(2-methoxyethyl)benzene

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 May 2018 to 05 Oct 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Test material information

Constituent 1

Reference substance name:

(2-methoxyethyl)benzene

EC Number:

222-619-7

EC Name:

(2-methoxyethyl)benzene

Cas Number:

3558-60-9

Molecular formula:

C9-H12-O

IUPAC Name:

(2-methoxyethyl)benzene

Test material form:

liquid

Method

Target gene:

hprt gene

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: CHO-K1 (ATCC CCL-61, Lot 4765275)
- Suitability of cells: This cell line has been demonstrated to be sensitive to the mutagenic activity of a variety of chemicals.
- Absence of Mycoplasma contamination: Yes
- Cell cycle length, doubling time or proliferation index : population doubling time of 12 to 14 h
- Modal number of chromosomes: 20
- Periodically checked for karyotype stability: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: Cells were grown in tissue culture flasks at 37±1°C in a humidified carbon dioxide incubator (5 ± 0.2 % CO2 in air).
Ham's F-12 medium supplemented with sodium bicarbonate, antibiotics, and L-glutamine was the basic medium.
Basic medium supplemented with 10% fetal bovine serum (FBS) was the complete medium and was used for the growth and multiplication of cells as well as in detaching and diluting the cells.
Basic medium supplemented with 5% FBS was the treatment medium and was used for target cell exposure to the test item and controls.
Cloning medium was basic medium supplemented with 20 % FBS and was used for the determination of cell viability or plating/cloning efficiency.
Selective medium was basic medium supplemented with 20 % FBS and the selective agent 6-Thioguanine (6-TG) at 35 µM and was used for the selection of mutants.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Aroclor 1254 induced rat liver S9 homogenate
- method of preparation of S9 mix: The S9 homogenate was prepared from male Wistar rats induced with a single intra-peritoneal injection of Aroclor 1254 (0.7 mL/rat ready to use solution), 5 days prior to sacrifice. The S9 homogenate was prepared in batches and stored in a deep freezer maintained at -68 to -86 °C.
- concentration or volume of S9 mix and S9 in the final culture medium: 1.5 mL of S9 mix was added to the respective flasks to achieve a final concentration of 1% S9 (v/v) in the medium.
- quality controls of S9: Each batch of S9 homogenate was characterized for its ability to metabolize the pro-mutagens 2-aminoanthracene and benzo(a)pyrene to mutagens using Salmonella typhimurium TA100 strain and for the protein content using modified Lowry Assay (Sword and Thomson, 1980). To check the sterility, the liver homogenate was streaked onto nutrient agar plates, in duplicate, and incubated for approximately 48 hours at 37 ± 1 °C.

Test concentrations with justification for top dose:

Based on the results of the preliminary cytotoxicity test, the following test concentrations were selected for testing in the gene mutation test: 50, 151, 454 and 1362 µg/mL

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: two replicates (with two flasks per replicate)
- Number of independent experiments: 1 preliminary cytotoxicity test and 2 gene mutation experiments

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 4.5 x 10e6 cells / 75 cm2 flask
- Test substance added in medium

TREATMENT SCHEDULE:
- Preincubation period: Exponentially growing CHO-KI cells were plated in two replicates and each replicate has two flasks containing 15 mL of complete medium at a density of approximately 4.5 x 10e6 cells / 75 cm2 flask and incubated for approximately 24 hours.
- Exposure duration: 3 hours (with and without S9)

