Sample 2
Neg. control	35	35	45	38
Pos. control	33	46	55	54
0.5 mM	46	33	43	45
1.0 mM	42	33	40	32
2.5 mM	47	41	41	34
5.0 mM	46	49	42	36

 

Please note that in this first line of testing, the incubation with TG for 14 days did not work as it should. This specific part of the experiment were performed once again at a later stage (see below).

 

Table 10. ADDITIONAL STAINING AND COUNTING OF COLONIES

 

Without metabolic activation and with TG

	Flask 1		Flask 2
	Sample 1	Sample 2	Sample 1	Sample 2
Neg. control	0	0	0	0
Pos. control	8	7	2	4
0.5 mM	0	0	0	0
1.0 mM	0	0	0	0
2.5 mM	0	0	0	0
5.0 mM	0	0	0	0

 

 

With metabolic activation and with TG

	Flask 1		Flask 2
	Sample 1	Sample 2	Sample 1	Sample 2
Neg. control	0	0	0	0
Pos. control	0	0	0	0
0.5 mM	0	0	0	0
1.0 mM	0	0	0	0
2.5 mM	0	0	0	0
5.0 mM	0	0	0	0

Conclusions:

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

Executive summary:

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of test chemical 1 -Phenylethanol (CAS: 98 -85 -1, E.C.: 202 -707 -1) when administered to Chinese Hamster Ovary (CHO) cells. The test was performed according to OECD test guideline 476 adopted 21st July 1997.

In the genotoxicity test, test chemical was administered to CHO cells for 3 hrs at the dose levels of 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. pH and osmolality was not determined in the gene mutation test.

Only the positive control ENU gave a clear indication of gene mutations occurring while no other treatment gave rise to gene toxicity. One very diffuse colony were seen in one well out of four at 5 mM and in the presence with 4% S9 liver microsomal fraction. This diffuse colony is not regarded to be relevant since the single spot was only mildly colored by crystal violet, thus indicating that it was a small cluster of apoptotic cells taking their last breath instead of cells surviving the TG-selection. This is further supported by the overall results of the tested concentrations of 1-phenylethanol, i.e. the test chemical did not show any evidence of diffuse or clear colonies present.

When the mutation frequency was determined, a frequency of 4.53 x 10 ^-4 was shown after a 3 hour exposure of ENU as the positive control and in the absence of S9 liver microsomal fraction. Since no other tested concentration of test chemical and in the absence or presence of S9 liver microsomal fraction resulted in colonies, we conclude that test chemical does not give rise to gene mutations when CHO cells are exposed in vitro to the test chemical at 0, 0.5, 1.0, 2.5 or 5.0 mM for 3 hrs.

Based on the results of the current study, we conclude that test chemical 1 -Phenylethanol does not give rise to gene mutations when CHO cells are exposed to the test chemical in vitro at 0, 0.5, 1.0, 2.5 or 5.0 mM for 3 hrs, in the presence or absence of metabolic activation. The test was performed according to GLP.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Gene mutation in vitro:

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

Ames assay:

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

1) The mutagenicity of the analogue substance, Methyl benzoate (CAS Number: 98-85-1, EC Number: 202-707-1) has been tested in Salmonella typhimurium tester strains (TA1535, TA1537, TA98, TA100 and TA102). The test was performed both in the presence and absence of liver microsomal activation (S9 mix). Based on the solubility and precipitation test results eight different concentrations viz., 0.0 (NC), 0.002, 0.005, 0.0158, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate were selected for pre-experiment. Based on the pre-experiment results, the test item was tested with the following concentrations 0.0 (NC), 0.0158, 0.050, 0.158, 0.501, 1.582 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9). The plates were observed for a dose dependent increase in the number of Histidine- independent (his+) colonies. The test chemical did not induce gene mutation in Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 in the presence and absence of S9 metabolic activation system. The positive controls produced expected increases in the number of revertant colonies, thus confirming the validity of the assay. Hence, the chemical is not likely to classify as a gene mutant in vitro.

2) The mutagenicity of the analogue substance, Methyl benzoate (CAS Number: 98-85-1, EC Number: 202-707-1) has been tested in Salmonella typhimurium tester strains (TA1535, TA1537, TA98, TA100 and TA102). The test was performed according to OECD test guideline 471. The test was performed both in the presence and absence of liver microsomal activation (S9 mix). Based on the solubility and precipitation test results eight different concentrations viz., 0.0 (NC), 0.002, 0.005, 0.0158, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate were selected for pre-experiment. Based on the pre-experiment results, the test item was tested with the following concentrations 0.0 (NC), 0.0158, 0.050, 0.158, 0.501, 1.582 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9). The plates were observed for a dose dependent increase in the number of Histidine- independent (his+) colonies. The test chemical did not induce gene mutation in Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 in the presence and absence of S9 metabolic activation system. The positive controls produced expected increases in the number of revertant colonies, thus confirming the validity of the assay. Hence, the chemical is not likely to classify as a gene mutant in vitro. The test was performed according to GLP.

