Dodecyl methacrylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Read across from structural analogue substance Isodecyl methacrylate CAS: 29964-84-9:

For the assessment of dodecyl methacrylate there are peer reviewed data of a review available and data of the structurally related substance isodecyl methacrylate (three in vitro studies).

One reverse mutation assay in bacteria with isodecyl methacrylate, one chromosome aberration micronucleus test and one HPRT test with the structurally related substance isodecyl methacrylate are available.

Bacterial reverse mutation assay

No study is available on dodecyl methacrylate but there is a study with the structurally related substance isodecyl methacrylate.

In a reverse gene mutation assay in bacteria according to OECD guideline 471 Salmonella typhimurium TA98, TA100, TA1535, TA1537 and TA102 strains were exposed to isodecyl methacrylate in THF at concentrations up to 5000 µg/plate in the presence and absence of mammalian metabolic activation. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

 

In vitro Chromosome aberration test in human lymphocytes

No study is available on dodecyl methacrylate but there is a study with the structurally related substance isodecyl methacrylate.

 

In a chromosome aberration test in human lymphocytes according to OECD guideline 473 (adopted May 26, 1983), human lymphocyte cultures were exposed to Isodecyl methacrylate (98.9%) in THF with and without metabolic activation (S9 mix).

The following concentrations were evaluated:

Experiment I:

22 hrs prep. interval, 4 h treatment without metabolic activation: 14.5, 25.4, 44.4, 77.7, 136.0, 238.1, 416.7, 729.1, 1276.0, 2233 µg/ml

22 hrs prep. interval, 4 h treatment with metabolic activation: 14.5, 25.4, 44.4, 77.7, 136.0, 238.1, 416.7, 729.1, 1276.0, 2233 µg/ml

Experiment II:

22 hrs prep. interval, 22 h treatment without metabolic activation: 44.4, 77.7, 136.0, 238.1, 416.7, 729.1, 1276.0, 2233 µg/ml

22 hrs prep. interval, 4 h treatment with metabolic activation: 44.4, 77.7, 136.0, 238.1, 416.7, 729.1, 1276.0, 2233 µg/ml

In Experiment I, visible precipitation of the test item in the culture medium was observed at 136.0 µg/ml and above in the absence and presence of S9 mix. In addition, precipitation occurred in Experiment II, in the absence of S9 mix, at 136.0 µg/ml and above and in the presence of S9 mix at 416.7 µg/ml and above. No relevant increase in the osmolarity or pH value was observed. In Experiment I, in the absence and presence of S9 mix, and in Experiment II, in the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In Experiment II, in the absence of S9 mix, a single clearly reduced mitotic index was observed at the highest dose evaluated for cytogenetic damage.

Isodecyl methacrylate was tested up to cytotoxic concentration.

 

In the absence and presence of S9 mix neither a statistically nor a biologically relevant increase in the number of cells carrying structural chromosome aberrations were observed after treatment with the test item.

HPRT-test in mammalian cells

No study is available on dodecyl methacrylate but there is a study with the structurally related substance isodecyl methacrylate.

Isodecyl methacrylate was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster according to OECD 476.

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

The cell cultures were evaluated at the following concentrations:

Experiment I:

without S9 mix:  0.1; 0.3; 0.5; 1.0; and 2.0 µg/ml

with S9 mix: 37.5; 75; 150; 300; and 1200 µg/ml

 

Experiment II:

without S9 mix: 18.8 ;37.5; 75.0; 150; and 600 µg/ml

with S9 mix: 37.5; 75.0; 150; 300; and 600 µg/ml

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 5.7 up to 24.0 mutants per 10⁶cells; the range of the groups treated with the test item was from 3.3 up to 34.1 mutants per 10⁶cells.

 

EMS(150 µg/mL in experiment I and 75 µg/mL in experiment II) and DMBA (2.0 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies. This showed the sensitivity of the test system and the activity of the S9 mix.

 

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

other information

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Peer reviewed data. No more information available.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Genotoxicity:

negative

Conclusions:

Interpretation of results: negative

Using a valid scientific method, Dodecyl methacrylate was negative for genotoxicity in the Salmonella typhimurium reverse mutation assay.