FOR GENE MUTATION:
- Expression time: For expression of the mutant phenotype, the cells from the replicate cultures were sub-cultured in complete medium in duplicate, at a density of approximately 2 x 10e6 cells / 75 cm2 flasks and incubated. The cells were sub-cultured as above at a 2-3-day interval and carried out for the 9-day expression period.
- Selection time: For selection of the 6-Thioguanine (6-TG) resistant phenotype, cells from each of the replicate cultures were plated in to 5 flasks at a density of approximately 2 x 10e5 cells / 25 cm2 flask (total of 10e6 cells/replicate) in selective medium and incubated for 7 days.
- Fixation time: 16 days
- Method used: in medium.
- Method to enumerate numbers of viable and mutants cells: The colonies were stained with 0.5 % methylene blue and counted for both cloning efficiency and mutant selection after 7 days of incubation.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cloning efficiency and relative survival (RS)
- Any supplementary information relevant to cytotoxicity: Preliminary cytotoxicity assessment: One hundred fifty microliters (150 µL) of the vehicle or the stock / dilutions of the test item were mixed with the medium in the respective flasks to get the required test concentrations per mL of the medium as well as the vehicle control. The flasks were incubated for 3 hours to expose the cells to treatment. After the treatment period, about 200 cells from the control and each treatment level were plated into T-25 cm2 flasks with 5 mL cloning medium in triplicate and incubated for 10 days. After incubation, medium from each flask was aspirated and the cells were stained with methylene blue and the colonies counted manually to determine the Cloning Efficiency (CE) and cytotoxicity was determined by estimating the RS.
- Initial cloning efficiency: About 200 cells from each replicate of the vehicle control, positive control (from Experiment 1) and each treatment level were plated into T-25 cm2 flasks in triplicate with cloning medium to determine CE and to express parallel cytotoxicity based on RS. After 9 days of incubation, the colonies were stained with methylene blue and counted for CE.
- Cloning efficiency at time of selection: For cloning efficiency determination at the time of selection, cells from each of the replicate cultures were plated at approximately 200 cells / 25 cm2 flask in triplicate in cloning medium and incubated for 7 days.

Evaluation criteria:

EVALUATION AND INTERPRETATION OF RESULTS
When all the validity criteria are fulfilled:
1. A test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
- At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control
- The increase is concentration-dependent when evaluated with an appropriate trend test
- Any of the results are outside the distribution of the historical vehicle control data
When all of these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.
2. A test chemical is considered to be clearly negative if, in all experimental conditions examined:
- None of the test concentrations exhibits a statistically 'significant increase compared with the concurrent vehicle control
- There is no concentration-related increase when evaluated with an appropriate trend test
- All results are inside the distribution of the historical vehicle control data The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.
3. There is no requirement for verification of a clearly positive or negative response.
4. The results will be considered equivocal if they do not meet all the criteria specified for a positive or negative response respectively. Additional evaluation and/or experimental work may be required to clarify such results and the activity, if any, will be documented in a study plan amendment.

Statistics:

A power transformation procedure was used with which, the observed mutant frequency was transformed using the formula:

Y = (X + A)B

where,
Y = transformed mutant frequency
X = observed mutant frequency
and A, B = constants.

Statistical analysis of the experimental data was carried out using validated copies of SYSTAT Statistical package version 12.0. In cases where analysis of variance was significant at p < 0.05, a Dunnett's test was conducted, comparing each treatment group and the positive control to the vehicle control (p < 0.05).

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: At the end of 3-hour exposure, the pH of the test medium in the presence of metabolic activation ranged from 7.09 to 7.15 with 7.12 in the DMSO control while in the absence of metabolic activation it was between 7.16 and 7.21 with 7.21 in the DMSO control, respectively.
- Data on osmolality: At the end of 3-hour exposure, osmolality of the test medium at the highest test item treatment level as well as the highest soluble test item level (1362 µg/mL) was 0.461 and 0.440 OSMOL/kg in the presence and absence of metabolic activation, respectively. The corresponding osmolality in the DMSO control was 0.455 and 0.463 OSMOL/kg, in the presence and absence of metabolic activation, respectively.
- Water solubility: The test item was insoluble in sterile water and soluble in DMSO at 136 mg/mL.
- Precipitation and time of the determination: At the end of 3-hour exposure, the test item did not precipitate in the test medium at any of the tested concentrations.

RANGE-FINDING/SCREENING STUDIES: In the initial cytotoxicity test, the test item did not exhibit evidence of significant cell growth inhibition as Relative Survival (10 to 20% RS compared to vehicle control) at any of the tested concentrations either in the presence or in the absence of metabolic activation. Based on these observations the highest OECD 476-recommended concentration of 1362 µg/mL (equivalent to 10 mM) was tested in the gene mutation assay both in the presence and absence of metabolic activation.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: The frequency of mutants in the DMSO control was within the range of the in-house historical control data. Under similar conditions the positive control 3-methyicholanthrene (3-MCA) induced statistically significant increases in the mutant frequency as compared with the vehicle control.

