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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation test (Ames test)

not mutagenic in test strains TA1535; TA97/ TA97a,TA1537; TA98; TA100; TA102 (and further strains outside OECD TG 471, TA104 and TA1538)

(NTP method, Zeiger et al. 1987), (Schweikl, 1994)

Cytogenicity in mammalian cells

In vitro cytogenetic test in mammalian cells (Chromosomal aberration test in CHO cells): not mutagenic (Anderson et al. 1990)

Gene mutation in mammalian cells
Mouse lymphoma assay: weakly positive with and without metabolic activation (Cifone/Litton 1981)
HPRT test, V79 cells: Ambiguous to weakly positive without metabolic activation (Schweikl et al. 1998)
Mouse lymphoma assay, clastogenicity tested: positive without metabolic activation, primarily small colony mutants indicating deletion mutations instead of gene mutations (Moore et al. 1988)



Link to relevant study records

Referenceopen allclose all

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:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Only 4 strains of Salmonella typhimurium were tested
Principles of method if other than guideline:
Only 4 strains of Salmonella typhimurium were tested
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Supplied by Rohm and Haas; 99+% pure
Target gene:
his+
Species / strain / cell type:
other: S. typhimurium TA 1535, TA97, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
AROCLOR 1254 induced rat and Syrian hamster liver S9 mix.
Test concentrations with justification for top dose:
33 - 6666 ug/plate
Test concentrations: 0, 33, 100, 1000, 3333, 6666 µg/plate
At 6666 µg/plate slight cytotoxicity was observed
Vehicle / solvent:
Not specified: vehicles used included water, DMSO, ethanol or acetone
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: without S9: 4-nitro-o-phenylenediamine (TA98). With S9: 2-aminoanthracene (all strains)
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
- Solvent: DMSO
- Cell density at seeding (if applicable):
- After addition of soft agar, the contents of each tube was poured onto minimal medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 minutes, 37 °C
- Exposure duration/duration of treatment: 48 hours, 37 °C
Evaluation criteria:
An individual trial was judged (+) mutagenic if a dose related increase over the corresponding solvent control was seen, and it was judged weakly mutagenic (+W) if a low dose response was seen. A trial was considered questionable (?) if a dose related increase was judged insufficiently high to justify a call of "+W" if only a single dose was elevated over control or if a non-dose related increase was seen. A chemical was judged weakly mutagenic "+W' or mutagenic "+" if it produced a reproducible, dose related increase in his+ revertants over the corresponding solvent controls in replicate trials.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Mutagenicity of Methyl methacrylate in Salmonella Typhimurium

 

 

Revertants per plate (a,b)

Strain

Dose

(µg/plate)

-S9

+S9 (rat)

+S9 (hamster)

TA100

0

140 +/- 6.7

141 +/- 8.0

131 +/- 4.7

 

33

118 +/- 5.6

-

-

 

100

122 +/- 21.1

157 +/- 10.2

143 +/- 9.9

 

333

126 +/- 10.5

143 +/- 7.1

145 +/- 4.1

 

1000

136 +/- 5.0

138 +/- 11.6

130 +/- 8.5

 

3333

129 +/- 8.8

121 +/- 10.7

116 +/- 6.2

 

6666

-

(c) 48+/- 16.3

(c) 65+/- 3.5

 

 

 

 

 

TA1535

0

26 +/- 4.4

9 +/- 1.5

6 +/- 0.6

 

33

22 +/- 2.2

-

-

 

100

18 +/- 1.7

6 +/- 1.2

143 +/- 9.9

 

333

17+/- 2.5

8 +/- 2.1

145 +/- 4.1

 

1000

16 +/- 2.3

7 +/- 1.0

130 +/- 8.5

 

3333

16 +/- 2.9

7 +/- 1.2

116 +/- 6.2

 

6666

-

(c) 4+/- 0.9

(c) 65+/- 3.5

 

 

 

 

 

TA97

0

145 +/- 4.5

180 +/- 9.4

248 +/- 105.5

 

33

145 +/- 13.6

-

-

 

100

151 +/- 16.6

175 +/- 11.6

152 +/- 9.1

 

333

138 +/- 14.0

190 +/- 2.3

157 +/- 10.6

 

1000

149 +/- 1.0

195 +/- 4.3

158 +/- 6.3

 

3333

156 +/- 3.8

166 +/- 7.3

169 +/- 4.8

 

6666

-

(c) 64 +/- 31.8

(c) 89+/- 7.7

 

 

 

 

 

TA98

0

22 +/- 2.3

2.9 +/- 4.9

26 +/- 2.0

 

33

20 +/- 2.4

-

-

 

100

18 +/- 1.5

32 +/- 2.2

24 +/- 2.6

 

333

23 +/- 3.0

26 +/- 3.2

25 +/- 1.0

 

1000

17 +/- 1.0

27 +/- 1.5

22 +/- 2.1

 

3333

19 +/- 0.3

16 +/- 2.3

18 +/- 1.2

 

6666

-

(c) 0 +/- 0.0

(c) 5 +/- 2.1

a)     S9 fractions prepared from the liver of Acrolor 1254-induced male Sprague-Dawley rats and male Syrian hamsters

b)    Mean +/- standard error

c)     Slightly toxic

Conclusions:
Methyl methacrylate was tested in bacterial reverse mutation test in Salmonella typhimurium (Ames test) in 4 strains (TA97, TA98, TA100 and TA1535) at test concentrations of 33 - 6666 µg/plate. Slight cytotoxicity was observed at 6666 µg/plate.
Methyl methacrylate was not mutagenic in bacterial reverse mutation test in Salmonella typhimurium (TA97, TA98, TA100 and TA1535).
Executive summary:

Methyl methacrylate was tested in  bacterial reverse mutation test in Salmonella typhimurium (Ames test) in 4 strains (TA97, TA98, TA100 and TA1535) at test concentrations of 33 - 6666 µg/plate. Slight cytotoxicity was observed at 6666 µg/plate. Methyl methacrylate was not mutagenic in  bacterial reverse mutation test in Salmonella typhimurium strains TA97, TA98, TA100 and TA1535.

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:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
other: according to Maron D.M. and Ames B.N.; Mutation Res. 113: 173 - 215 (1983)
Deviations:
yes
Remarks:
Test Strains: Salmonella typhimurium TA97a, TA98, TA100, TA102, TA104
Principles of method if other than guideline:
Only 4 of the 5 tested strains are the recoomended strains accordin to OECD 477
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Supplier: E Merck, M 800 590, Darmstadt, Germany
Target gene:
his+
Species / strain / cell type:
other: Salmonella typhimurium TA 97a, TA 98, TA 100, TA 102 and TA 104
Metabolic activation:
with and without
Metabolic activation system:
prepared from rat liver microsomes
Test concentrations with justification for top dose:
0 - 25 mg/plate
Vehicle / solvent:
DMSO (eluates with S9)
Physiological saline (eluates with and without S9)
Negative solvent / vehicle controls:
yes
Positive control substance:
sodium azide
cumene hydroperoxide
other: ICR-191 for TA97a (10 µg/plate); 2,4,7-TNFone for TA98 (0.5 µg/plate); glutaraldehyde for TA104a (100 µg/plate)
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation
NUMBER OF REPLICATIONS: 3
Evaluation criteria:
according to Maron D.M. and Ames B.N.; Mutation Res. 113: 173 - 215 (1983)
Species / strain:
S. typhimurium, other: TA97a
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at the highest tested dose (25mg/plate)
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at the highest tested dose (25mg/plate)
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at the highest tested dose (25mg/plate)
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA104
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid

Mutagenicity of DMSO Eluates of MMA in Absence of Liver Microsomes (S9)

Dose
(mg/plate)
TA97a TA98 TA100 TA102 TA104
0.000 146 +/-8 28 +/- 2 149 +/-9 239 +/- 23 362 +/- 7
0.005       248 +/- 8 364 +/- 13
0.025       267 +/- 2 319 +/- 15
0.050 154 +/-10 26 +/- 3 130 +/-6 264 +/- 3 357 +/- 23
0.250       256 +/- 1 343 +/- 2
0.500 141 +/-4 24 +/- 5 129 +/-15 256 +/- 15 328 +/- 22
1.250       223 +/- 15 339 +/- 18
2.500       221 +/- 33 318 +/- 2
2.750       234 +/- 8 329 +/- 6
5.000 127 +/-7 24 +/- 5 112 +/-7 258 +/- 10 327 +/- 12
12.500       274 +/- 13 301 +/- 8
25.000       230 +/- 3 188 +/- 32
Conclusions:
Methyl methacrylate is not mutagenic in bacterial reverse mutaion test in Salomonella typhimurium strains TA97a, TA98, TA100 and TA102 and in the additionally tested not in OECD 471 recommended strain TA104.
Executive summary:

Methyl methacrylate was tested in bacterial reverse mutation test in Salmonella typhimurium (Ames test) in 4 strains which are recommended in OECD 471 (TA97a, TA98, TA100 and TA102) and additionally in strain TA104 at test concentrations of 0.005 – 25 mg/plate µg/plate. Slight cytotoxicity was observed at 5 mg/plate in strains TA97a, TA98 and TA100.

