Benzyl methacrylate

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:

2011-08-23 to 2011-10-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

dated May 30, 2008

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 21 July 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

certificate dated 30 March 2009

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Benzyl methacrylate

EC Number:

219-674-4

EC Name:

Benzyl methacrylate

Cas Number:

2495-37-6

Molecular formula:

C11H12O2

IUPAC Name:

benzyl methacrylate

Test material form:

liquid

Specific details on test material used for the study:

- Name of test material (as cited in study report): Benzyl methacrylate

Method

Target gene:

HPRT

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM containing Hank’s salts supplemented with 10% FBS, 5 µg/mL neomycin, 1% amphotericin B
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
- Other: each batch screened for spontaneous mutant frequency

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Sß mix

Test concentrations with justification for top dose:

range finding pre-experiment: 13.8 to 1760 mg/L (approx. 10 mM, the guideline limit concentration)

Doses applied:
Experiment I:
13.8, 27.5, 55, 110, 220, 330, 440 mg/L without S9 mix, exposure period 4 h; 13.8, 27.5, 55, 110 mg/L chosen for mutation rate analysis
55, 110, 220, 440, 880, 1760 mg/L with S9 mix, exposure period 4 h; 55, 110, 220, 440, 880 mg/L chosen for mutation rate analysis

Experiment II:
13.8, 27.5, 55, 110, 165, 220 mg/L without S9 mix, exposure period 24 h; 13.8, 27.5, 55, 110, 165 mg/L chosen for mutation rate analysis
27.5, 55, 110, 220, 440, 880 mg/L with S9 mix, exposure period 4 h; 27.5, 55, 110, 220, 440 mg/L chosen for mutation rate analysis

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (final concentration in medium: 0.5%)
- Justification for choice of solvent/vehicle: solubility properties, relative non-toxicity to cell cultures

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

other: (without metabolic activation)

Controls 2

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

other: (with metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Experiment I: 4 h with and without metabolic activation
Experiment II: 4 h with metabolic activation, 24 h without metabolic activation
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 15 days

SELECTION AGENT (mutation assays): 11 mg/L 6-thioguanine (6-TG)

NUMBER OF REPLICATIONS: 2 replicates per experiment

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

OTHER:
- colonies used to determine the cloning efficiency (survival) were fixed and stained approx. 7 days after treatment
- the colonies were stained with 10 % methylene blue in 0.01 % KOH solution; colonies with more than 50 cells were counted.
- Following the expression time of 7 days five cell culture flasks were seeded with about 3 - 5x10E05 cells each in medium containing 6-TG; 2 additional flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability.

Evaluation criteria:

The assay is considered acceptable if it meets the following criteria:
- the numbers of mutant colonies per 10E06 cells found in the solvent controls fall within the laboratory historical control data range
- the positive control substances must produce a significant increase in mutant colony frequencies
- the cloning efficiency (absolute value) of the solvent controls must exceed 50%

A positive response is described as follows:
- reproducible induction of a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment
- reproducible concentration-related increase of the mutation frequency; such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
Biological and statistical significance were considered together.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: solvent control: 7.33; 1760 mg/L BNMA: 7.31 -> no relevant shift
- Effects of osmolality: solvent control: 365 mOsm; 1760 mg/L BNMA: 344 -> no relevant shift
- Water solubility:
Phase separation was observed in the first experiment at 220 mg/L and above with metabolic activation and at 110 mg/L and above without metabolic activation. In the second experiment phase separation was observed at 110 mg/L and above with and without metabolic activation.