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements: There was no evidence of excessive cytotoxicity (i.e., <10 % RS) at any of the tested concentrations either in the presence or absence of metabolic activation. The RS values in the presence of metabolic activation, ranged from 86 to 58 % while in the absence of metabolic activation, RS values ranged from 90 to 62 % compared to the vehicle control.
- Genotoxicity results: The test item did not cause a significant increase in the frequencies of mutants compared to the vehicle control in the presence or absence of metabolic activation at any of the tested concentrations.

Any other information on results incl. tables

TABLE 1. Parallel Cytotoxicity Test Results from Experiment 1

Treatment µg/mL	No. of Colonies /Flask			CE *	ACE	RS %
	1	2	3
DMSO	186	188	184	0.94	0.91	100
	186	189	191
50	175	171	173	0.86	0.78	86
	168	170	177
151	160	158	161	0.80	0.64	70
	164	157	154
454	150	148	161	0.77	0.60	66
	160	157	148
1362	140	139	136	0.69	0.53	58
	135	137	134
3-MCA	152	162	147	0.77	0.69	76
	149	156	159

 Baseline cell count (No. of cells at the beginning of treatment): 15.75 x 10⁵cells/mL

Vehicle Control: DMSO

Positive Control: 8 µg/mL 3-MCA

CE: Cloning Efficiency                               ACE: Adjusted CE                    RS: Relative Survival

* calculated from the mean values of the replicates of each group and rounded off to

two decimal places

CE = No. of colonies / No. of cells plated

ACE = CE x [(No. of cells at the end of treatment) / (No. of cells at the beginning of treatment)]

RS = [(Adjusted CE in treated culture) / (Adjusted CE in the vehicle control)] x 100

TABLE 2. Parallel Cytotoxicity Test Results from Experiment 2

Treatment ug/mL	No. of Colonies /Flask			CE*	ACE	RS %
	1	2	3
DMSO	189	187	188	0.95	0.90	100
	191	189	190
50	178	179	181	0.89	0.81	90
	170	178	175
454	157	162	160	0.80	0.66	73
	162	159	160
	155	156	148	0.75	0.63	70
	150	146	143
1362	137	145	140	0.70	0.56	62
	136	141	134

 

Baseline cell count (No. of cells at the beginning of treatment): 15.75 x 10⁵cells/mL Vehicle Control: DMSO

CE: Cloning Efficiency                                         ACE: Adjusted CE                    RS: Relative Survival

* calculated from the mean values of the replicates of each group and rounded off to two decimal places

CE = No. of colonies / No. of cells plated

ACE = CE x [(No. of cells at the end of treatment) / (No. of cells at the beginning of treatment)]

RS = [(Adjusted CE in treated culture) / (Adjusted CE in the vehicle control)] x 100

 

TABLE 3. Summary Results of the Gene Mutation Assay in the Presence of Metabolic Activation (Experiment 1)

Treatment µg/mL	Mutation Assay Flasks						Cloning Efficiency of Mutant Colonies	Cloning Efficiency Flasks				6-TG Mutants per 10^6 Clonable Cells (MF)
	TG Colonies/Flask							No. of Colonies/Flask
	1	2	3	4	5	Total		1	2	3	CE*
DMSO	1	2	2	1	0	14	0.000007	185	187	188	0.93	7.53
	3	0	0	2	3			189	185	183
50	0	1	3	3	1	16	0.000008	178	180	175	0.89	8.99
	0	2	4	2	0			184	176	180
151	2	1	1	1	3	13	0.000007	163	159	160	0.81	8.02
	1	3	0	1	0			161	166	167
454	2	2	3	0	2	17	0.000009	149	144	144	0.73	11.64
	3	0	0	2	3			150	140	152
1362	3	0	3	1	2	17	0.000009	151	154	150	0.74	11.49
	3	3	0	2	0			152	145	141
3-MCA	35	32	29	31	30	321	0.000161	156	161	164	0.80	200.63
	37	32	29	31	35			152	165	157