Methyl methacrylate was not mutagenic in bacterial reverse mutation test in Salmonella typhimurium TA97a, TA98, TA100 and TA102 and in the additionally tested not in OECD 471 recommended strain TA104.

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:
comparable to guideline study
Remarks:
Method and results sufficient described
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Principles of method if other than guideline:
As described by Galloway SM, Bloom, A, Resnick, M, Margolm, B, Nakamura, F, Archer, P, Zeiger, E (1985) Development of a standard protocol for in vitro cytogenetic testing with CHO cells: Comparison of results for 22 compounds in two laboratories. Environ Mutagen 7:1-52
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
Supplier: Rohm & Haas
Purity: > 99 %
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
Cloned at Litton Bionetics
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced male Sprague-Dawley rats
Test concentrations with justification for top dose:
without S9-mix: 750 - 3000 µg/mL
with S9-mix: 160 - 5000 µg/mL
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: without S9: 0.05 µg/mL mitomycin C; With S9: 50 µg/mL cyclophosphamide
Remarks:
A low dose postive control was included to determine if the assay could detect 20-60% increase over the solvent control.
Details on test system and experimental conditions:
Approximately 24 hrs before treatment cells were initiated at a density of 1.2-1.75x10e+6 cells/75 cm2 flask. For the trials without S9 the cells were incubated with the appropriate control or chemical for 8 hrs. For the trials with S9 the cells were treated with S9 and test chemical in serum free medium for 2 hrs, washed, resuspended in medium containing serum and incubated for an additional 8-10 hrs.
One hundred cells per dose were scored.
Evaluation criteria:
If the low dose positive control was less than 20% and the test concentrations did not show significant increases, the trial was to be concluded a "no test".
If a trial had a positive trend and no significant doses, or if there was no trend and only one significant dose the trial was considered equivocal (?); if a trial had significant trend and one significant dose it was judged weak positive (+W) and if the trial had two significant doses it was judged positive (+), whether or not a positive trend was obtained. If only one dose was significant and the increase over the control was P<0.0005 the trial was denoted (+W).
Statistics:
ABS data were evaluated for both trend and dose point increase over the solvent control. A binomial sampling assumption was used to evaluate an absolute increase in aberrations over solvent control. Dose points with P values adjusted by Dunnett's method were considered significant if <0.05, whereas a trend of P<0.003 was significant.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
ambiguous
Remarks:
positive without S9 (lowest effective dose = 1600 µg/ml); weak positiv with S9 (lowest effective dose =5000 µg/ml)
Cytotoxicity / choice of top concentrations:
other: yes, but not specified. At least the 5000 µg/mL dose has to be considered as cytotoxic dose
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
negative
Remarks:
weak positiv with S9 (lowest effective dose =5000 µg/ml)
Cytotoxicity / choice of top concentrations:
other: yes, but not specified. At least the 5000 µg/mL dose has to be considered as cytotoxic dose
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid

Results of Anderson et al. 1990:

Without S9-mix

Harvest Time 11.5 hrs

 

 

       Percent cells with aberrations

 Dose (µg/mL)

 Cells

 Total

 Simple

 Complex

 0

100 

 0

 0

 0

 15

 100

 3

 3

 0

 50

 100

 2

 2

 0

 150

 100

 0

 0

 0

 500

 100

 2

 2

 1

 1000

 100

 10

 10

 0

 MMC 0.0050

 100

 37

 24

 19

Harvest Time 11.5 hrs

 

 

       Percent cells with aberrations

 Dose (µg/mL)

 Cells

 Total

 Simple

 Complex

 0

100 

 0

 0

 0

 750

 100

 1

 0

 1

1000

 100

 2

 1

 1

1500

 100

 5

 0

 5

3000

 100

 5

 5

 1

 MMC 0.0050

 100

 56

 39

 39

 

With S9-mix

Harvest Time 12 hrs

 

 

       Percent cells with aberrations

 Dose (µg/mL)

 Cells

 Total

 Simple

 Complex

 0

100 

 1

 1

 0

 150

 100

 0

 0

 0

500

 100

 3

 1

2

1500

 100

 2

 2

 0

5000

 100

 30

 27

 5

CPA 50

 100

 63

 46

 43

 

---

Results of NTP 1986:

Without S9-mix

 

    

 Dose (µg/mL)

  

Aberrations per 100 cells

  

Percent cells with aberrations

 

 0

 0

 0

 

 750

 1

 0

 

1000

 2

 1

 

1600

 5

 0

 

3000

 6

 5

 

 MMC 0.0050

 113

 58

 

 

With S9-mix

Harvest Time 12 hrs

 

  

 Dose (µg/mL)

 Aberrations per 100 cells

 

Percent cells with aberrations

 

 0

 1

 1

 

 160

 0

 0

 

500

 3

3

 

1600

 2

 2

 

5000

 44

30

 

CPA 50

 117

63

 

 

---

The result data differ slightly between the two sources; however the mutagenic pattern is the same.

Conclusions:
Methyl methacrylate is not mutagenic in a cytogenetic test with CHO cells of chromosomal aberrations. Methyl methacrylate seems to be a high toxicity clastogen, i. e., the induction of chromosomal aberrations is bound to highly toxic doses.
Executive summary:

In a cytogenetic test with CHO cells induction of chromosomal aberrations was bound to high doses which are assumed to be strongly cytotoxic (Anderson et al., 1990). With S-9 mix, treatment was for 2 h followed by 8 to 10 h recovery. Doses up to 1600 μg/ml were negative, at 5000 μg/ml the frequency of aberrant cells was 30%; only one experiment was performed. Without S-9 mix, treatment time was 8 hours with 2.0 to 2.5 h recovery. Doses up to 500 μg/ml were negative; at 1600 and 3000 μg/ml aberration frequencies ranging from 5 to 6 % were found. Data on cytotoxic effects were not given, however, it can be assumed from the data presentation and the general approach of the authors that the highest doses tested led to strong cytotoxic effects. Thus, methyl methacrylate seems to be a high toxicity clastogen, i. e., the induction of chromosomal aberrations is bound to highly toxic doses.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1981-05-05 to 1981-08-31
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
other: In vitro mammalian cell gene mutation test / mouse lymphoma assay
Version / remarks:
pre guideline study
(OECD guidline 476 was at first adopted 1997)
Principles of method if other than guideline:
Mouse Lymphoma Forward Mutation Assay (Rohm and Haas Protocol No. 81P-259)
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
Lot 354058
- Name of test material (as cited in study report): Methyl methacrylate (TD-80-254)
- Physical state: Clear, colorless liquid
- Analytical purity: no data
- Lot/batch No.: 354058
Target gene:
TK
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
AROCLOR 1254 induced rat liver S9 mix.
Test concentrations with justification for top dose:
up to cytotoxic concentrations of 300 nl/ml
First trial: 3.91-62.5 nl/ml without S9; 15.6-250 nl/ml with S9
Second trial: 50-150 nl/ml without S9; 100-300 nl/ml with S9
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Ethylmethane sulfonate (EMS) for nonactivation studies; Dimethylnitrosamine (DMN) for studies with activation
Details on test system and experimental conditions:
3 tests at ranges of concentrations up to cytotoxic levels.

