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Toxicological information

Genetic toxicity: in vitro

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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 guidelineopen allclose all
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
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

Constituent 1
Chemical structure
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 2-methylprop-2-enoate
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
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
other: (without metabolic activation)
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.

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