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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Not mutagenic in in vitro bacterial reverse mutation (OECD 471) test with and without metabolic activation

- Not mutagenic in in vitro gene mutation study in mammalian cells, OECD 476, Chinese hamster lung fibroblasts (V79), with and without metabolic activation

- Not mutagenic in vitro cytogenicity / chromosome aberration study in mammalian cells, OECD 473, cells of Chinese hamster lung, with and without metabolic activation

Link to relevant study records

Referenceopen allclose all

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
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
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
Target gene:
Gene mutations at the HPRT locus
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:
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.
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

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

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.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Japanese reference, only abstract and tables in English available, sited in SIDS
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
Mitsui chemicals, Inc., Lot No.710130
Purity: >= 99.5%
Species / strain / cell type:
Chinese hamster lung (CHL/IU)
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
-S9 mix (continuous treatment): 0, 0.23, 0.45, 0.90 (10mM) mg/mL
-S9 mix (short-term treatment): 0, 0.23, 0.45, 0.90 (10mM) mg/mL
+S9 mix(short-term treatment): 0, 0.23, 0.45, 0.90 (10mM) mg/mL
Vehicle / solvent:
Solvent: distilled water
Key result
Species / strain:
mammalian cell line, other: a clone of normal chinese hamster lung cell line
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: Toxicity was not observed up to 0.90 mg/mL (10mM).
Vehicle controls validity:
valid
Positive controls validity:
valid
Conclusions:
In a Chromosomal aberration test with CHL/IU cells according to OECD 473 in the presence and absence of mammalian activation mutagenicity
was not observed up to 0.90 mg/mL (10mM).
Executive summary:

In a mammalian cell cytogenetics assay Chromosome aberration , CHL/IU cell cultures were exposed to Methacrylamide (>= 99.5% in destilled water) at concentrations of 0, 0.23, 0.45, 0.90 (10.0 mM) mg/ml with and without metabolic activation (S9 -mix from rat liver, induced with phenobarbital and 5,6 -benzoflavone).

Methacrylamide was tested to limit concentration 0.90 mg/ml (10.0 mM).

Positive controls induced the appropriate response.  There was no evidence of Chromosome aberration induced over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 473 for in vitro cytogenetic mutagenicity data.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
. Japanese reference, only abstract and tables in English available. Japanese government peer-reviewed the documents, audited selected studies.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
JAPAN: Guidelines for Screening Mutagenicity Testing of Chemicals
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Mitsui chemicals, Inc., Lot No.710130
Purity: >= 99.5%
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
-S9 mix; 0, 313, 625, 1250, 2500, 5000 µg/plate
+S9 mix; 0, 313, 625, 1250, 2500, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: distilled water
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
not specified
Details on test system and experimental conditions:
Test conditions:
System of testing:
-Metabolic activation system: S9 from rat liver, induced with phenobarbital and 5,6-benzoflavone. Administration:
-Number of replicates: 2
-Plate per test: 3
-Application: pre-incubation
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: Toxicity was not observed up to 5000 µg/plate
Vehicle controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: Toxicity was not observed up to 5000 µg/plate
Vehicle controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: Toxicity was not observed up to 5000 µg/plate
Vehicle controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: Toxicity was not observed up to 5000 µg/plate
Vehicle controls validity:
valid
Key result
Species / strain:
E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: Toxicity was not observed up to 5000 µg/plate
Vehicle controls validity:
valid
Conclusions:
Negative in the presence and absence of mammalian metabolic activation
Methacrylamide has to be judged as nonmutagenic up to 5000 µg/plate in the presence and absence of mammalian metabolic activation in bacterial reverse mutation assay.
Executive summary:

In a reverse gene mutation assay in bacteria (Ames test) acc. OECD 471 strains TA1535, TA1537, TA98 and TA100 of S. typhimurium and Escherichia coli WP2 uvrA were exposed to Methacrylamide at concentrations of up to 5000 µg/plate in the presence and absence of mammalian metabolic activation. 

Methacrylamide was tested to limit concentrations 5000 µg/plate. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background. 