Nonactivation Assay
The procedure used is based on that reported by Clive and Spector (1975) and is summarized as follows: Cultures exposed to the test chemical for four hours at the preselected doses are washed and placed in growth medium for two or three days to allow recovery, growth and expression of the induced TK-/- phenotype. Cell counts are determined daily and appropriate dilutions are made to allow optimal growth rates. At the end of the expression period, 3*10e8 cells for each selected dose are seeded in soft agar plates with selection medium and resistant (mutant) colonies are counted after approximately 10 days of incubation. To determine the actual number of cells capable of forming colonies, a portion of the cell suspension is also cloned in normal medium (nonselective). The ratio of resistant colonies to total viable cell number is the mutant frequency.

Activation Assay
The activation assay can be run concurrently with the nonactivation assay. The only difference is the addition of the S9 fraction of rat liver homogenate and necessary cofactors
(CORE) during the four-hour treatment period. CORE consists of NADP (sodium salt) and isocitric acid.
Evaluation criteria:
Minimum criteria for mutagenesis was a mutant frequency exceeding:
Trials 1 and 2, without S9: 40.1E-6 and 27.4E-6, respectively.
Trials 1 and 2, with S9: 33.4E-6 and 40.4E-6, respectively.
for further details, see any other informations below
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test substance induced significant increases in mutant frequency at the TK locus of L5178Y mouse lymphoma only in the presence of S9. Without activation, concentrations up 100 nl/ml were highly toxic without inducing significant increases in the mutant frequency. In the presence of S9 microsomal activation concentrations of the test substance ranging from 100 nl/ml to 200 nl/ml induced moderate to high toxicity and dose dependent increases in the mutant frequency.
---
In the first trial without activation, the test material was lethal at concentrations as low as 125 nl/ml. Therefore five treatments ranging from 3.91 nl/ml to 52.5 nl/ml were selected for mutant analysis in order to cover a wide range of toxic action. The percent relative growths ranged from 21.7 to 75.2% which indicates a moderate range of toxic action, but does not include treatments in the highly toxic range (10% to 15% relative growth). The minimum criterion for mutagenesis in this assay was a mutant frequency exceeding 40.1x10e-6. None of the treatments induced mutant frequencies that approached this level of mutant action. However, since none of the treatments were in the highly toxic range, the test material required further analysis.
A second trial without activation was initiated that included treatments ranging from 50 nl/ml to 150 nl/ml with some duplicate concentrations. The 150 nl/ml treatment was lethal after 24 hours, therefore eight treatments ranging from 50 nl/ml to 100 nl /ml were selected for mutant analysis. The minimum criterion for mutagenesis in this assay was a mutant frequency exceeding 27.4x10e-6. None of the assayed treatments induced this level of mutant action,
The percent relative growths ranged from 2.1% to 17.5% which includes the highly toxic range. Since no evidence of mutagenesis was demonstrated over a wide range of toxic action in two trials of the nonactivation assay, the test material was considered nonmutagenic in this assay.
In the presence of S9 microsomal activation, the test material was slightly less toxic indicating some interaction of the test material with the activation mix. In the first trial the test material was excessively toxic at 50 nl/ml and 1000 nl/ml but demonstrated only moderate inhibition of cell growth at 250 nl/ml. Five treatments ranging from 15.6 nl/ml to 250 nl/ml were selected for mutant analysis. The minimum criterion for mutagenesis in this assay was a mutant frequency of 33.4x10e-6. A positive response was just detectable at 125 nl/ml and 250 nl/ml (mutant frequencies 37.3x10e-6 and 41.4x10e-6 respectively). Among the assayed treatments the percent relative growths ranged from 30.6 to 127.4%. Since treatments in the highly toxic range (below 15% relative growth) were not available for cloning and two treatments were weakly positive, confirmation was required and a second trial with activation was initiated in addition, the positive control treatment was excessive1y toxic. An assay with an unacceptable positive control but where positive doses are observed is not discarded but is used as secondary evidence for mutagenicity.
In the second trial with S9 microsomal activation, duplicate treatments at 100 nl/ml, 200 nl/ml and 300 nl/ml were assayed for mutant induction. Higher concentrations of test material (400 nl/ml and 500 nl/ml) were excessively toxic. The percent relative growths of the assayed treatments ranged from 1% to 55.7% which covers a range of toxic action that includes the highly toxic range. The minimum criterion for mutagenesis was a mutant frequency exceeding 40.4x10e-6. Very weak mutagenesis was detectable at 100 nl/ml and 200 nl/ml in one duplicate at each concentration. The average of the duplicates at each dose exceeded the minimum criterion and small but observable increases in total mutant clones were also noted. The two treatments assayed at 300 nl/ml both induced significant increases in the mutant frequency (10.1-fold and 7.9-fold above background) but were accompanied by very high toxicities and lowered cloning efficiencies.
In order to clarify the mutagenic response in more detail a third trial was initiated. Duplicate treatments from 100 nl/ml to 275 nl/ml were analyzed for mutant induction in that trial. The test material was excessively toxic above 200 nl/ml and duplicate treatments were not consistent, therefore, these treatments were not considered in the analysis of this test material. The minimum criterion in this assay (a mutant frequency exceeding 73.7x10e-6) was just exceeded by one of the 100 nl/ml treatments. A dose-dependent increase in mutant frequency
was observed at 150 nl/ml and 200 nl/ml and reached an average of 5.7-fold above the background (average of solvent and untreated control mutant frequencies). The test material was therefore considered mutagenic in the presence of S9 activation in this assay.
The average cloning efficiencies for the solvent and untreated negative controls varied from 79.3% and 95% without activation to 87.9%, 82.1% and 84.2% with activation, which demonstrated good cloning conditions for the assays. The negative control mutant frequencies were all in the normal range and the positive control compounds yielded normal mutant frequencies that were greatly in excess of the background. The relative cloning efficiencies of the DMN control cultures were low but this has been observed with this lot of S9 and has not appeared to alter the efficacy of the assays. The suspension growth in the solvent control cultures was only 53.3% of the untreated control value. When an unusual solvent is used this could mean that the solvent is toxic. However, in this study, no effect was observed in other trials using the same solvent and DMSO is not known to cause toxic effects at the concentrations used. The differences observed therefore are likely due to normal variations in cell growth.
Remarks on result:
other: other: L5178Y TK+/- cells
Remarks:
Migrated from field 'Test system'.
Executive summary:

In an pre guideline in vitro mammalian cell gene mutation test (Cifone 1981) with mouse lymphoma L5178Y tk +/- cells, methyl methacrylate at concentrations of 100 - 300 nl/ml (0.9 -2.7 mM) significantly increased the frequency of tk +/- mutations in a dose dependent manner but only in the presence of S9 external metabolic activation, however, clear effects were observed only at doses with high toxicity below 20% relative growth.

Without activation, concentrations up 100 nl/ml were highly toxic without inducing significant increases in the mutant frequency.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Method and results sufficient described, pre guideline study ofOECD-guideline 476. Test performed only in the absence of a metabolically active microsomal fraction (S9)
Qualifier:
equivalent or similar to guideline
Guideline:
other: In vitro mammalian cell gene mutation test / mouse lymphoma assay
Version / remarks:
pre guideline study
(OECD guidline 476 was at first adopted 1997)
Deviations:
yes
Remarks:
Test performed only in the absence of a metabolically active microsomal fraction (S9)
Principles of method if other than guideline:
In addition to the mutation test at the TK locus, analyses for chromosomal aberrations and induction of micronuclei in vitro were run.
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
Supplier: Rohm & Haas Company (Spring House, PA, USA)
Purity: no data
10 ppm methyl hydrochinone was present as inhibitor
Target gene:
TK
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
TK +- -3.7.2C heterozygote of L5187Y mouse lymphoma cells
Metabolic activation:
without
Metabolic activation system:
-
Test concentrations with justification for top dose:
1000 - 3000 µg/ml (10 - 30 mM)
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Details on test system and experimental conditions:
Duplicate cultures were treated for four hours with test substance without metabolic activation. The cultures were split for use in a mutagenicity assay, aberration analysis and micronucleus assay. For the mutation assay, colonies were incubated for 9-11 days at 37°C, counted and colony size distribution determined.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
cytotoxicity
Positive controls validity:
valid
Additional information on results:
Methyl methacrylate produced a positive mutagenic response at the tk locus with responses variable at 2-4 times background but consistently positive at doses from 2300 to 3000 ug/ml. The highest mutant frequency occurred at 2800 ug/ml with 298 mutants/10E6 survivors. Because the background frequency was relatively high after the analysis of 100 metaphases, an additional 100 metaphases were scored. Weak positives were recorded at 2200, 2500, 2700, 2800 and 3000 ug/ml. The highest response was at 2800 ug/ml with 45 aberrations in 39 of 200 metaphases (background = 16 aberrations in 15 of 200 metaphases scored). It should be noted that the high background was due to an increase in the number of chromosome rearrangements and breaks in the control culture. No concurrent increase in background TK mutant or micronucleus frequency was observed. The MMA treated cells showed a significant increase in chromatid events with no change in the number of chromosome events. Micronucleus responses were also variable with weak positive responses recorded at 2200, 2400, 2500, 2800, 2900 and 3000 ug/ml. The greatest response was at 2900 ug/ml with micronuclei in 25 of 1000 binucleates (background = 9 /1000). The 2 lowest doses, 1000 and 1750, were negative for mutagenicity, aberrations and micronuclei. All test concentrations, except the lowest, are above the guideline recommendation of 10 mM in OECD 476 (1000 µg/ml = 10 mM).