Executive summary:

Using a valid scientific method, Dodecyl methacrylate was negative for genotoxicity in the Salmonella typhimurium reverse mutation assay.

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27-03-2008 - 08-04-2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD 471, GLP. Study according to relevant guideline.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted May 26, 1983

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): Isodecyl methacrylate
- Supplier: Evonik Röhm GmbH, Darmstadt, Germany
- Substance type: organic
- Physical state at room temperature: liquid
- Expiration date of the lot/batch: Aug 26, 2008
- Stability under test conditions: Stability in water: Several days at room temperature, refrigerated and in the freezer
- Storage condition of test material: At room temperature

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix fraction of Aroclor 1254-induced, male Sprague-Dawley rats and male Syrian hamster livers

Test concentrations with justification for top dose:

Main experiment:
-S9 mix; 0; 33; 100; 333; 1000; 2500; 5000 µg/plate
+S9 mix; 0; 33; 100; 333; 1000; 2500; 5000 µg/plate

According to the results of the pre-experiment the concentrations applied in the main experiments were chosen.
The maximum concentration was 5000.0 µg/plate. The concentration range include two logarithmic decades. Six adequately spaced concentrations
were tested. Two independent experiments were performed.


Each chemical was tested initially at half-log dose intervals up to a dose that elicited toxicity, or to a dose immediately below one which was toxic in
the preliminary toxicity test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Tetrahydrofuran (THF)
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Untreated negative controls:

yes

Remarks:

Concurrent untreated and solvent control: DMSO were performed

Negative solvent / vehicle controls:

yes

Remarks:

vehicle: THF

True negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

for TA 1535, TA 100

Positive control substance:

sodium azide

Remarks:

sodium azide (purity: >= 99.0%, supplier: Serva, D-69042 Heidelberg, Germany) dissolved in aqua dest.; concentration: 10 µg/plate

Positive controls:

yes

Remarks:

for TA 1537, TA 98

Positive control substance:

other: 4-nitro-o-phenylenediamine, without metabolic activation

Remarks:

4-nitro-o-phenylenediamine (purity: > 99.9%, supplier: Sigma, D-82041 Deisenhofen, Germany) dissolved in DMSO; concentration: 10 µg/plate

Positive controls:

yes

Remarks:

for TA 102

Positive control substance:

methylmethanesulfonate

Remarks:

methylmethanesulfonate (purity: > 99.0%, supplier: Merck-Schuchardt, D-85662 Hohenbrunn, Germany) dissolved in aqua dest.; concentration: 1.0 µl/plate Migrated to IUCLID6: without metabolic activation

Positive controls:

yes

Remarks:

for TA 1535, TA 1537, TA 98, TA 100, TA 102

Positive control substance:

other: 2-aminoanthracene, with metabolic activation

Remarks:

2-aminoanthracene (purity: > 99%, supplier: MERCK, D-64293 Darmstadt, Germany) dissolved in DMSO; concentration: 2.5 µg/plate (10 µg/plate in TA 102)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation assay as described by Haworth et al. 1983, with some differences.

- Salmonella typhimurium strains were obtained from Dr. Bruce Ames (University of California, Berkeley, U.S.A.) and were stored as recommended (Maron and Ames, 1983).
- Cultures were grown overnight with shaking at 37 °C in Oxoid No. 2 broth, and their phenotypes were analyszed prior to their use for mutagenicity assays.

Test conditions:
System of testing:
-Metabolic activation system: S9 from rat liver, induced with phenobarbital and 5,6-benzoflavone.
Administration:
-Number of replicates: 2
-Plate per test: 3
-Application: pre-incubation

Evaluation criteria:

EVALUATION OF RESULTS
The generally accepted conditions for the evaluation of the results are:

- corresponding background growth on both negative controls and test plates
- normal range of spontaneous reversion rates.

Range of spontaneous reversion frequencies*
------------------------------------------------------------------------------------
TA 135 TA 1537 TA 98 TA 100 TA 102
10 - 29 5 - 28 15 - 57 77 - 189 121 - 293

* These values refer to the negative control without metabolic activation and represent our historical control range in 1993.