This study is classified as acceptable. This study satisfies the requirement for tests for in vitro mutagenicity (bacterial reverse gene mutation) data.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

- Not mutagenic in in vivo Mouse bone marrow micronucleus test, OECD 474.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04-Feb.-2003 - 08-Apr.- 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
9 th addendum to OECD Section 4, No. 474 adopted July 21, 1997
Deviations:
no
Principles of method if other than guideline:
Pre-Experiment for Toxicity
A preliminary study on acute toxicity was performed with two animals per sex under identical conditions as in the mutagenicity study concerning:
starvation period, animal strain; vehicle; route, frequency, and volume of administration.
The animals were treated orally (200, 350, 500, 1000 and 1800 mg/kg bw in 1% CMC) with the test item and examined for acute toxic symptoms at
intervals of around 1 h, 2-4 h, 6 h, 24 h, 30 h, and 48 h after administration of the test item.

Deviations from the study plan:
In the main experiment not all the animals treated with the high and mid dose (350 and 175 mg/kg bw) were observed for clinical signs of toxicity at
the 6h post treatment interval.
This deviation, however, does not affect the validity of the experiment.
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay
Specific details on test material used for the study:
- Supplier: Evonik Röhm GmbH
- Purity: 99.3% (HPLC analysis)
- Lot/batch No.: 11121211 of December 20, 2002
- Expiration date of the lot/batch: December 2003
- Stability under test conditions: at least 15 hours in aqua deionised
- Storage condition of test material: Refrigerator (+4°C), light protected
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd., 4414 Füllinsdorf, Switzerland
- Number of animals. 72 (36 males/36 females)
- Age at study initiation: Males: 5 - 7 weeks; Females: 7 - 9 weeks
- Weight at study initiation: males mean value 33.9 g (SD ± 2.2 g)
females mean value 25.6 g (SD ± 1.7 g)
- Assigned to test groups randomly: yes
- Fasting period before study: 18 hours before treatment
- Housing: single
- Diet: pelleted standard diet,ad libitum
- Water: tap water, ad libitum
- Acclimation period: minimum 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ±3°C
- Humidity (%): 30 - 70 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs artifical light
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: 1% CMC (carboxymethyl cellulose)
- Justification for choice of solvent/vehicle: vehicle was chosen to its relative non-toxicity for animals. All animals received a sigle standard volume of 10 mL/kg body weight orally.
- Concentration of test material in vehicle:
- Supplier: Fluka; SIGMA-Aldrich Vertiebs-GmbH, 82041 Deisenhofen, Germany
- Lot/batch no.: no data
- Catalogue no.: 21902
- Purity: no data
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment, Methacrylamide was formulated in 1% CMC. All animals received a single standard volume of 10 ml/kg body weight
orally.

DIET PREPARATION
- Rate of preparation of diet (frequency): no data
- Mixing appropriate amounts with (Type of food): no data
- Storage temperature of food: no data
Duration of treatment / exposure:
24h and 48h
Frequency of treatment:
single dose
Dose / conc.:
87.5 mg/kg bw/day (nominal)
Remarks:
24 h
Dose / conc.:
175 mg/kg bw/day (nominal)
Remarks:
24 h
Dose / conc.:
350 mg/kg bw/day (nominal)
Remarks:
24 h
Dose / conc.:
350 mg/kg bw/day (nominal)
Remarks:
48 h
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
CPA; cyclophosphamide
- Supplier: Fluka; SIGMA-Aldrich Vertiebs-GmbH, 82041 Deisenhofen, Germany
- Purity: > 98%
- Dissolved in: deionised water
- Route of administration: orally, once
- Doses / concentrations: 40 mg/kg bw
Volume administered: 10 mL/kg bw

The stability of CPA at room temperature is sufficient. At 25 °C only 3.5% of its potency is lost after 24 hours.

Historical Controls
------------------
1999 - 2001
Negative controls:
Percent micronucleated polychrornatic erythrocytes
Range:0.010 to 0.150
Mean: 0.066* ± 0.032
Positive controls:
Cyclophosphamide; 40 mglkg b.w. [CPA]
Percent micronucleated polychromatic erythrocytes
Range:0.910 to 2.975
Mean: 1.644*± 0.446
*= mean of 95 experiments
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly or
2000 mglkg as the upper limit for non-toxic test items.
The maximum tolerated dose level is determined to be the dose that causes toxic reactions without having major effects on survival within 48
hours.
The volume to be administered should be compatible with physiological space available.
Three adequate spaced dose levels spaced by a factor of 2 were applied at the central sampling interval 24 h after treatment. For the highest dose
level an additional sample was taken at 48 h after treatment.

The highest dose (350 mg/kg) was estimated by a pre-experiment to be suitable.