Comparison of results: Chromosome aberrations, micronucleus induction and mutations at the TK locus, small colony mutants and survival.

MMA [µg/ml] Cells w/Ab. Cells w/MN Total MF/106cells Small colonies MF/106 % Small colonies Survival [%]
0 15 9 68 39 57% 1OO
1000 17 6 74 60 81% 53
1750 24 10 121 88 73% 34
2102 28 22 143 115 80% 26
2400 21 20 168 144 86% 22
2499 33 18 226 180 80% 15
2601 27 17 179 140 78% 19
2700 30 12 169 147 87% 19
2799 39 20 298 260 87% 11
2901 16 25 255 227 89% 12
3000 35 22 254 222 87% 12

Increases in any type of mutations correlate strongly with a decrease in survival. All test concentrations, except the lowest, are above the guideline recommendation of 10 mM in OECD 476 (1000 µg/ml = 10 mM). The increase in mutations at the TK locus is primarily due to small colony mutants with a trend to more small colonies at higher concentrations, indicating that these are primarily due to deletions (chromosome mutations) rather than gene mutations.

Conclusions:
In pre guideline mouse lymphoma assay which was only run without S-9 mix, weak effects were obtained for doses producing high toxicity (Moore et al., 1988) The vast majority of induced colonies were small ones, indicating that the genetic effect was derived from clastogenicity and not from gene mutations.
Executive summary:

In pre guideline mouse lymphoma assay which was only run without S-9 mix, weak effects were obtained for doses producing high toxicity (Moore et al., 1988). According to the authors, 2000 μg/ml was positive in both experiments (92 and 98 mutants per 106 survivors vs. 54 and 68 in the negative controls), relative survival was approximately 20% and 30%; in one experiment the highest dose of 499 μg/ml induced 143 mutants at 10% relative survival; in the second experiment the highest dose of 3100 μg/ml induced 220 mutants with 11% relative survival. The vast majority of induced colonies were small ones, indicating that the genetic effect was derived from clastogenicity and not from gene mutations.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Method and results sufficient described. Test performed only in the absence of a metabolically active microsomal fraction (S9)
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
Test performed only in the absence of a metabolically active microsomal fraction (S9)
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
Methylmethacrylate (CAS: 80-62-6)
purchased from Merck
Purity analyzed HPLC and MS
Target gene:
hprt
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
V79B
Metabolic activation:
without
Metabolic activation system:
rat liver
Test concentrations with justification for top dose:
0.0, 10, 20 mM
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 9,10-dimethyl 1,2-benzanthracene and ethyl methane sulfonate
Details on test system and experimental conditions:
1.5E+6 cells were plated onto cell culture plates with culture medium and incubated for 20-24 hours. Cells were exposed to varying concentrations of the test substance for 24 hrs in the absence of S9 mix or 4 hrs with or without S9 mix. The cells were subcultured after four days, cell numbers counted and expressed relative to cell counts in solvent control cultures or the plating efficiency. 3E+6 cells were subcultured for eight days and mutant isolation started on day 10 by replating the cells in selective media containing 6-thioguanidine, and the cloning efficiency determined.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
ambiguous
Remarks:
taking into account the range of control values
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>20 mM/l
Additional information on results:
A very weak mutagenic response in V79 cells was observed at the concentrations tested after a direct exposure for 24 hours. Without metabolic activation, the mutant frequencies in the tested concentrations of 10 and 20 mM were 6 and 16 per million surviving cells, while in the control treatment 3 mutants per million surviving cells were observed. Other control data from parallel experiments reported in the same paper range from 2 to 10 mutants per million surviving cells. The cell numbers of the low and high dose treatment were 71 and 49% of the control, respectively. Data from the trial with metabolic activation were not reported in detail. The authors interpret the data as a very weakly positive finding. In the light of the range of control data, the result is interpreted as ambiguous.

The authors interpret the data as a very weakly positive finding. In the light of the range of control data reported in the paper (2 - 10 / mio cells), the result in the higher concentration (16 / mio. cells) is interpreted as ambiguous.

Conclusions:
Incubation of V79 cells with 10 to 20 mM MMA for 24 h in the absence of S9 produced very small increases in Hprt mutaions without serious toxicity determined by cell counts post treatment and relative cloning efficiency. Although cell survivals were greater than 20 %, the authors refrained from calling this response unequivocally poisitive.
Executive summary:

Incubation of V79 cells with 10 to 20 mM MMA for 24 h in the absence of S9 produced very small increases in Hprt mutations without serious toxicity determined by cell counts post treatment and relative cloning efficiency. Although cell survivals were greater than 20 %, the authors refrained from calling this response unequivocally positive.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

in vivo
Dominant lethal assay, mouse: negative (Zeneca/ICI 1976)
Micronucleus assay, mouse: negative (Hachiya et al. 1982)

Chromosome aberration assay, rat: negative/inconclusive (Zeneca/ICI 1976, 1979)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
Method and results sufficient described,pre guideline study similar to OECD-guideline 478
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 478 (Genetic Toxicology: Rodent Dominant Lethal Test)
Version / remarks:
pre guideline study. OECD 478 was first adopted 1984
Deviations:
no
GLP compliance:
no
Type of assay:
rodent dominant lethal assay
Specific details on test material used for the study:
Methylmethacrylate (CAS: 80-62-6)
Purity: not reported
Species:
mouse
Strain:
CD-1
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 10-12 w
- Diet: Alderley Park mouse cubes
Route of administration:
inhalation
Vehicle:
dry clean air
Details on exposure:
During exposure, male CD-1 mice were individually housed in chambers made of stainless steel and glass with an internal capacity of three liters. Seven groups of mice, previously shown to be fertile, were treated according to the scheme presented below.
Fertility testing: Prior to the five-day inhalation exposures, male mice were each mated with two virgin adult female mice for five days. After a five-day mating period, the females were transferred to other cages. The females were sacrificed 15 days following the first day of placement with the males and examined for pregnancy. Only males successful in mating were used on the test.
Experimental mating and necropsy: After treatment, male mice were individually housed. Two virgin female mice were placed in each cage. After a five-day mating period, the females were removed and pair-housed. After a two-day rest period, two new virgin female mice were housed with each male for a five-day mating period. This process was repeated until the male mice had been mated for eight weeks. The male mice were then sacrificed and discarded without necropsy. It was assumed the females were fertilized within two to three days after mating pairs were set up. Thirteen days after the fertilization date, each female was sacrificed and examined for pregnancy, living fetuses and early and late fetal development. 
Duration of treatment / exposure:
5 days, 6 hours/day
Frequency of treatment:
Daily
Dose / conc.:
0.405 mg/L air
Remarks:
corresponding to 100 ppm