According to international guidelines a statistical evaluation of the results is recommended. However, no evaluated statistical procedure can be
recommended for analysis of data from the bacterial assays at this time.
A test article is considered positive if either a dose related and reproducible increase in the number of revertants or a significant and reproducible
increase for at least one test concentration is induced.
A test article producing neither a dose related and reproducible increase in the number of revertants nor a significant and reproducible positive
response at any one of the test points is considered non-mutagenic in this system.

A significant response is described as follows:
A test article is considered mutagenic if in strain TA 100 and TA 102 the number of reversions is at least twice as high and in strains TA 1535,
TA 1537, and TA 98 it is at least three times higher as compared to the spontaneous reversion rate. Also, a dose-dependent and reproducible
increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the
highest dose induced the above described enhancement factors or not.

Statistics:

According to OECD guideline 471, a statistical analysis of the data is not mandatory.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Conclusions:

Interpretation of results: negative

In conclusion, it can be stated that during the decribed mutagenicity test and under the experimental conditions reported, the test item did not
induce gene mutations by base pair changes or frameshifts in the genome of the strains tested.
Therefore, Isodecyl methacrylate has to be judged as nonmutagenic in the presence and absence of mammalian metabolic activation
according to the Ames test results.

Executive summary:

In a reverse gene mutation assay in bacteria (Ames test), strains TA1535, TA1537, TA98, TA100, and TA102 of Salmonella typhimurium were exposed to Isodecyl methacrylate at concentrations of up to 5000 µg/plate in the presence and absence of mammalian metabolic activation S9 -mix. 

No toxic effects occurred in the test groups with and without metabolic activation.The plates incubated with the test article showed normal background growth up to ug/plate with and without S9-mix in all strains used. No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with at any concentration level, either in the presence or absence of metabolic activation (S9 -mix). There was also no tendency to higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls.

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

 

There was no evidence of induced mutant colonies over background.

Therefore, Isodecyl methacrylate is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

 

This reliable and valid study satisfies the requirement for tests for in vitro mutagenicity (bacterial reverse gene mutation) data.

 

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.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10-03-2008 - 30-04-2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD 473, GLP. Study according to relevant guideline.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted May 26, 1983 and EEC Directive 92/69, L 383 A, Annex V, B 10, dated December 29, 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

- Name of test material (as cited in study report): Isodecyl methacrylate
- Supplier: Evonik Röhm GmBH, Darmstadt, Germany
- Substance type: organic
- Physical state at room temperature: liquid
- Expiration date of the lot/batch: Aug 26, 2008
- Stability under test conditions: Stability in water: Several days at room temperature, refrigerated and in the freezer
- Storage condition of test material: At room temperature

Species / strain / cell type:

lymphocytes: human, short-term culture

Details on mammalian cell type (if applicable):

Blood samples were obtained from healthy donors not receiving medication.

Metabolic activation:

with and without

Metabolic activation system:

Mammalian rat liver microsomal fraction induced with phenobarbital / ß-naphthoflavone (S9 mix)

Test concentrations with justification for top dose:

Experiment I: 22 hrs prep. interval, 4 hrs exposure period: without S9 mix: 14.5; 25.4; 44.4; 77.7; 136.0; 238.1; 416.7; 729.1; 1276.0; 2233 µg/ml
22 hrs prep. interval, 4 hrs exposure period: with S9 mix: 14.5; 25.4; 44.4; 77.7; 136.0; 238.1; 416.7; 729.1; 1276.0; 2233 µg/ml




Experiment II: 22 hrs prep. interval, 22 hrs exposure period: without S9 mix: 44.4; 77.7; 136.0; 238.1; 416.7; 729.1; 1276.0; 2233 µg/ml
22 hrs prep. interval, 4 hrs exposure period: with S9 mix: 44.4; 77.7; 136.0; 238.1; 416.7; 729.1; 1276.0; 2233 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Tetrahydrofuran (THF) (E. Merck, D-64293 Darmstadt, Germany; purity: >= 99.5%)
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties and its relatrive non-toxicity to the cells.
The final concentration of DMSO in the culture medium was 0.5 % (v/v).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

concurrent negative and solvent controls

True negative controls:

no

Positive controls:

yes

Remarks:

dissolved in: nutrient medium

Positive control substance:

ethylmethanesulphonate

Remarks:

Final concentration: 825 µg/ml Migrated to IUCLID6: without metabolic activation

Positive controls:

yes

Remarks:

dissolved in: nutrient medium

Positive control substance:

cyclophosphamide

Remarks:

Final concentration: 15 µg/ml in Experiment I and 30 µg/ml in Experiment II Migrated to IUCLID6: with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium


DURATION
- Preincubation period: 50 - 80 hrs
- Exposure duration: 4 h (with S9 mix); 22 hours (without S9 mix)




SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays): Giemsa (Merck, D-64293 Darmstadt, Germany)


NUMBER OF REPLICATIONS: 2


NUMBER OF CELLS EVALUATED: 100 metaphases were scored


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; in addition the number of polyploid cells in 250 metaphase cells was scored (% polyploid metaphases)

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY
The chromosomal aberration assay is considered acceptable if it meets the following criteria:

a) The number of aberrations found in the negative and/or solvent controls falls within the range of historical laboratory control
data: 0.00 % - 4.00 %.
b) The positive control substances should produce significant increases of the number of cells with structural chromosomal aberrations.


EVALUATION OF RESULTS

A test article is classified as mutagenic if it induces reproducibly either a significant concentration-related increase in the number of structural
chromosomal aberrations or a significant and reproducible positive response for at least one of the test points.

A test article producing reproducibly neither a significant concentration-related increase in the number of structural chromosomal aberrations nor
a significant and reproducibly positive response at any one of the test points is considered nonmutagenic in this system.

Statistical significance was confirmed by means of the Fisher's exact test (p < 0.05).

However, both biological and statistical significance should be considered together.

Statistics:

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the Fisher's exact test. Evaluation was performed only for cells
carrying aberrations exclusive gaps.

Key result

Species / strain:

lymphocytes: human, short-term culture

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

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxicity was only observed in Experiment II, in the absence of S9 mix, where a single clearly reduced mitotic index was observed at the highest dose
evaluated for cytogenetic damage

Remarks on result:

other: strain/cell type: Human Lymphocytes

Conclusions:

Interpretation of results: negative

In the Chromosomal aberration test with human lymphocytes in vitro according to OECD 473 in the presence and absence of
mammalian metabolic activation structural chromosomal aberrations were not observed when tested up to cytotoxic
and/or precipitationg concentrations.
Therefore, the test substance is considered to be non-mutagenic under the experimental conditions reported.

Executive summary:

The test item Isodecyl methacrylate, dissolved in THF, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro in two independant experiments.

 

The study design was performed as follows:

 

Experiment I: 22 hrs preparation interval, 4 hrs exposure period without and with S9 mix

                      

Experiment II: 22 hrs prep. interval, 22 hrs exposure period without S9 mix and 22 hrs prep. interval, 4 hrs exposure period with S9 mix.

 

In each experimental group, two parallel cultures were analysed. Per culture 100 metaphase plates were scored for structural chromosomal aberrations, except for the positive controls in Experiment I, in the presence of S9 mix and Experiment II, in the absence of S9 mix, where 50 metaphase plates were scored due to strong clastogenic effects.

 

The highest applied concentration in Experiment I (2333.0 µg/ml of the test item, approx. 9.9 mM) was chosen with regard to the solubility of the test item in an appropriate solvent (THF) and with respect to the current OECD Guideline 473.

 

Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation in accordance with OECD Guideline 473.

In Experiment I, visible precipitation of the test item in the culture medium was observed at 136.0 µg/ml and above in the absence and presence of S9 mix. In addition, precipitation occurred in Experiment II, in the absence of S9 mix, at 136.0 µg/ml and above and in the presence of S9 mix at 416.7 µg/ml and above. No relevant increase in the osmolarity or pH value was observed. In Experiment I, in the absence and presence of S9 mix, and in Experiment II, in the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In Experiment II, in the absence of S9 mix, a single clearly reduced mitotic index was observed at the highest dose evaluated for cytogenetic damage.

 

In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item.

 

No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls.

 

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

 

 

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations as determined by the chromosome aberration test in human lymphocytes in vitro.

 

Therefore, the test substance is considered to be non-clastogenic under the experimental conditions reported.

