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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:
Jun 27 - Aug 22, 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted July 21, 1997
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

Constituent 1
Chemical structure
Reference substance name:
Methacrylamide
EC Number:
201-202-3
EC Name:
Methacrylamide
Cas Number:
79-39-0
Molecular formula:
C4H7NO
IUPAC Name:
methacrylamide
Test material form:
solid
Specific details on test material used for the study:
- Supplier: Evonik Röhm GmbH, Darmstadt, Germany
- Purity: 99.9%
- Lot/batch No.: 11171114
- Expiration date of the lot/batch:
- Stability under test conditions: stable
- Storage condition of test material: At room temperature (+15 to +25°C), light protected

Method

Target gene:
Gene mutations at the HPRT locus
Species / strain
Species / strain / cell type:
Chinese hamster lung (CHL/IU)
Details on mammalian cell type (if applicable):
V79
Metabolic activation:
with and without
Metabolic activation system:
Mammalian liver microsomal fraction S9 mix
Test concentrations with justification for top dose:
Experiment I:
-S9 mix; 26.9, 53.8, 107.5, 215.0, 430.0, 860.0 µg/ml
+S9 mix; 26.9, 53.8, 107.5, 215.0, 430.0, 860.0 µg/ml

Experiment II:
-S9 mix; 26.9, 53.8, 107.5, 215.0, 430.0, 860.0 µg/ml
+S9 mix; 26.9, 53.8, 107.5, 215.0, 430.0, 860.0 µg/ml

In both main experiments the cultures at the lowest concentration without metabolic activation (26.9 µg/mL) were not continued since a minimum of only four analysable concentrations is required by the guidelines.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle:
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Remarks:
concurrent solvent controls (deionised water)
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension

DURATION
- Exposure duration: Experiment I: 4 hours with and without metabolic activation
Experiment II: 4 hours with and 24 hours without metabolic activation. The experimental parts of the second experiment with
and without metabolic activation were performed in two separate experiments (experiment II and IIA) for technical reasons. The
results are combined and reported as experiment II.
NUMBER OF REPLICATIONS:


NUMBER OF CELLS EVALUATED: The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation
microscope (Nikon, 40407 Düsseldorf, Germany).
Evaluation criteria:
Acceptability of the Assay
The gene mutation assay is considered acceptable if it meets the following criteria:
- the numbers of mutant colonies per 10exp+6 cells found in the negative and/or solvent controls fall within the laboratory historical control data
range of 2001 – 2006.
- the positive control substances must produce a significant increase in mutant colony frequencies (Historical data).
- the cloning efficiency II (absolute value) of the negative and/or solvent controls must exceed 50 %.

Evaluation of Results
A test item is regarded as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive
response at one of the test points.
A test item producing neither a concentration- related increase of the mutant frequency nor a reproducible positive response at any of the test
points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is regarded as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency
at least at one of the concentrations in the experiment.
The test item is regarded as mutagenic if there is a 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.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low
spontaneous mutation rate in the range normally found (0.5 – 31.8 mutants per 10exp+6 cells) a concentration-related increase of the mutations
within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.
Statistics:
Statistical Analysis
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT®11 (SYSTAT
Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software. 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. However, both, 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
Remarks:
in forward gene mutations in mammalian cells
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: strain/cell type: V79

Any other information on results incl. tables

Results and Disscussions

Methacrylamide was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.

The assay was performed in two independent experiments with identical experimental procedures, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was performed with a treatment period of 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation.

No precipitation of the test item was observed up to the maximal concentration of 860.0 µg/mL (corresponding to a molar concentration of about 10 mM) in all experiments.

No relevant toxic effects indicated by a reduction of the relative cloning efficiency 1 to values below 50 % of the corresponding control occurred in any part of the project.

No relevant and reproducible increase in mutant colony numbers/106cells was observed in the main experiments up to the maximum concentration. The induction factor did not reach the threshold of 3.0 at any experimental point and no statistically trend occurred.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT ® statistics software. No significant dose dependent trend of the mutation frequency indicated by a probability value of < 0.05 was determined in any of the experimental groups.

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 11.5 up to 31.3 mutants per 106 cells; the range of the groups treated with the test item was from 4.9 up to 36.0 mutants per 106cells. The highest solvent control value (31.3 mutant colonies per 106cells) slightly exceeded the range of historical data (1.1-29.1 mutant colonies per 106 cells). The mean value of both parallel cultures however, (31.3 and 15.6 equal to 23.5 colonies per 106 cells) is fully acceptable.

EMS (150 µg/mL in experiment I and 75 µg/mL in experiment II) and DMBA (1.3 µg/mL in experiment I and 1.1 µg/mL in experiment II) were used as positive controls and showed a distinct increase in induced mutant colonies. This showed the sensitivity of the test system and the activity of the S9 mix.

Summary of results

relative

relative

mutant

relative

relative

mutant

Conc.