Dose / conc.:
4.05 mg/L air
Remarks:
corresponding to 1000 ppm
Dose / conc.:
36.45 mg/L air
Remarks:
corresponding to 9000 ppm
No. of animals per sex per dose:
total number of animals: control: 35; test groups: 20; positive controls: 13 (CTX in water), 5 (EMS in water) and (12 HN2 in saline)
Control animals:
yes, concurrent no treatment
Positive control(s):
200 mg cyclophosphamide in water/kg bw once by i.p. injection on day 5; 150 mg ethylmethane sulphonate in water/kg bw orally once a day for 5 days ; 2.5 mg meclorethamine in saline once 
Tissues and cell types examined:
1) total implants/pregnancy; early deaths/pregnancy; and early deaths/total implants/pregnancy. 
Statistics:
A simple 2X2 Chi-square was used to analyze the data. Also, a week-by-week hierarchical analysis of variance was applied. The following three responses on each female were analyzed: 1) total  implants/pregnancy; early deaths/pregnancy; and early deaths/total  implants/pregnancy. For response 2, the Freeman-Tukey Poisson variance stabilizing transformation was used. Non-pregnant females were taken as missing data. Dunnett's t-test was used for multiple comparisons.
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Mortality was observed in the three dose groups exposed to the test substance. One animal died in the 100-ppm group the week following exposure, one animal died (95% survival) in the 1000-ppm group and six animals died (70% survival rate) in the 9000-ppm group during exposure. Five animals from the cyclophosphamide positive control group died within eight weeks after dosing.  
Fertility successful mating frequency: No effects observed in the MMA-exposed groups. Positive controls showed appropriate reduction in fertility.
Pregnancy frequency: Reduction in the 1000-ppm group in week 6 only was not considered related to MMA toxicity. Positive controls showed a  decrease in frequency.
Total implantations: No effects observed in the MMA-exposed groups. Positive controls showed appropriate reduction in implant numbers.
Early deaths: Percentages of early deaths were not affected in the MMA-exposed groups. Positive controls showed an appropriate increase in the number of early deaths.
Mean number of early deaths: No effects observed in the MMA-exposed  groups. Positive controls showed an appropriate increase in the number of early deaths.
Percentage of total implantations per pregnancy that were early deaths: No effects observed in the MMA-exposed groups.  
Late deaths: No effects were observed in this study.

Mean early death per pregnancy

 

 

Treatment Group

 

 

 

 

 

Methyl methacrylate [ppm]

 

 

 

Period

Week

Negative control

100

100

9000

CTX

E.M.S

HN2

Before treatment

0.66

0.39

0.46

0.54

0.71

0.40

0.6

1

0.75

0.72

0.52

0.72

5.53***

5.29***

1.2

2

0.67

0.61

0.61

0.85

3.80***

2.75***

2.2

3

0.78

0.84

1.11

0.87

1.70**

1.00

1.5

4

0.62

0.75

0.61

0.50

0.60

0.86

0.8

5

0.55

0.75

0.40

0.67

0.42

1.11*

0.5

6

0.66

0.65

0.85

0.67

1.00

0.56

0.1

7

0.92

0.74

0.40

0.77

0.83

0.60

0.7

8

0.97

1.00

0.79

0.78

0.93

0.80

0.90

 

Dunnett‘ t-test of transformed values significant at

*           5 %

*           1 %

***       0.1 % level

Percentage of early death / total implants / pregnancy

 

 

Treatment Group

 

 

 

 

 

Methyl methacrylate [ppm]

 

 

 

Period

Week

Negative control

100

100

9000

CTX

E.M.S

HN2

Before treatment

5.6

3.7

4.0

4.3

5.8

3.1

9.5

1

6.6

6.0

4.9

9.4

58.3***

61.2***

14.8***

2

5.3

4.8

5.0

8.7

36.5***

21.2***

21.6***

3

7.3

6.7

9.4

7.4

14.8**

13.8

13.1*

4

5.2

6.9

5.3

4.5

4.7

8.5

6.8

5

5.1

6.6

4.0

5.6

3.5

9.5*

4.0

6

5.3

6.4

7.0

6.2

7.7

5.6

0.9

7

7.9

5.9

3.7

8.1

7.9

6.6

6.0

8

7.8

8.8

7.4

6.8

7.4

7.3

6.8

 

Dunnett‘ t-test of transformed values significant at

*           5 %

*           1 %

***       0.1 % level

Conclusions:
Methyl methacrylate was negative in vaild in vivo germ cell study in mice (rodent dominat lethal test).
Executive summary:

Methyl methacrylate was tested in an in vivo pre guideline dominat lethal test in mice. This test evaluates mutation induction, usually at the chromosome level, in sperm by assessing viability of embryos in subsequent matings. Male CD-1 mice were exposed by inhalation to methyl methacrylate at doses of 100, 1000 and 9000 ppm 6 hours/days for five consecutive days. Specific data on toxicity were not given, however, in the 9000 ppm group 6 out of 20 males died.

After exposure the males were mated to females for 5 days.

Following removal, the females were sacrificed 15 days later and products of conception analyzed. No effects were seen as regards number of early embryo death, mean number of early death per pregnancy or pre-implantation egg loss. This test result was negative indicating no in vivo germ cell mutagenesis. The cyclophosphamid control gave clearly positive results under the conditions of the test.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Method and results sufficient described, similar to OECD-guideline 475.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Version / remarks:
pre guideline study. Guideline study, guideline first adopted 1984
Deviations:
yes
Remarks:
The conditions of OECD TG 475 include: the collection of BM at two different times after single treatment; the analysis of min 200 metaphases/ animal. Here, the BM was collected only once after single treatment; max 50 cells/ animal were analysed.
GLP compliance:
no
Type of assay:
chromosome aberration assay
Specific details on test material used for the study:
Supplied by ICI;
Stabilizer: 11 ppm hydroquinone
Species:
rat
Strain:
other: Alderley Park
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Alderley Park
- Age at study initiation: 8-10 weeks
- Weight at study initiation: 150-200 g
- Housing: individually
- Diet (e.g. ad libitum): Alderley Park rat cubes
- Water (e.g. ad libitum): unspecified
Route of administration:
inhalation
Vehicle:
unchanged
Details on exposure:
Atmosphere of various concentrations of test substance and conrol were generated by a controlled fluid-feed atomiser technique (Gage 1959)
Atmospheres were monitored by infra-red spectrophotometer (Wilks Moran 1A)
Duration of treatment / exposure:
single treatment: 2 hrs
repeated treatment: five hrs a day for 5 consecutive days
Frequency of treatment:
single treatment: once
repeated treatment: daily for 5 days
Post exposure period:
Animals were sacrificed 24 hr following the last exposure period.
Dose / conc.:
0.4 mg/L air
Remarks:
corresponding to 100 ppm
Dose / conc.:
4.1 mg/L air
Remarks:
corresponding to 1000 ppm
Dose / conc.:
36.9 mg/L air
Remarks:
corresponding to 9000 ppm
No. of animals per sex per dose:
single treatment: 2-4 rats per group or 5 rats per group
repeated treatment: 7 rats per group.
Control animals:
yes, concurrent no treatment
Positive control(s):
10, 750 and 7500 ppm benzene
Tissues and cell types examined:
bone marrow samples were collected and processed
Details of tissue and slide preparation:
Animals were sacrificed 24 hours after expsosure
Preparation of bone marrows according to the method of Sugiyama (1971) but with slight modifications:
- i.p. injection of colchinine two hours prior to sacrifice to arrest dividing cells in metaphase
-killing rats by an overdose of Fuothane
- after femurs were removed from animals bone marrow was harvested by aspiartion with Hanks' Basis Salt solution
-cells tearted with hpotinic solution followed by fixation in glacial acetic acid:methanol, 1:3
-Slides were prepared by air-drying and stained with Giemsa
- 50 cells from each animal were examined

Following abnormaties were assigend:
- chromatid and chromosome gaps
- chromatid breaks
- fragments
- other complex abnormalties
Statistics:
The data were transformed using a variance stabilizing transformation; the data were analysed using an analysis of variance and a one sided students t test was used on the transformed data
Sex:
male
Genotoxicity:
ambiguous
Toxicity:
not specified
Vehicle controls validity:
other: air was vehivle and negative control
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The study was negative for chromosomal aberration frequencies when gaps were excluded as usual.
When the data from the two 2 hr exposure experiments were combined and gaps were included, 1000 and 9000 ppm MMA groups were significantly different from controls; there was evidence of a dose response relationship.
The group exposed to 9000 ppm for five repeated exposures was significantly different from controls; there was evidence of a dose response relationship.