Thus, based on the above summarized studies on test chemical, it can be concluded that the given test chemical did not induce mutation in the Salmonella typhimurium strains both in the presence and absence of S9 metabolic activation system and hence is not likely to be mutagenic under the conditions of this study.

In vitro mammalian chromosome aberration study:

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

1) The mutagenicity of the analogue substance, Methyl benzoate (CAS Number: 98-85-1, EC Number: 202-707-1) has been tested in Salmonella typhimurium tester strains (TA1535, TA1537, TA98, TA100 and TA102). The test was performed according to OECD test guideline 471. The test was performed both in the presence and absence of liver microsomal activation (S9 mix). Based on the solubility and precipitation test results eight different concentrations viz., 0.0 (NC), 0.002, 0.005, 0.0158, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate were selected for pre-experiment. Based on the pre-experiment results, the test item was tested with the following concentrations 0.0 (NC), 0.0158, 0.050, 0.158, 0.501, 1.582 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9). The plates were observed for a dose dependent increase in the number of Histidine- independent (his+) colonies. The test chemical did not induce gene mutation in Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 in the presence and absence of S9 metabolic activation system. The positive controls produced expected increases in the number of revertant colonies, thus confirming the validity of the assay. Hence, the chemical is not likely to classify as a gene mutant in vitro. The test was performed according to GLP.

2) This study was conducted to determine the chromosomal aberration induction potential of test chemical in human peripheral blood lymphocyte cultures. The methods followed were as per OECD guideline No. 473, adopted on 29^thJuly 2016 “In VitroMammalian Chromosome Aberration Test. The experiment was conducted using human peripheral blood lymphocytes. Blood was drawn from a healthy volunteer, by venous puncture using heparinised syringe. The experiment was performed both in the presence and in the absence of metabolic activation system after 48 h mitogenic stimulation. The test chemical was dissolved in DMSO and used at dose level of 0, 0.00025, 0.0005 and 0.001 mg/mL.in the presence and absence of S9 metabolic activation system in phase 1 and phase 2. Phase I of experiment was performed by short term treatment method both in the presence and absence of metabolic activation system(1%). Phase II of experiment was performed by short term treatment as well as long term treatment method. Long term treatment was performed in absence of metabolic activation to confirm the negative results obtained in the absence of metabolic activation in Phase I. Short term treatment method was performed with increased metabolic activation (2%) condition to confirm the negative results obtained in the presence of metabolic activation in Phase I. The doses for the main study were based on the cytotoxicity study conducted both in the presence and absence of metabolic activation system. 3 test concentrations (0.5, 1 and 2mg/mL of culture media) based on the solubility, precipitation and pH test of the test item were tested. Cytotoxicity was determined by reduction in the mitotic index in comparison with negative control. The medium of the proliferatingblood culture was removed by centrifugation at 1500 rpm for 10 minutes. The cells were suspended in plain medium (medium without serum) mixed with S9 mix (Phase I - 1 % and Phase II - 2 % v/v) and in complete media mixed with phosphate buffer for the treatment in presence and in absence of metabolic activation system respectively. A volume of 7.92 mL of proliferating culture was dispensed to individual sterile culture tubes/flasks. Each tube/flask according to treatment groups was identified. Negative control tubes were treated with 80 µL of RPMI media and treatment group were treated with 80 µL of respective test item stock solution. The cultures were incubated at 37 ± 2 °C for duration (exposure period). For Phase I, after incubation cells were spun down by gentle centrifugation at 1500 rpm for 10 minutes. The supernatant with the dissolved test item was discarded and the cells were re-suspended in Phosphate Buffer Saline (PBS). The washing procedure was repeated once again. After washing the cells were re-suspended in complete culture medium (RPMI-1640 with 10 % serum) and cultured at 37 ± 2 °C for 1.5 normal cell cycle lengths (22 - 25 hours). The cultures were harvested at the end of incubation of 24 hours after treatment. Before 3 hours of harvesting, 240 µL of colcemid (10 µg/mL) (final concentration: 0.3 µg/mL) was added to each of the culture tube, and kept under incubation at 37 ± 2 °C. The cultures were harvested 24 hours after beginning of treatment by centrifugation at 1500 rpm for 10 minutes. The supernatant was discarded and the cells were re-suspended in 7 mL of freshly prepared, pre-warmed (37 ± 2 °C) hypotonic solution of potassium chloride (0.075 M KCl). Then the cell suspension was allowed to stand at 37 ± 2 °C for 30 minutes in water bath. After hypotonic treatment, the culture was centrifuged and supernatant was removed. After that 5 mL of freshly prepared, chilled Carnoy’s fixative (3:1 methanol: acetic acid solution) was added and left for 5 min. The cells were collected by centrifugation and washed twice with Carnoy’s fixative. After the final centrifugation, the supernatant was removed completely, and the cell pellet resuspended in 0.5 mL of Carnoy’s fixative. The slides were prepared by dropping the cell suspension onto a clean ice-chilled microscope slide. The slides were dried over a slide warmer and labelled. At least two slide was made from each sample. The cells were stained with 5 % fresh Giemsa stain in phosphate buffer and mounted using DPX mountant. Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. A minimum of 1000 cells were counted in different fields of slide per culture and the number of metaphases were recorded for mitotic index (MI) calculation.300 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides. Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. Chromosomal and chromatid breaks, acentric fragments, deletions, exchanges, pluverization, polyploidy (including endoreduplication) and disintegrations were recorded as structural chromosomal aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. Only metaphases with 46± 2 centromere regions were included in the analysis. Test chemical is non-clastogenic at the highest tested concentration of 0.001mg/ml both in the presence (1% and 2%) and in the absence of metabolic activation under the specified conditions and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Thus, based on the above summarized studies on test chemical, it can be concluded that the given test chemical did not induce chromosome aberrations in the mammalian cell line in the presence and absence of S9 metabolic activation system and hence it is not mutagenic in the chromosome aberration study performed.