                          

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.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11-03-2008 - 12-06-2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD 476, GLP. Study according to relevant guideline.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

adopted July 21, 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): Isodecyl methacrylate
- Supplier: Evonik Röhm GmBH, Darmstadt, Germany
- Substance type: organic
- Physical state at room temperature: liquid
- Expiration date of the lot/batch: Aug 26, 2008
- Stability under test conditions: Stability in water: Several days at room temperature, refrigerated and in the freezer
- Storage condition of test material: At room temperature

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

Mammalian liver microsomal fraction S9 mix

Test concentrations with justification for top dose:

Experiment I:
without S9 mix: 0.1; 0.3; 0.5; 1.0; and 2.0 µg/ml
with S9 mix: 37.5; 75; 150; 300; and 1200 µg/ml

Experiment II:
without S9 mix: 18.8 ;37.5; 75.0; 150; and 600 µg/ml
with S9 mix: 37.5; 75.0; 150; 300; and 600 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Tetrahydrofuran (THF)
- Justification for choice of solvent/vehicle: relative non-toxicity towards the cells and solubility properties of the test item

Negative solvent / vehicle controls:

yes

Remarks:

concurrent solvent controls (THF)

Positive controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Purity > 98 % ; Supplier :ACROS Organics, Geel, Belgium

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

Purity 99.5 %; Supplier: Merck, Darmstadt, Germany Migrated to IUCLID6: with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension

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



NUMBER OF CELLS EVALUATED: The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation
microscope (Nikon, 40407 Düsseldorf, Germany).

Evaluation criteria:

Acceptability of the Assay
The gene mutation assay is considered acceptable if it meets the following criteria:
- the numbers of mutant colonies per 10exp+6 cells found in the negative and/or solvent controls fall within the laboratory historical control data
range of 2001 – 2006.
- the positive control substances must produce a significant increase in mutant colony frequencies (Historical data).
- the cloning efficiency II (absolute value) of the negative and/or solvent controls must exceed 50 %.

Evaluation of Results
A test item is regarded as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive
response at one of the test points.
A test item producing neither a concentration- related increase of the mutant frequency nor a reproducible positive response at any of the test
points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is regarded as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency
at least at one of the concentrations in the experiment.
The test item is regarded as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be
considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low
spontaneous mutation rate in the range normally found ( mutants per 10exp+6 cells) a concentration-related increase of the mutations
within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.

Statistics:

Statistical Analysis
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT®11 (SYSTAT
Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software. The number of mutant colonies obtained for the
groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability
value) is below 0.05. However, both, biological and statistical significance were considered together.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

in forward gene mutations in mammalian cells

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1200 µg/mL with S9 mix; at 1.0 µg/mL and above in Experiment I and 37.5 µg/mL in Experiment II without S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Results and Discussion

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

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

The cell cultures were evaluated at the following concentrations:

Experiment I:

without S9 mix:  0.1; 0.3; 0.5; 1.0; and 2.0 µg/ml

with S9 mix: 37.5; 75; 150; 300; and 1200 µg/ml

 

Experiment II:

without S9 mix: 18.8 ;37.5; 75.0; 150; and 600 µg/ml

with S9 mix: 37.5; 75.0; 150; 300; and 600 µg/ml

Phase separation of the test item was observed at 300 µg/mL and above in the first experiment with metabolic activation and at 150 µg/mL and above in the second experiment without metabolic activation. In the second experiment with metabolic activation phase separation was noted at 300 µg/mL and above.

Relevant toxic effects indicated by a relative cloning efficiency 1 below 50 % occurred at 1.0 µg/mL and above in the first experiment without metabolic activation and at 1200.0 µg/mL and above with metabolic activation. In the second experiment toxic effects as described above occurred at 37.5 µg/mL without metabolic activation and at 1200 µg/mL with metabolic activation. The striking difference of toxic concentrations with and without metabolic activation is probably based on protein or lipid binding effects. In the presence of metabolic activation the protein and lipid concentration is higher due to the S9-mix added.This fact is furthermore supported by the considerably less severe cytotoxicity following 24 h treatment without metabolic activation. During long term exposure 10 % FCS have to be added increasing the protein and lipid concentration of the medium.