Combined abnormalities of the two single treatment assays

 

Treatment

% abnormal cells (out of 50 cells)

Ctrl

5.1

100 ppm MMA

5.5

1000ppm MMA

8.0*

9000 ppm MMA

9.4**

10 ppm B

10.3**

750 ppm B

18.0***

7500 ppm B

26.5***

 B=Benzene

Abnormalities of the repeated treatment assay

 

Treatment

% abnormal cells (out of 50cells)

Ctrl

3.1

100 ppm MMA

1.4

1000ppm MMA

6.3

9000 ppm MMA

6.9*#

10 ppm B

7.4**

750 ppm B

10.0***

7500 ppm B

14.0***

 # Statistically significant, however, compared to the control in the single-treatment segment (5.1 % ab.) the increase is biologically unimportant and does not comprise a positive effect.

Mean % abnormalities (excluding gaps)

 

Treatment

Single treatment A

Single treatment B

Repeated treatment

Ctrl

1.0

1.2

0.3

100 ppm MMA

0.7

0.8

0.3

1000ppm MMA

3.3

2.0

0.3

9000 ppm MMA

1.0

2.0

1.2

10 ppm B

5.0

1.6

2.0*

750 ppm B

8.0*

4.0

2.0*

7500 ppm B

15.3**

11.6*

3.7*

 

*    = significance level: 5%

**  = significance level: 1%

*** = significance level: 0.1%

Conclusions:
No increase in chromosome aberrations excl. gaps. An allegedly significant increase in chromosome aberrations incl. gaps is due to an - in comparison - low control value and biologically unimportant compared to other control values. In addition, the biological significance of chromosome gaps is unclear and, as an isolated finding, would not be regarded as a positive test result.
Executive summary:

Methyl methacrylate was tested in a pre guideline cytogenetic bone marrow test in rats which were exposed by inhalation (ICI, 1976). The test was performed by single application for 2 hours (7animals per dose group) and for repeated application (7 animals per dose group) for 5 hours on 5 consecutive days. Exposure concentrations were 100, 400, 700 and 1000 ppm in both tests. Positive control was 7500 ppm benzene.

As far as possible 50 metaphases were analysed per animal.

When the data from the two 2 hr exposure experiments were combined and gaps were included, 1000 and 9000 ppm MMA groups were significantly different from controls; there was  evidence of a dose response relationship.

The group exposed to 9000 ppm for five repeated exposures was significantly different from controls; there was evidence of a dose response relationship.

The study was negative for chromosomal aberration frequencies when gaps were excluded as usual.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
12.01.1977 - 16.01.1977
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Method and results sufficiently described, with significant technical problems
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Deviations:
yes
Remarks:
The conditions of OECD TG 475 include: the collection of BM at two different times after single treatment; the analysis of min 200 metaphases/ animal. Here, the BM was collected only once after single treatment; max 50 cells/ animal were analysed.
GLP compliance:
no
Type of assay:
chromosome aberration assay
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Animal Breeding Unit, Alderley Park, Cheshire
- Age at study initiation: 8-10 weeks
- Weight at study initiation: 150-200 g
- Housing: four/cage
- Diet (e.g. ad libitum): Alderley Park rat cubes
- Water (e.g. ad libitum): unspecified


ENVIRONMENTAL CONDITIONS
- Air changes (per hr): minimum 8
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation
Vehicle:
unchanged
Details on exposure:
whole body
Duration of treatment / exposure:
single treatment: 2 hours
repeated treatment: 5 hours
Frequency of treatment:
repeated treatment: daily for 5 days
Post exposure period:
24 h after the last exposure
Dose / conc.:
0.4 mg/L air
Remarks:
corresponding to 100 ppm
Dose / conc.:
1.6 mg/L air
Remarks:
corresponding to 400 ppm
Dose / conc.:
2.8 mg/L air
Remarks:
corresponding to 700 ppm
Dose / conc.:
4 mg/L air
Remarks:
corresponding to 1000 ppm
No. of animals per sex per dose:
12 animals in the negative control group; 8 animals in each of the treated and positive control groups.
Control animals:
yes, concurrent no treatment
Positive control(s):
Benzene, 7500 ppm
Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
Animals were sacrificed 24 hours after expsosure
Preparation of bone marrows according to the method of Sugiyama (1971) but with slight modifications:
- i.p. injection of colchinine two hours prior to sacrifice to arrest dividing cells in metaphase
-killing rats by an overdose of Fuothane
- after femurs were removed from animals bone marrow was harvested by aspiartion with Hanks' Basis Salt solution
-cells tearted with hpotinic solution followed by fixation in glacial acetic acid:methanol, 1:3
-Slides were prepared by air-drying and stained with Giemsa
- 50 cells from each animal were examined

Following abnormaties were assigend:
- chromatid and chromosome gaps
- chromatid breaks
- fragments
- other complex abnormalties
Statistics:
Proportion of cells with any abnormalities or proportion of cells with any abnormalities other than gaps: Analysis of Variance after transformation of the original data using a double arcsine transformation.
Proportion of rats which had any cells with any abnormality other than gaps: One-sided Fischers Exact test.
Sex:
male
Genotoxicity:
ambiguous
Toxicity:
no effects
Vehicle controls validity:
other: air was vehivle and negative control
Negative controls validity:
valid
Positive controls validity:
valid

The maximum concentration tested caused significant reductions in mitotic activity in the bone marrow of the
exposed animals. Small and non-dose related increases in the percentages of cells with chromosomal aberrations were
observed in the animals exposed to MMA. However, these increases were almost exclusively due to gap-type aberrations and when these were excluded from the data small increases were only observed at 400 ppm. Such small 
increases, observed at lower, but not at higher concentrations tested, are not considered to be 
biologically significant.

For further details, see attached document including 11 tables.

Executive summary:

Methyl methacrylate was tested in a pre guideline cytogenetic bone marrow test in rats which were exposed by inhalation (ICI, 1976). The test was performed by single application for 2 hours (5 animals per dose group) and for repeated application (7 animals per dose group) for 5 hours on 5 consecutive days. Exposure concentrations were 100, 1000 and 9000 ppm in both tests. Positive control was benzene.

The study demstratates some methological problems: Analysis of 50 metaphases was not possible for 10 out of 27 animals in the acute and 10 out 26 in the subacute test.

MMA may cause weak, but statistically significant, increases in chromosome damage in rat at some exposure levels at the sampling times investigated by comparison with the negative control values both after single and multiple exposures. However, there is no constistent dose response and the effects are of an indeterminate toxicological significance in view of the overall negative toxicological profile of the substance.

A clear conclusion cannot be drawn this study.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Similar to OECD-guideline 474, all relevant study details available
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
The conditions of OECD 474 include: the collection of BM at two different times after single treatment; the scoring of at least 4000 immature erythrocytes/ animal. Here, the BM was collected only once; only 2000 erythrocytes were evaluated per animal.
GLP compliance:
not specified
Type of assay:
micronucleus assay
Specific details on test material used for the study:
purity: > 99 %
Species:
mouse
Strain:
other: ddy
Sex:
male
Route of administration:
oral: gavage
Vehicle:
olive oil, 25 ml/kg
Duration of treatment / exposure:
4 doses
Frequency of treatment:
3 doses: once, 24 h before terminal sacrifice
1 dose: 4 split doses every 24 h, the last one 24 h before terminal sacrifice, total duration 5 d
Post exposure period:
24 h
Dose / conc.:
25 mg/kg bw/day (nominal)
Dose / conc.:
1 130 mg/kg bw/day (nominal)
Dose / conc.:
2 260 mg/kg bw/day (nominal)
Dose / conc.:
4 520 mg/kg bw/day (nominal)
Remarks:
Dose corresponds to 50% of LD50
Remarks:
4 x 1130 mg/kgbw per dose
No. of animals per sex per dose:
6 (repeated treatment: 5)
Control animals:
yes, concurrent vehicle
Positive control(s):
3 mg Mitomycin C, single dose by i.p. administration 24 h prior to preparation
Tissues and cell types examined:
Sampling time for bone marrow: 3 single doses - 24 h post-administration; for repeated administration: 5 days after first administration.
Statistics:
according to Kastenbaum/Bowman
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
at all doses, single and repeated
Vehicle controls validity:
valid
Positive controls validity:
valid

The substance has been administered by gavage as a solution in olive oil in 3 single doses ranging from 1130 mg/kg to 4520 mg/kg (0.5 LD50) 24 h prior to preparation of the bone marrow. A separate group of 5 animals was administered 4 doses of 1130 mg/kg 96, 72, 48 and 24 h prior to preparation. Olive oil (25 ml/kg) was used as the solvent control and mitomycin C (3 mg/kg, i.p.) as the positive control. 2000 erythrocytes were evaluated per animal (12000/10000 per dose). No increase in micronucleated polychromatic erythrocytes was observed at any dose, while an induction of micronuclei was seen in the positive control. MMA was not mutagenic in vivo under test conditions.