RANGE-FINDING/SCREENING STUDIES: pre-test for cytotoxicity, cytotoxicity at 110 mg/L and above without metabolic activation, and 880 mg/L and above with metabolic activation

COMPARISON WITH HISTORICAL CONTROL DATA: mutant frequencies were within the historical range of solvent controls

Any other information on results incl. tables

 

	Conc. [mg/L]	S9 mix	Rel. CE [%]	Mutant colonies /10E06 cells	Induction factor	Rel. CE [%]	Mutant colonies /10E06 cells	Induction factor
Experiment I, 4 h treatment			Culture I			Culture II
Solvent control		-	100.0	16.9	1.0	100.0	18.3	1.0
P.C. (EMS)		-	97.9	132.8	7.0	102.5	147.1	8.0
Test item	13.8	-	99.3	8.1	0.4	104.0	20.9	1.1
Test item	27.5	-	99.9	10.4	0.6	104.3	9.7	0.5
Test item	55	-	97.9	12.4	0.7	111.0	14.5	0.8
Test item	110	-	89.5	8.0	0.4	72.8	10.4	0.6
Test item	220	-	0.0	not continued		0.0	not continued
Test item	330	-	0.0	not continued		0.0	not continued
Test item	440	-	0.0	not continued		0.0	not continued
Experiment I, 4 h treatment			Culture I			Culture II
Solvent control		+	100.0	29.1	1.0	100.0	14.6	1.0
P.C. (DMBA)		+	45.5	983.2	33.7	60.0	675.1	46.1
Test item	55	+	95.9	47.4	1.6	96.0	29.9	2.0
Test item	110	+	97.3	21.0	0.7	99.5	26.1	1.8
Test item	220	+	94.0	17.5	0.6	97.3	8.6	0.6
Test item	440	+	90.9	10.1	0.3	94.5	16.9	1.2
Test item	880	+	61.0	16.8	0.6	75.3	14.1	1.0
Test item	1760	+	31.3	not continued		55.2	not continued
Experiment II, 24 h treatment			Culture I			Culture II
Solvent control		-	100.0	4.6	1.0	100.0	20.4	1.0
P.C. (EMS)		-	99.4	136.3	29.7	87.6	153.1	7.5
Test item	13.8	-	95.3	5.6	1.2	84.6	10.1	0.5
Test item	27.5	-	102.8	6.1	1.3	79.6	8.8	0.4
Test item	55	-	99.0	8.4	1.8	82.3	7.7	0.4
Test item	110	-	84.0	7.5	1.6	79.9	18.6	0.9
Test item	165	-	40.2	13.8	3.0	55.9	6.8	0.3
Test item	220	-	5.2	not continued		11.3	not continued
Experiment II, 4 h treatment			Culture I			Culture II
Solvent control		+	100.0	7.0	1.0	100.0	3.6	1.0
P.C. (DMBA)		+	48.5	487.9	70.1	38.2	444.0	125.1
Test item	27.5	+	100.4	6.8	1.0	101.3	5.3	1.5
Test item	55	+	97.8	12.6	1.8	99.5	6.8	1.9
Test item	110	+	96.9	10.9	1.6	98.8	9.0	2.5
Test item	220	+	100.0	11.0	1.6	102.2	7.0	2.0
Test item	440	+	101.1	13.8	2.0	100.4	5.8	1.6
Test item	880	+	71.6	not continued		86.2	not continued

 

 

 

P.C. = positive control

CE = cloning efficiency

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, BNMA is considered to be non-mutagenic in this HPRT assay.

Executive summary:

In a mammalian cell gene mutation assay according to OECD guideline 476, adopted 21 July 1997 (HPRT test), V79 cells cultured in vitro were exposed to BNMA (98.37% a.i.) at the following concentrations in the presence and absence of mammalian metabolic activation (S9 mix).

Experiment I:

Without metabolic activation: 0 (control), 13.8, 27.5, 55, 110 mg/L

With metabolic activation: 0 (control), 55, 110, 220, 440, 880 mg/L

 

Experiment II:

Without metabolic activation: 0 (control), 13.8, 27.5, 55, 110, 165 mg/L

With metabolic activation: 0 (control), 27.5, 55, 110, 220, 440 mg/L

BNMA was tested up to cytotoxic or phase-separation concentrations. The positive controls induced the appropriate response. There was no evidence of induced mutant colonies over background.

In this HPRT test, BNMA was found to be not mutagenic.

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.