Positive Control: 8 tig/mL 3-MCA                Vehicle Control: DMSO                 CE: Cloning Efficiency                MF: Mutant Frequency

* calculated from the mean values of the replicates of each group and rounded off to two decimal places
Mutant frequency of 6-TG mutants is significantly higher than the concurrent vehicle control value (p < 0.05)

CE = Total no. of colonies / No. of cells plated

MF = [(CE of mutant colonies in selective medium) / (CE in non-selective medium)] x 10⁶

 

TABLE 4. Summary Results of the Gene Mutation Assay in the Absence of Metabolic Activation (Experiment 2)

Treatment µg/mL	Mutation Assay Flasks						Cloning Efficiency of Mutant Colonies	Cloning Efficiency Flasks				6-TG Mutants per 10^6 Clonable Cells (MF)
	TG Colonies/Flask							No. of Colonies/Flask
	1	2	3	4	5	Total		1	2	3	CE*
DMSO	1	1	2	3	1	15	0.000008	185	191	187	0.94	7.98
	2	1	0	3	1			192	189	185
50	3	1	2	0	1	16	0.000008	178	180	175	0.89	8.99
	2	1	3	1	2			184	176	180
151	2	1	1	1	0	11	0.000006	163	159	160	0.81	6.79
	1	1	1	2	1			160	165	166
454	2	2	0	3	1	17	0.000009	160	158	164	0.79	10.76
	1	3	2	1	2			150	162	152
1362	1	2	1	1	1	12	0.000006	153	154	157	0.78	7.69
	2	1	0	2	1			160	158	150

Vehicle Control: DMSO                 CE: Cloning Efficiency         MF: Mutant Frequency

* calculated from the mean values of the replicates of each group and rounded off to two decimal places

CE = Total no. of colonies / No. of cells plated

MF = [(CE of mutant colonies in selective medium) / (CE in non-selective medium)] x 10⁶

Applicant's summary and conclusion

Conclusions:

It is concluded that the test item does not have the potential to induce gene mutation in CHO-K 1 cells up to the OECD 476-recommended top concentration of 1362 mg/mL (equivalent to 10 mM) under the conditions of testing employed.

Executive summary:

In this GLP compliant OECD 476 study, the mutagenic potential of the test item to induce gene mutation in mammalian cells was evaluated using Chinese Hamster ovary (CHO-K1) cells. The study consisted of a preliminary cytotoxicity test and a definitive gene mutation test. The gene mutation test comprised of two independent experiments, one each in the presence and absence of metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver).

The test item was soluble in Dimethyl sulfoxide (DMSO) at 136 mg/mL. The test item was found to be stable in DMSO for 24 hours at room temperature at fortification levels of 4 and 140 mg/mL. In a preliminary cytotoxicity test for the selection of test concentrations for the gene mutation assay, the test item did not exhibit evidence of significant cell growth inhibition as Relative Survival (10 to 20% RS compared to vehicle control) at any of the tested concentrations either in the presence or absence of metabolic activation. The test item did not precipitate in the test medium and did not cause any appreciable change in the pH and osmolality of test medium. Based on these observations the highest OECD 476-recommended concentration of 1362 µg/mL (equivalent to 10 mM) was tested in the gene mutation assay. In the gene mutation test, CHO-K 1 cells were exposed to the test item in duplicate at concentrations of 50, 151, 454 and 1362 µg/mL for 3 hours in the presence (Experiment 1) and absence (Experiment 2) of metabolic activation. In a similar way, a concurrent vehicle control (DMSO) and a positive control, 3-methylcholanthrene (Experiment 1) were also tested in duplicate. There was no evidence of induction of gene mutations in any of the test item treated cultures either in the presence or absence of metabolic activation. The positive control in Experiment 1 produced a statistically significant increase in the frequencies of mutants, under identical conditions. The results of the concentration analysis of the dose formulation samples of the gene mutation assay confirmed that the top dose level of 1362 µg/mL was achieved and the results support the validity of the study conclusion.

In conclusion, the results of the forward gene mutation test at the hprt locus with the test item indicated that the test item was non-mutagenic up to the highest OECD 476-recommended concentration of 1362 µg/mL (equivalent to 10 mM) under the conditions of this study.