No relevant and reproducible increase in mutant colony numbers/10⁶cells was observed in the main experiments up to the maximum concentration.

The induction factor reached or exceeded the threshold of three times the corresponding solvent control in experiment I at 37.5 µg/mL in the first culture with metabolic activation and at the same concentration in the first culture in experiment II without metabolic activation. However, both effects were judged as biologically irrelevant fluctuations since no increase was observed at higher concentrations or in the parallel cultures under identical conditions. Furthermore, the effects were not dose-dependent as indicated by the lacking statistical significance.

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 5.7 up to 24.0 mutants per 10⁶cells; the range of the groups treated with the test item was from 3.3 up to 34.1 mutants per 10⁶cells.

 

EMS(150 µg/mL in experiment I and 75 µg/mL in experiment II) and DMBA (2.0 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies. This showed the sensitivity of the test system and the activity of the S9 mix.

 

Conclusion

 

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

 

Therefore, Isodecyl methacrylate is considered to be non-mutagenic in this HPRT assay.

 

Conclusions:

Interpretation of results: negative

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.
Therefore, Isodecyl methacrylate is considered to be non-mutagenic in this HPRT assay.

Executive summary:

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

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

The cell cultures were evaluated at the following concentrations:

Experiment I:

without S9 mix:  0.1; 0.3; 0.5; 1.0; and 2.0 µg/ml

with S9 mix: 37.5; 75; 150; 300; and 1200 µg/ml

 

Experiment II:

without S9 mix: 18.8 ;37.5; 75.0; 150; and 600 µg/ml

with S9 mix: 37.5; 75.0; 150; 300; and 600 µg/ml

Phase separation of the test item was observed at 300 µg/mL and above in the first experiment with metabolic activation and at 150 µg/mL and above in the second experiment without metabolic activation. In the second experiment with metabolic activation phase separation was noted at 300 µg/mL and above.

Relevant toxic effects indicated by a relative cloning efficiency 1 below 50 % occurred at 1.0 µg/mL and above in the first experiment without metabolic activation and at 1200.0 µg/mL and above with metabolic activation. In the second experiment toxic effects as described above occurred at 37.5 µg/mL without metabolic activation and at 1200 µg/mL with metabolic activation. The striking difference of toxic concentrations with and without metabolic activation is probably based on protein or lipid binding effects. In the presence of metabolic activation the protein and lipid concentration is higher due to the S9-mix added.This fact is furthermore supported by the considerably less severe cytotoxicity following 24 h treatment without metabolic activation. During long term exposure 10 % FCS have to be added increasing the protein and lipid concentration of the medium.

 

No relevant and reproducible increase in mutant colony numbers/10⁶cells was observed in the main experiments up to the maximum concentration.

 

The induction factor reached or exceeded the threshold of three times the corresponding solvent control in experiment I at 37.5 µg/mL in the first culture with metabolic activation and at the same concentration in the first culture in experiment II without metabolic activation. However, both effects were judged as biologically irrelevant fluctuations since no increase was observed at higher concentrations or in the parallel cultures under identical conditions. Furthermore, the effects were not dose-dependent as indicated by the lacking statistical significance.

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 5.7 up to 24.0 mutants per 10⁶cells; the range of the groups treated with the test item was from 3.3 up to 34.1 mutants per 10⁶cells.

 

EMS(150 µg/mL in experiment I and 75 µg/mL in experiment II) and DMBA (2.0 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies. This showed the sensitivity of the test system and the activity of the S9 mix.

 

Conclusion

 

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

 

Therefore, Isodecyl methacrylate is considered to be non-mutagenic in this HPRT assay.

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 single key study has been selected for in vitro genetic toxicity; instead, all available studies have been used in a weight-of-evidence approach. Read-across in vitro: In three different reliable (Klimisch score: 1, with GLP) in vitro genetic toxicity studies Isodecyl methacrylate (structurally related substance of Dodecyl methacrylate) received negative results. The absence of a mutagenic potential of Dodecyl methacrylate in vitro was supported by peer reviewed data of the review (Johannsen et al., 2008). Genetic toxicity data in vivo: Mouse micronucleus test, OECD 474: negative (Klimisch score: 1, GLP, (RCC-CCR, 1989))

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09-July-1989 - 31-Aug.- 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD 474, GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Principles of method if other than guideline:

Pre-Experiment for Toxicity
A preliminary study on acute toxicity was performed with 2 animals per sex under identical conditions as in the mutagenicity study concerning:
starvation period, animal strain; vehicle; route, frequency, and volume of administration.
The animals were treated orally with a single dose of 5000 mg/kg bw in 1% CMC with the test item and examined for acute toxic symptoms.

