Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

OECD 471, Salmonella typhimurium TA98, TA 100, TA 1535, TA 1537 and TA 102, with and without metabolic activation: negative

No further data available for MAAH.

Read across data from methyl methacrylate, MMA (donor substance for the primary metabolite MAA; read across justification see attached document) are negative in vivo so that further in vitro testing with MAAH is not required.

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

Genetic toxicity in vivo

Description of key information

No data available for MAAH.

Read across data from methyl methacrylate, MMA (donor substance for the primary metabolite MAA; read across justification see attached document) are negative in vivo:

Dominant lethal assay, mouse: negative (Anderson and Hodge, 1996)

Chromosome aberration assay, rat: negative/inconclusive (Anderson et al., 1976, 1979)

Micronucleus assay, mouse: negative (Hachiya et al. 1982)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Remarks:
Type of genotoxicity: 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, similar to OECD-guideline 478
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 478 (Genetic Toxicology: Rodent Dominant Lethal Test)
GLP compliance:
no
Type of assay:
rodent dominant lethal assay
Species:
mouse
Strain:
CD-1
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 10-12 w
- Diet: Alderley Park mouse cubes
Route of administration:
inhalation
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, 5 and 12
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: 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.
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
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:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
GLP compliance:
no
Type of assay:
chromosome aberration assay
Species:
rat
Strain:
other: Alderley Park
Sex:
male
Details on test animals 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
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.
Remarks:
Doses / Concentrations:
ca. 0.4, 4.1 and 36.9 mg/L (corresponding to 100, 1000 and 9000 ppm)
Basis:
nominal conc.
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:
Preparation according to the method of Sugiyama (1971) but with slight modifications.
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:
not examined
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 50cells)

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:
Interpretation of results (migrated information): negative
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.
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
supporting study
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:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
not specified
Type of assay:
micronucleus assay
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:
Doses 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 conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Data availability: For methacrylic anhydride a bacterial mutation test is available. For all other required endpoints data from methyl methacrylate are used by read-across as methacrylic anhydride and methyl methacrylate (MMA) have the same metabolite: Methacrylic anhydride as well as methyl methacrylate will be metabolised/hydrolysed very fast to methacrylic acid (MAA; see attached read across justification).

In vitro

Gene mutation in bacteria

In an OECD 471 (bacterial reverse mutation assay) using 5 strains (Salmonella typhimurium TA98, TA 100, TA 1535, TA 1537 and TA 102 methacrylic anhydride was not mutagenic with and without metabolic activation.

Gene mutation in mammalian cells

There are no gene mutation studies available in mammalian cells. The EU ESR for methacrylic acid (2002) concluded "Further testing on methacrylic acid is lacking. However, taking into consideration the data on the structurally related substance methyl methacrylate - which indicate that this substance does not express a genotoxic potential in vivo - there is no need for further testing. " Supporting evidence can be drawn by category read-across from a negative gene mutation test with ethylhexyl methacrylate (Harlan, 2009). Although the dominant lethal assay of Anderson (1976) and micronucleus assay of Hachiya (1982) with methyl methacrylate are strictly speaking not gene mutation assays, the absence of positive findings is also supporting the absence of concern for genotoxicity of methacrylic acid.

Cytogenicity in mammalian cells

In vitro

Methacrylic acid, by analogy to methyl methacrylate, has the potential for induction of mutagenic effects, esp. clastogenicity; however, this potential seems to be limited to high doses with strong toxic effects.

In vivo

Because the in vitro data for methyl methacrylate are somewhat ambiguous the in-vivo study data are used by read-across. Quote from the methyl methacrylate ESR (ECB, 2002): “Two chromosomal aberration tests were conducted by Anderson et al. (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 methyl methacrylate 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. ”

Methacrylic acid ESR concluded“In vitro methyl methacrylate has the potential for induction of mutagenic effects, esp. clastogenicity; however, this potential seems to be limited to high doses with strong toxic effects. Furthermore, the negative in vivo micronucleus test - and to some extent the negative dominant lethal assay - indicates that this potential is probably not expressed in vivo. ”

Overall, it may be concluded from mammalian cell culture assays with methyl methacrylate that methacrylic acid has the potential to be a high-toxicity clastogen (i. e. induction of chromosomal aberrations is bound to highly toxic doses). Therefore the in vivo data for methyl methacrylate are used to finally assess the genotoxic potential. The weight of evidence approach indicates that methyl methacrylate is not mutagenic. Methacrylic acid , having a reduced reactivity at the double bond compared to methyl methacrylate, is not expected to be mutagenic.

 

Gene mutation in mammalian cells

The EU ESR for Methacrylic aacid (2002) concluded "Further testing on methacrylic acid is lacking. However, taking into consideration the data on the structurally related substance methyl methacrylate - which indicate that this substance does not express a genotoxic potential in vivo - there is no need for further testing. "

Cytogenicity in mammalian cells

see in vivo data

Dominant lethal assay, mouse: negative (Anderson and Hodge, 1996)

Chromosome aberration assay, rat: negative/inconclusive (Anderson et al., 1976, 1979)

Micronucleus assay, mouse: negative (Hachiya et al. 1982)

Value used for CSA:Genetic toxicity: negative


Justification for selection of genetic toxicity endpoint
For methacrylic anhydride a bacterial mutation test is available, which is negative. For all other required endpoints data from methyl methacrylate are used by read-across as methacrylic acid - the primary metabolite of methacrylic anhydride - and methyl methacrylate have the same metabolites.
The chosen key study is GLP compliant anf of high quality ( Klimisch score 1).

Justification for classification or non-classification

Methacrylic anhydride was negative in a bacterial gene mutation test. Additional information is gained by read-across from methyl methacrylate. From 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 toxic doses). The effect is not dependent on presence of S-9 mix. In vivo an 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. A dominant lethal assay with male mice led to a negative result. In vitro methyl methacrylate has the potential for induction of mutagenic effects, esp. clastogenicity; however, this potential seems to be limited to high doses with strong toxic effects. Furthermore, the negative in vivo micronucleus test and the negative dominant lethal assay indicate that this potential is probably not expressed in vivo.

The absence of the ester group in methacrylic acid reduces the reactivity of the double bond in comparison to the esters.

Therefore, methacrylic anhydride has not to be classified by read across with methacrylic acid /methyl methacrylate for its mutagenic potential according to CLP (1272/2008/EEC) and UN-GHS requirements.