 In vitro 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:

1) An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of test chemical 1 -Phenylethanol (CAS: 98 -85 -1, E.C.: 202 -707 -1) when administered to Chinese Hamster Ovary (CHO) cells. The test was performed according to OECD test guideline 476.

In the genotoxicity test, test chemical was administered to CHO cells for 3 hrs at the dose levels of 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. pH and osmolality was not determined in the gene mutation test.

Only the positive control ENU gave a clear indication of gene mutations occurring while no other treatment gave rise to gene toxicity. One very diffuse colony were seen in one well out of four at 5 mM and in the presence with 4% S9 liver microsomal fraction. This diffuse colony is not regarded to be relevant since the single spot was only mildly colored by crystal violet, thus indicating that it was a small cluster of apoptotic cells taking their last breath instead of cells surviving the TG-selection. This is further supported by the overall results of the tested concentrations of 1-phenylethanol, i.e. the test chemical did not show any evidence of diffuse or clear colonies present.

When the mutation frequency was determined, a frequency of 4.53 x 10^-4was shown after a 3 hour exposure of ENU as the positive control and in the absence of S9 liver microsomal fraction. Since no other tested concentration of test chemical and in the absence or presence of S9 liver microsomal fraction resulted in colonies, we conclude that test chemical does not give rise to gene mutations when CHO cells are exposed in vitro to the test chemical at 0, 0.5, 1.0, 2.5 or 5.0 mM for 3 hrs.

Based on the results of the current study, we conclude that test chemical 1 -Phenylethanol does not give rise to gene mutations when CHO cells are exposed to the test chemical in vitro at 0, 0.5, 1.0, 2.5 or 5.0 mM for 3 hrs, in the presence or absence of metabolic activation. The test was performed according to GLP.

2) An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of the given test chemical Phenethyl phenylacetate(CAS no.: 102 -20 -5, E.C.: 203 -013 -1) when administered to Chinese Hamster Ovary (CHO) cells. The test was performed according to OECD test guideline 476.

A preliminary dose-finding study was conducted prior to the main study. A range of different 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, 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. No cytotoxic effects were observed when CHO cells were exposed to test chemical for 3 hrs.

Based on the results of the current 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 hence it is not induce cytotoxic effects at concentrations of ≤ 5.0mM. The test was performed according to GLP.

Thus, based on the above summarized studies on test chemical, 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 data available and applying weight of evidence approach, the given test chemical does not exhibit gene mutation in vitro by Ames assay, In vitro mammalian chromosome aberration study and In vitro gene mutation study in mammalian cells. Hence, the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.