TREATMENT AND SAMPLING TIMES:

Treatment:
Approximately 18 hours before treatment the animals received no food but water ad libitum. At the beginning of the treatment the animals (including
the controls) were weighed and the individual volume to be administered was adjusted to the animals body weight. The animals received the test item, the vehicle or the positive control substance once. Twelve animals, six males and six females, were treated per dose group and sampling time. The
animals of the highest dose group were examined for acute toxic symptoms at intervals of around 1 h, 2-4h, 6 h and 24 h after administration of the
test item.
Sampling of bone marrow was done 24 and 48 hours after treatment,respectively.


DETAILS OF SLIDE PREPARATION:

Preparation of the Animals:
The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal
calf serum, using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small
drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Griinwald (MERCK, D-64293
Darmstadt, Germany)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-79110 Freiburg, Germany). At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
Analysis of Cells:
Evaluation of the slides was performed using NlKON microscopes with 100x oil immersion objectives. At least 2000 polychromatic erythrocytes (PCE)
were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and total erythrocytes was determined in thesame sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides.
Ten animals (5 males, 5 females) per test group were evaluated as described.
Evaluation criteria:
The study was considered vailid as the following criteria are met:
- the negative controls are in the range of our historical control data (0.01 - 0.15 %; mean= 0.066 ± 0.032 PCEs with micronuclei).
- the positive controls are in the range of our historical control data (0.91 - 2.975 %; mean = 1.644 ± 0.446 PCEs with micronuclei).
- at least 80 % of animals are evaluable
Statistics:
Statistical methods (nonparametric Mann-Whitney test) will be used as an aid in evaluating the results. However, the primary point of consideration
is the biological relevance of the results.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY (see Remarks on results including tables and figures)
- Dose range: 200 - 1800 mg/kg bw

Results:  

Pre-Experiment for Toxicity In the pre-experiment 4 animals (2 males, 2 females) per dose group received orally a single dose of 1800, 1000, 500, 350 and 200 mg/kg b.w. Methacrylamide formulated in 1% CMC. The volume administered was 10 ml/kg b.w.. The animals treated with Methacrylamide expressed toxic reactions as shown in the table:

toxic

reactions

hours post-treatment (male/female) 1800 mg/kg bw

hours post-treatment (male/female) 1000 mg/kg bw

hours

1

2-4

6

24

30

48

1

2-4

6

24

30

48

Reduction of spontaneous activity

2/2

2/2

2/2

0/0

-

-

2/2

2/2

2/2

0/0

-

-

Abdominal position

1/1

2/2

2/2

0/0

-

-

0/0

1/1

1/1

0/0

-

-

Eyelid closure

2/1

2/2

2/2

0/0

-

-

2/1

2/1

2/1

0/0

-

-

Ruffled fur

2/2

2/2

2/2

0/0

-

-

2/2

2/2

2/2

0/0

-

-

Apathy

1/0

1/1

1/1

0/0

-

-

0/0

1/1

1/1

0/0

-

-

death

0/0

0/0

0/0

2/2

-

-

0/0

0/0

0/0

2/2

-

-

toxic

reactions

hours post-treatment (male/female) 500 mg/kg bw

hours post-treatment (male/female) 350 mg/kg bw

hours

1

2-4

6

24

30

48

1

2-4

6

24

30

48

Reduction of spontaneous activity

2/2

2/2

2/2

2/0

0/-

0/-

1/1

2/2

2/2

0/0

0/0

0/0

Eyelid closure

0/0

2/2

2/2

0/0

0/-

0/-

1/0

0/0

0/0

0/0

0/0

0/0

Ruffled fur

2/0

2/2

2/2

2/0

0/-

0/-

1/1

1/1

1/1

0/0

0/0

0/0

death

0/0

0/0

0/0

0/2

0/-

0/-

n.d.

n.d.

n.d.

n.d

n.d.

n.d.

toxic

reactions

hours post-treatment (male/female)

200 mg/kg bw

hours

1

2-4

6

24

30

48

Reduction of spontaneous activity

2/1

0/0

0/0

0/0

0/0

0/0

Ruffled fur

1/1

1/0

0/0

0/0

0/0

0/0

Toxic symptoms in the Main Experiment

In the main experiment for the highest dose group 24 animals (12 males, 12 females) received orally a single dose of 350 mg /kg b.w.

Methacrylamide formulated in 1% CMC. The volume administered was 10 ml/kg b.w.. The animals treated with 350 mg /kg b.w. expressed

toxic reactions as shown in the table:

Toxic reactions

hours post treatment

male/female

Hours [hr]

1

2-4

6*

24

Reduction of spontaneous activity

9/8

9/10

3/5

0/1

Abdominal position

1/0

0/0

0/0

0/0

Eyelid closure

4/4

5/7

3/5

2/1

Ruffled fur

6/1

7/5

3/3

7/1

*: at the 6h post treatment interval only 6 of the 12 animals were observed.

