Methacrylic anhydride

Endpoint summary

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.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Ninth Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No. 471, "Bacterial Reverse Mutation Test", adopted 21st July, 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

Commission Regulation (EC) No. 440/2008 B.13/14:"Mutagenicity - Reverse Mutation Test using Bacteria", dated May 30, 2008.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: his D 3052; rfa-; uvrB-; R-factor: frame shift mutations

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: his G 46; rfa-; UV1-B"; R-factor: base-pair substitutions

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

other: his G 46; rfa-; UV1-B": base-pair substitutions

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

other: his C 3076; rfa-; uvrB-: frame shift mutations

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

other: his G 428 (pAQI); rfa-; R-factor: base-pair substitutions

Test concentrations with justification for top dose:

The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment .
2.5 µL/plate was selected as the maximum concentration, The concentration range covered two logarithmic decades. Two independent experiments were performed with the following concentrations:

Experiment I:
0.00100,0.00316,0.0100,0.0316,0.100,0.316 and 1.0 µL/plate (TA 1535, TA 1537 and TA 102 without metabolic activation)
0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0 and 2.5 µL/plate
(with metabolic activation and TA 98, TA 100 without metabolic activation)
Experiment II:
0.00200, 0.00632, 0.0200, 0.0632, 0.200, 0.632 and 1.0 µL/plate
(without metabolic activation)
0.00632, 0.0200, 0.0632, 0.200, 0.632, 1.0 and 2.0 µL/plate
(with metabolic activation)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

Preparation of Bacteria
SampIes of each tester strain were grown by culturing for 12 h at 38,5 °C in Nutrient Broth to the late exponential or early stationary phase of growth (approx, 10E+9 cells/mL),
The nutrient medium consists per litre:
8 g Nutrient Broth
5 g NaCI
A solution of 125 µL ampicillin (10 mg/mL) (TA 98, TA 100, TA 102) was added in order to retain the phenotypic characteristics of the strain,

Agar Plates
The Vogel-Bonner Medium E agar plates with 2% glucose used in the Ames Test were prepared by BSL BIOSERVICE GmbH or provided by an
appropriate supplier. Quality controls were performed.
Vogel-Bonner-salts contain per litre:
10 g MgS04x 7H2O
100 g citric acid
175 g NaNH4HP04 x 4 H2O
500 g K2HP04

Sterilisation was performed at 121°C in an autoclave.
Vogel-Bonner Medium E agar plates contain per litre:
15 g Agar Agar
20 mL Vogel-Bonner salts
50 mL glucose-solvent (40%)
Sterilisation was performed at 121°C in an autoclave.

Overlay Agar
The overlay agar contains per litre:
7.0 g Agar Agar
6.0 g NaCI
10.5 mg L-histidine x HCl x HzO
12.2 mg biotin
Sterilisation was performed at 121°C in an autoclave.

Mammalian Microsomal Fraction S9 Mix
The bacteria most commonly used in these reverse mutation assays do not possess the enzyme system which, in mammals, is known to convert
promutagens into active DNA damaging metabolites. In order to overcome this major drawback an exogenous
metabolic system was added in form of mammalian microsome enzyme activation mixture.

S9 Homogenate
The S9 liver microsomal fraction was preparcd at BSL BIO SERVICE GmbH. Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and
ß-naphthoflavone (100 mg/kg bw) for three consecutive days by oral route.
The following quality control determinations are performed:
a) Biological activity in:
- the Salmonella typhimurium assay using 2-aminoanthracene
-the mouse lymphoma assay using benzol [ a ]pyrene
-the chromosome aberration assay using cyclophosphamide.
b) Sterility Test
A stock of the supernatant containing the microsomes was frozen in aliquots of 2 and 4.5 mL and stored at <-75 °C.
The protein concentration in the S9 preparation (Lot: 110310) was 31 mg/mL. The S9 mix preparation was performed according to Ames et al.
Preparation of S9 Mix
100 mM of ice-cold sodium-Ortho-phosphat-buffer, pH 7.4, was added to the following pre-weighed sterilised reagents to give final concentrations in the S9 mix of:
8 mM MgCl2; 33 mM KCI; 5 mM glucose-6-phosphate; 4 mM NADP
This solution was mixed with the liver 9000 x g supernatant fluid in the following proportion:
co-factor solution 9.5 parts
liver preparation 0.5 parts
During the experiment the S9 mix was stored on ice.