Conclusions:
Interpretation of results (migrated information): negative
The substance has been administered by gavage as a solution in olive oil in 3 single doses ranging from 1130 mg/kg to 4520 mg/kg (0.5 LD50) 24 h prior to preparation of the bone marrow. A separate group of 5 animals was administered 4 doses of 1130 mg/kg 96, 72, 48 and 24 h prior to preparation. Olive oil (25 ml/kg) was used as the solvent control and mitomycin C (3 mg/kg, i.p.) as the positive control. 2000 erythrocytes were evaluated per animal (12000/10000 per dose). No increase in micronucleated polychromatic erythrocytes was observed at any dose, while an induction of micronuclei was seen in the positive control. The substance has been administered by gavage as a solution in olive oil in 3 single doses ranging from 1130 mg/kg to 4520 mg/kg (0.5 LD50) 24 h prior to preparation of the bone marrow. A separate group of 5 animals was administered 4 doses of 1130 mg/kg 96, 72, 48 and 24 h prior to preparation. Olive oil (25 ml/kg) was used as the solvent control and mitomycin C (3 mg/kg, i.p.) as the positive control. 2000 erythrocytes were evaluated per animal (12000/10000 per dose). No increase in micronucleated polychromatic erythrocytes was observed at any dose, while an induction of micronuclei was seen in the positive control. MMA was not mutagenic in vivo under test conditions.
Executive summary:

The substance has been administered by gavage as a solution in olive oil in 3 single doses ranging from 1130 mg/kg to 4520 mg/kg (0.5 LD50) 24 h prior to preparation of the bone marrow. A separate group of 5 animals was administered 4 doses of 1130 mg/kg 96, 72, 48 and 24 h prior to preparation. MMA was not mutagenic in vivo under test conditions.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
unclear dose/exposure time response; inhalative exposure "to simulate a working environment" without details than an estimated value of 150 ppm/ 8 hrs exceeding the current OEL by a factor of 3
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
no reference was made to the OECD GL in the publication so that it is assumed that there are several deviations to the GL
Principles of method if other than guideline:
The aim of this study was to evaluate the genotoxic potential of methyl methacrylate (MMA) vapor by simulating standard occupational exposure of 8 hours per day and using the micronucleus test.
GLP compliance:
not specified
Type of assay:
mammalian erythrocyte micronucleus test
Specific details on test material used for the study:
not specified
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals or test system and environmental conditions:
adults
Weight: 200 to 250 g
Route of administration:
inhalation: vapour
Details on exposure:
TYPE OF INHALATION EXPOSURE: / whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: large rectangular cages measuring 49 × 34 × 16 cm
- biotherium controlled for temperature and humidity and subjected to a 12-hour dark-light cycle.

MMA exposure
was controlled via amber glass bottles (Nadir Figueiredo Indústria e Comércio, São Paulo, Brazil) with a capacity of 100 mL, an opening of 2 cm in diameter and a perforated cover; 10 mL of MMA
was added to each bottle (99.9% Classic - Indústria Brasileira Ltda., São Paulo, Brazil). The bottles
were placed inside each box and fixed to the upper grid. The evaporation rate of MMA was approximately 0.5 mL per day (estimated to be 150 ppm). The rats in the control group were also subjected to poor ventilation but were not exposed to MMA.

Duration of treatment / exposure:
Animals were devided into 3 groups:
- Group A: 16 rats exposed for 8 hrs daily, foregoing feeding and drinkig (8 rats were expsoed only for 1 day, the other8 rats were exposed for repeated exposue simulating a 40 hours/week occupational exposure)
- Group B (positive control): - 8 rats receiving cyclophosphamide (Cytoxan, Baxter Oncology GmbH, Halle, Germany) in single subcutaneous doses (50 mg/kg) on the first day of the experiment
- Group C (negative control): 8 rats receiving water and food ad libitum

8 animals of goups A and animals of group B and C were sacrifieced 24 hrs after start of test. The repeated dose group after 5 days.
Euthanasia: sodium pentobarbital
Frequency of treatment:
8 hours daily
Dose / conc.:
150 ppm
Remarks:
estimated by a predicted evaporation rate of MMA (3fold value of the long-term OEL/ MAK value)
No. of animals per sex per dose:
8
Control animals:
yes
Positive control(s):
- cyclophosphamide
- Route of administration: subcutaneous
- Doses / concentrations: 50 mg/kg
Tissues and cell types examined:
erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:

TREATMENT AND SAMPLING TIMES: Bone marriows collected from femur at time of sacrifice. 2 sample slides per animal

DETAILS OF SLIDE PREPARATION: Slides stained with Giemsa

METHOD OF ANALYSIS: 200 polychromatic slides/animal (100 on each slide)

Statistics:
Kruskal-Wallis-Test followed by multiple comparison of the ranking using Dunn test.
Significance level: 5 %

MMA did significantly induce micronuclei in bone marrow cells over control levles withe the short interval exposure but not withe the long term exposure. With only a single exposure concentration, dose responses cannot be determined. Although the authors recognize that their finding is biologically unexplained (positive short term and negative long term), they speculate that stimulation of DNA repair blunts the response long-term.

Moreover, rats were exposed to undefined concentrations of MMA. Moreover, the estimated concentration of 150 ppm for 8 hrs is the 3 -fold value of the current long-term OEL (MAK) value, thus the rats were expposed to irregular high MMA concentrations from an occupational perspective (and in contrast to the intended workplace relevance).

Executive summary:

Rats were exposed by inhalation for eight hours for one day or on five consecutive days for a total of 40 hours, with sacrifice 24 hrs after exposure for asssessment of micronuclei in 2000 bone marrow cells per animal. Exposure technique consisted of evaporation from container in the cages that contanied 10 ml Methyl methacrylate without analytical confirmation, just estimating that evaporation of 0.5 ml/day resulted in a concentration in air of 150 ppm , i.e. the three-fold long-term OEL value. Here, MMA did significantly induce MN in BM cells over control level with short interval exposure but not with the long term exposure. With only a single exposure conc. dose-responses cannot be determined. Although the authors recognize that their finding is biologically unexplained (positive short term and negative long-term), they speculate that stimulation of DNA repair blunts the response long term. Moreover, the rats were exposed to irregular high MMA concentrations from an occupational perspective (and in contrast to the intended workplace relevance), so that in total the study is considered as not reliable for hazard or risk assessment.

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

Additional information

Data availability: For MMA mutagenicity data are available for all required endpoints.

in vitro

Gene mutation in bacteria

In a standardized according to NTP protocol, methyl methacrylate was not mutagenic in S. typhimurium strains TA97, TA98, TA100, TA1535 with and without metabolic activation (Zeiger et al. 1987). Additionally, methyl methacrylate was not mutagenic in an Ames test with the tester strain S. typhimurium TA102 (below others in that study) which is sensitive for crosslinking and oxidising agents (Schweikl et al. 2001). No mutangenicity was also observed in additional tested, not in OECD 471 recommended test strains TA104 (Schweikl et al. 2001) and in test strain TA1538 (Waegemaekers 1984).

Gene mutation in mammalian cells

Litton Bionetics (1981) reported on a mouse lymphoma assay which was weakly positive in the TK locus in presence and negative in absence of S-9 mix. Without S-9 mix doses up to 100 nl/ml were tested, higher doses led to total toxicity. With S-9 mix methyl methacrylate was positive in the dose range 100 nl/ml to 250 nl/ml, however, clear effects were observed only at doses with high toxicity below 20% relative growth.