For the mid dose group 12 animals (6 males, 6 females) received orally a single dose of 175 mg /kg b.w. Methacrylamide formulated in

1% CMC. The volume administered was 10 mllkg b.w..

The animals treated with 175 mg /kg b.w. expressed toxic reactions as shown in the table:

Toxic reactions

hours post treatment

male/female

Hours [hr]

1

2-4

6*

24

Reduction of spontaneous activity

2/2

4/2

-*/1

0/0

Eyelid closure

2/0

2/2

-*/2

0/0

Ruffled fur

2/0

4/0

-*/0

0/0

*: at the 6h post treatment interval the 6 males were not observed.

For the low dose group 12 animals (6 males, 6 females) received orally a single dose of 87.5 mg /kg b.w. Methacrylamide formulated in

1% CMC. The volume administered was 10 mllkg b.w..

The animals treated with 87.5 mg /kg b.w. did not express any toxic reactions.

Summary of Micronucleus Test Results

Test group

Dose

mg/kg bw.

Sampling

Time [hr]

PCEs with micro-nuclei [%]

Range

PCE per 2000

erythocytes

Vehicle control versus test group

Signifi-cance

p

Vehicle

0

24

0.030

0-2

1025

n.d.

n.d.

Methacrylamide

87.5

24

0.035

0-2

1037

-

0.5000

Methacrylamide

175

24

0.040

0-2

1041

-

0.4000

Methacrylamide

350

24

0.035

0-3

1023

-

0.5000

Positive control CPA

40

24

1.220

9-46

1015

+

0.0001

Methacrylamide

350

48

0.055

0-2

1014

-

0.1356

n.d.: not determined

-: not significant; +: significant

Statistical significance at the five per cent level (p 0.05) was evaluated by means of the non-parametric Mann-Whitney test.


The mean number of polychromatic erythrocytes was not increased after treatment with the test item as compared to the mean value of polychromatic erythotrcytes (PCEs) of the vehicle control indicating that Methacrylamide had no cytotoxic properties in the bone marrow.
In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with Methacrylamide were below or near to the value of the vehicle control group.
40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a statistically significant increase of induced micronucleus frequency.
Conclusions:
Interpretation of results: negative
During the study decribed and under the experimental conditions reported, Methacrylamide did not induce micronuclei. This was determined by the
micronucleus test with bone marrow cells of the mouse (OECD guideline 474).
Therefore, Methacrylamide is considered to be non-mutagenic in this micronucleus assay.
Executive summary:

In a NMRI mouse bone marrow micronucleus assay, 5 animals/male/female/dose were treated orally with Methacrylamide (99.3%) at doses of 0, 87.5, 175 and 350 mg/kg bw. Bone marrow cells were harvested at 24 and 48 hours post-treatment. The vehicle was 1% CMC (carboxymethyl cellulose). The animals received orally a single dose of Methacrylamide.  

There were no signs of toxicity during the study. Methacrylamide was tested at an adequate dose up to 350 mg/kg bw. The positive control induced the appropriate response.  There was not a significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time.

This study is classified as accceptable. This study satisfies the requirement for Test Guideline OECD 474 for in vivo cytogenetic mutagenicity data.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Positive response was observed in OECD 473 (Mitsui Chem 1993) in concentrations above the max. exposure concentration of 10 mM.

At the dose levels less than 2.50 mg/mL response was not significantly different from the negative control group. In the metabolic activation method with and without S9 -mix the frequency of structural aberrations at all doses were not significantly different from that of the negative control group.

Justification for classification or non-classification

Methacrylamide is not mutagenic in in vitro gene mutation tests OECD 471 (Ames test with Salmonella typhimurium TA100, TA1535, TA98 and TA1537, Escherichia coli  WP2 uvrA) and OECD 476 (in vitro mammalian gene mutation test with Chinese hamster lung cells, V79, HPRT-test) and not mutagenic in mammalian cytogenetic chromosome aberration test acc. OECD 473 with CHL/IU cells up to limit conc. of 10 mM.

Additionally methacrylamide was not mutagenic in vivo cytogenetic mouse micronucleus test acc. OECD 474 in NMRI mice.

Based on available data, the classification criteria are not met.