S9 Mix Substitution Buffer
The S9 mix substitution buffer was used in the study as a replacement of S9 mix,
without metabolic activation (-S9).
Phosphate-buffer (0.2 M) contains per litre:
0.2 M NaH2P04 x H20: 120 mL
0.2 M Na2HP04: 880 mL
The two solutions were mixed and the pH was adjusted to 7.4. Sterilisation was performed at 121°C in an autoclave.
This 0.2 M phosphate-buffer was mixed with 0.15 M KCI solution (sterile) in the following proportion:
0,2 M phosphate-buffer: 9.5 parts
0.15 M KCI solution: 0.5 parts
This S9 mix substitution buffer was stored at 4°C.

Experimental Performace
For the plate incorporation method the following materials were mixed in a test tube and poured over the surface of a minimal agar plate:
100 µL: Test solution at eaeh dose level, solvent control, negative control of reference mutagen solution (positive control),
500 µL: S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation),
100 µL: Bacteria suspension (cf. Preparation of Baeteria, pre-culture of the strain),
2000 µL: Overlay agar

For each strain and dose level, including the controls, three plates were used,
After solidification the plates were inverted and incubated at 37°C for at least 48 h in the dark.

Data Recording
The colonies were counted using a ProtocoL counter (Meintrup DWS Laborgeräte GmbH). If precipitation of the test item precluded automatic
counting the revertant colonies were counted by hand, in addition, tester strains with a low spontaneous mutation frequency like TA 1535 and TA 1537 were counted manually,

Evaluation criteria:

Cytotoxicity can be detected by a clearing or rather diminution of the background lawn
or a reduction in the number of revertants down to
a mutation factor of approximately <= 0.5 in relation to the solvent control

A test is considered acceptable if for each strain: the bacteria demonstrate their typical responses to ampicillin (TA 98, TA 100, TA 102) the control plates with and without S9 mix are within the following ranges (mean values of the spontaneous reversion frequency are within the historical control data
range):
- S9 +S9
TA 98: 18 18-46 18- 57
TA 100: 77 - 163 78 - 165
TA 1535: 5 -29 5-27
TA 1537: 5 -30 5 -36
TA 102: 164 -390 163 -472
- corresponding background growth on negative control, solvent control and test plates is observed
- the positive controls show a distinct enhancement of revertant rates over the control Plate

The Mutation Factor is calculated by dividing the mean value of the revertant counts
through the mean values of the solvent control
A test item is considered as mutagenic if:
a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.

Acc. to OECD guidelines, the biol. relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Statistics:

According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

It can be stated that during the described mutagenicity test and under the experimental conditions reported, Methacrylic anhydride did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, Methacrylic anhydride is considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

In a reverse gene mutation assay in bacteria (Ames test), strains TA1535, TA1537, TA98, TA100, and TA102 of Salmonella typhimuriumwere exposed to Methacrylic anhydride at concentrations of up to 2.0 µl/plate in the presence and absence of mammalian metabolic activation S9 -mix. 

No biologically relevant inereases in revertant colony numbers of any of the five tester strains were observed following treatment with Methacrylic anhydride at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II

The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiments.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, Methacrylic anhydride did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.

Therefore, Methacrylic anhydride is considered to be non-mutagenic in this bacterial reverse mutation assay. Appropriate reference mutagens were used as positive controls.

The positive controls induced the appropriate responses in the corresponding strains.

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

This study is classified as acceptable, it satisfies the requirements for testing bacterial reverse mutations.

 

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

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

Reference

Reference 1

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

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 or test system 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.

Reference 2

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

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 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

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.

Reference 3

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

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.