In a HPRT assay without metabolic activation, methyl methacrylate was weakly positive in V79 cells (Schweikl et al. 1998). Without metabolic activation, the mutant frequencies in the tested concentrations of 10 and 20 mM were 6 and 16 per million surviving cells, while in the control treatment 3 mutants per 10e6 surviving cells were observed. The cell numbers of the low and high dose treatment were 71 and 49% of the control, respectively.

In a further lymphoma assay which was only run without S-9 mix, weak effects were obtained for doses producing high toxicity (Moore et al., 1988). According to the authors, 2000 μg/ml was positive in both experiments (92 and 98 mutants per 106 survivors vs. 54 and 68 in the negative controls), relative survival was approximately 20% and 30%; in one experiment the highest dose of 499 μg/ml induced 143 mutants at 10% relative survival; in the second experiment the highest dose of 3100 μg/ml induced 220 mutants with 11% relative survival. The vast majority of induced colonies were small ones (indicating that the genetic effect was derived from clastogenicity and not from gene mutations).

In a supporting study methyl methacrylate was tested in the National Toxicology Program (NTP) in a pre guideline mouse local lymphnode assay with and without metabolic activation (S9). Myhr et al. (1990) reviewed the data. MMA was positive at concentrations of 14 mM without S9 but the results were inconclusive in the presence of S9. Cell survival data were not provided. The majority of the data were small colony, indicating slow growth and clastogenicity.

Cytogenicity in mammalian cells

In a cytogenetic test ( in vitro mammalian chromosome aberration test) with CHO cells induction of chromosomal aberrations was bound to high doses which are assumed to be strongly cytotoxic (Anderson et al., 1990). With S-9 mix, treatment was for 2 h followed by 8 to 10 h recovery. Doses up to 1600 μg/ml were negative, at 5000 μg/ml the frequency of aberrant cells was 30%; only one experiment was performed. Without S-9 mix, treatment time was 8 hours with 2.0 to 2.5 h recovery. Doses up to 500 μg/ml were negative; at 1600 and 3000 μg/ml aberration frequencies ranging from 5 to 6 % were found. Data on cytotoxic effects were not given, however, it can be assumed from the data presentation and the general approach of the authors that the highest doses tested led to strong cytotoxic effects. Thus, methyl methacrylate seems to be a high toxicity clastogen, i. e., the induction of chromosomal aberrations is bound to highly toxic doses.

in vivo

Quote from the MMA ESR (ECB, 2002): “A dominant lethal assay on male mice was negative after inhalation exposure to doses ranging from 0.4 to 36.5 mg/L (corresponding to 100 to 9000 ppm; Zeneca/ICI 1976). Specific data on toxicity were not given, however, in the 36.6 mg/L group 6/20 males died. Treated males were mated to 2 females each for 8 periods of 1 week each; females were killed 13 days after assumed dates of fertilization. There was no significant increase in dominant lethal mutations.

Two chromosomal aberration tests were conducted by ICI (1976, 1979) investigating the effect of inhalation exposure to methyl methacrylate for doses ranging from ca. 0.4 to 36.5 mg/L (100 to 9000 ppm).In both tests acute exposure was for 2 h (sampling 24 h after treatment) and subacute exposure for 5 h a day on 5 consecutive days (sampling 24 h after last treatment). Data on toxicity were not given. Group sizes varied from 2 to 9; as far as possible 50 metaphases were analysed per animal. The first study was negative for chromosomal aberration frequencies when - as usual - gaps were excluded. Including gaps and combining two acute experiments conducted independently some increases in aberration frequency were statistically significant.” This is also due to a particularly low control rate in this experimental segment. Compared to other, almost twofold higher control values in other segments of the report, this finding appears to be of little biological importance. Further from the MMA ESR (ECB, 2002): “In the second study frequencies of chromosomal aberrations excluding gaps were not given. Including gaps increases were recorded at some experimental entries. Furthermore, combined data on chromosomal aberration frequencies exclusively gaps from both studies were given, then weak increases were obtained for 400 and 700 ppm in the acute study (not for 100, 1,000 or 9,000 ppm) and 9,000 ppm in the subacute study. Both studies suffer from inadequate description; esp. the second study demonstrates severe methodological problems, e. g., analysis of 50 metaphases was not possible for 10 out of 27 animals in the acute and 10 out 26 in the subacute test. Altogether, a clear conclusion cannot be drawn from these studies.

Hachiya et al. (1982) reported on a negative bone marrow micronucleus assay with mice. In an acute test methyl methacrylate was given by gavage in doses ranging from 1,130 to 4,520 mg/kg, in a subacute assay daily doses of 1,130 mg/kg were given on 4 consecutive days. All groups consisted of 6 animals; sampling was done 24 h after (last) administration. There was no increase in the frequency of micronucleated polychromatic erythrocytes. The percentage of reticulocytes from all bone marrow cells was not affected data on general toxicity were not given.”

Other important information for hazard assessment based on weight-of-evidence considerations

Based upon the facts that MMA is rapidly metabolised within the body with a halflife in blood in the order of 4.4 minutes (Jones, 2002, see chapter "Toxicokinetics") it is extremely unlikely that MMA as intact ester reaches the germ cells, especially as the protection of germ cells against mutagenic insults is much more efficient than in the case of somatic cells. Regarding mutagenicity in somatic cells, MMA has been shown to be non-carcinogenic in several guideline carcinogenicity studies by multiple routes oral/drinking water and inhalation (see chapter "Carcinogenicity"

. This clearly establishes that MMA does not possess a toxicologically relevant mutagenic potential in somatic cells. As ECHA’s endpoint specific guidance (2017[2]) indicates that “there is considerable evidence of a positive correlation between the mutagenicity of substancesin vivoand their carcinogenicity in long-term studies with animals.” it scientifically justified that MMAs lack of carcinogenicity also demonstrates a lack of in vivo genotoxicity of this substance in somatic cells

A review of the mutagenicity of lower alkyl methacrylates was published in Albertini RJ (2017), The lower alkyl methacrylates: Genotoxic profile of non-carcinogenic compounds, Regulatory Toxicology and Pharmacology (2017), 84, 77-93 (see attachment)  

Justification for classification or non-classification

For the assessment of the mutagenic potential of Methyl methacrylate (MMA) in germ cells and somatic cells, in an weight-of-evidence approach data from mutagenicity studies as well as from toxicokinetic investigations, carcinogenicity studies in animals and epidemiological studies in humans were considered with reference to their individual reliability and consistency per line of evidence.

Regarding “germ cell mutagenicity”, the result in the fully valid Dominant Lethal Test was negative indicating no in vivo germ cell mutagenesis. This assessment is supported by toxicokinetic considerations: Based upon the facts that MMA is rapidly metabolised within the body with a halflife in blood in the order of 4.4 minutes, it is extremely unlikely to reach the germ cells, especially as the protection of germ cells against mutagenic insults is much more efficient than in the case of somatic cells.

Regarding “mutagenicity in somatic cells”, data from various in vitro and in vivo mutagenicity studies have to be considered, together with carcinogenicity studies in animals and epidemiological studies in humans. MMA was negative in bacterial gene mutation tests while from pre-guideline mammalian cell culture assays it may be concluded that methyl methacrylate is a high toxicity clastogen (i .e. induction of chromosomal aberrations is bound to highly cytotoxic doses). The effect is not dependent on presence of S-9 mix. These findings are in line with results from mouse lymphoma assays where positive findings seem to be due to the induction of small colonies. In vivo, a pre-guideline oral mouse bone marrow micronucleus test was negative for doses up to 4,520 mg/kg bw. No clear conclusion could be drawn from bone marrow chromosomal aberration assays with rats due to methodological deficiencies. MMA has been shown to be non-carcinogenic in several guideline carcinogenicity studies by multiple routes oral/drinking water and inhalation and in three species. Together with reliable epidemiological studies that show no increased cancer risk due to MMA exposure, this clearly establishes that MMA does not possess a toxicologically relevant mutagenic potential in somatic cells.

Therefore, methyl methacrylate has not to be classified for its mutagenic potential according to CLP, 1272/2008/EC and UN-GHS requirements, respectively.