S9 mix

cloning

cloning

Colonies/

induction

cloning

cloning

Colonies/

induction

µg/mL

Efficientcy 1

Efficientcy 2

106 cells

factor

106 cells

factor

%

%

%

%

Column

1

2

3

4

5

6

7

8

9

10

Exp.I
 4 hours

Culture I

Culture II

Solvent contr. water

-

100.0

100.0

18.9

1.0

100.0

100.0

25.4

1.0

Pos. contr.

150.0

-

100.0

106.6

95.1

5.0

100.0

103.5

119.4

4.7

Methacrylamide

26.9

-

89.1

Culture was not continued#

81.2

Culture was not continued#

Methacrylamide

53.8

-

118.6

110.9

20.2

1.1

92.5

115.2

17.2

0.7

Methacrylamide

107.5

-

103.2

108.1

14.3

0.8

99.2

108.6

27.2

1.1

Methacrylamide

215.0

-

109.8

108.7

19.3

1.0

89.7

111.3

26.0

1.0

Methacrylamide

430.0

-

108.0

110.3

31.3

1.7

91.3

109.6

24.4

1.0

Methacrylamide

860.0

-

120.0

117.6

22.0

1.2

84.5

108.1

33.6

1.3


Solvent contr. water

+

100.0

100.0

31.3

1.0

100.0

100.0

15.6

1.0

Pos.contr. DMBA

1.3

+

29.3

92.6

1397.2

44.6

31.3

81.2

1420.5

90.9

Methacrylamide

26.9

+

99.9

Culture was not continued#

99.0

Culture was not continued#

Methacrylamide

53.8

+

96.7

150.4

13.7

0.4

90.5

109.5

15.2

1.0

Methacrylamide

107.5

+

93.8

140.2

11.1

0.4

89.2

116.4

14.8

0.9

Methacrylamide

215.0

+

83.3

184.3

14.1

0.4

82.2

106.0

17.4

1.1

Methacrylamide

430.0

+

92.9

129.7

4.9

0.2

90.6

113.6

13.5

0.9

Methacrylamide

860.0

+

107.7

175.8

13.2

0.4

93.9

110.4

15.9

1.0

Exp.II
 24 hours

Culture I

Cluture II

Solvent contr. water

-

100.0

100.0

28.3

1.0

100.0

100.0

28.8

1.0

Pos. contr.

75.0

-

53.3

65.5

337.6

11.9

54.3

60.3

409.7

14.5

Methacrylamide

26.9

-

102.2

Culture was not continued#

94.2

Culture was not continued#

Methacrylamide

53.8

-

92.4

108.4

5.4

0.2

90.7

91.3

20.6

0.7

Methacrylamide

107.5

-

91.7

108.7

18.9

0.7

93.3

91.0

26.2

0.9

Methacrylamide

215.0

-

94.1

107.9

11.9

0.4

93.7

85.7

36.0

1.3

Methacrylamide

430.0

-

105.7

94.5

16.9

0.6

93.7

106.2

15.9

0.6

Methacrylamide

860.0

-

97.2

94.7

22.8

0.8

97.8

97.3

23.5

0.8


Solvent contr. water

+

100.0

100.0

11.5

0.4

100.0

100.0

22.1

0.8

Pos.contr. DMBA

1.1

+

13.1

103.7

1477.0

52.2

10.6

79.4

1438.5

50.8

Methacrylamide

26.9

+

95.3

Culture was not continued#

86.4

Culture was not continued#

Methacrylamide

53.8

+

85.2

133.0

24.8

0.9

88.0

71.2

20.1

0.7

Methacrylamide

107.5

+

83.5

147.6

21.8

0.8

101.4

88.3

12.0

0.4

Methacrylamide

215.0

+

91.0

148.4

16.3

0.6

87.8

72.8

18.4

0.6

Methacrylamide

430.0

+

89.9

125.4

24.6

0.9

96.9

86.3

14.4

0.5

Methacrylamide

860.0

+

88.4

138.2

19.7

0.7

78.0

82.1

17.2

0.6

# culture was not continued since a minimum of only four analysable concentrations is required

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 Methacrylamide did not induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.
Therefore, Methacrylamide is considered to be non-mutagenic in this HPRT assay.
Executive summary:

In a mammalian cell gene mutation assay HPRT locus using V79 cells of the Chinese hamster cultured in vitro were exposed to Methacrylamide (99.9%) dissolved in deionised water at concentrations of 53.8; 107.5; 215.0; 430.0 and 860.0 µg/mL in the presence and absence of mammalian metabolic activation S9 mix. 

The assay was performed in two independent experiments. The cells were exposed to Methacrylamide for 4 hours in the first experiment with and without metabolic activation. The second experiment was performed with a treatment period of 24 hours in the absence and 4 hours in the presence of metabolic activation.

The maximum dose was 860 μg/mL corresponding to a molar concentration of about 10 mM.

The positive controls did induce the appropriate response. No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiments up to the maximum concentration. There was no evidence of induced mutant colonies over background. 

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.

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