GLP compliance:

yes

Type of assay:

micronucleus assay

Specific details on test material used for the study:

- Supplier: Evonik Röhm GmbH, D-64293 Darmstadt, Germany

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL Tierfarm, Füllinsdorf, Switzerland
- Number of animals. 84 (42 males/42 females)
- Age at study initiation: minimum 11 weeks
- Weight at study initiation: approximately 30 g
- Assigned to test groups randomly: yes
- Fasting period before study: 18 hours before treatment
- Housing: single
- Diet: pelleted standard diet,ad libitum
- Water: tap water, ad libitum
- Acclimation period: minimum 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ±3°C
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs artifical light

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 1% CMC (carboxymethyl cellulose)
- Justification for choice of solvent/vehicle: vehicle was chosen to its relative non-toxicity for animals. All animals received a single standard volume
of 10 mL/kg body weight orally.
- Concentration of test material in vehicle:
- Supplier: Fluka; SIGMA-Aldrich Vertiebs-GmbH, 82041 Deisenhofen, Germany
- Lot/batch no.: no data
- Catalogue no.: 21902
- Purity: no data

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment, the test substance was formulated in 1% CMC. All animals received a single standard volume of 10 ml/kg body weight
orally.

DIET PREPARATION
- Rate of preparation of diet (frequency): no data
- Mixing appropriate amounts with (Type of food): no data
- Storage temperature of food: no data

Duration of treatment / exposure:

24h, 48h and 72 hours

Frequency of treatment:

single dose

Remarks:

Doses / Concentrations:
5000 mg/kg suspended in Carboxymethylcellulose (1 %)
Basis:
nominal conc.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

CPA; cyclophosphamide
- Supplier: SERVA, Heidelberg, Germany
- Purity: commercial grade
- Dissolved in: physiological saline
- Route of administration: orally, once
- Doses / concentrations: 40 mg/kg bw
Volume administered: 10 mL/kg bw

The stability of CPA at room temperature is sufficient. At 20 °C only 1% of CPA is hydrolysed per day in aqueous solution.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly or
the highest dose that can be formulated and administered reproducibly.
The maximum tolerated dose level is determined to be the dose that causes toxic reactions without having major effects on survival within 72
hours.
The volume to be administered should be compatible with physiological space available.

TREATMENT AND SAMPLING TIMES:

Treatment:
Approximately 18 hours before treatment the animals received no food but water ad libitum. At the beginning of the treatment the animals (including
the controls) were weighed and the individual volume to be administered was adjusted to the animals body weight. The animals received the test item, the vehicle or the positive control substance once. Twelve animals, six males and six females, were treated per dose group and sampling time.
Sampling of bone marrow was done 24, 48 and 72 hours after treatment, respectively.


DETAILS OF SLIDE PREPARATION:

Preparation of the Animals:
The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal
calf serum, using a syringe. The cell suspension was centrifuged at 1000 rpm for 5 minutes and the supernatant was discarded. A small
drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (Kindler, D-79110 Freiburg,
Germany/Giemsa. Cover slips were mounted with EUKITT (KINDLER, D-79110 Freiburg, Germany). At least one slide was made from each bone
marrow sample.

METHOD OF ANALYSIS:
Analysis of Cells:
Evaluation of the slides was performed using NlKON microscopes with 100x oil immersion objectives. At least 1000 polychromatic erythrocytes (PCE)
were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was
determined in the same sample and expressed in normochromatic erythrocytes per 1000 erythrocytes. The analysis was performed with coded
slides.
Ten animals (5 males, 5 females) per test group were evaluated as described. The remaining animal of each test group was evaluated in case an
animal had died in its test group spontaneously or due to gavage error.

Evaluation criteria:

A test article is classified as mutagenic if it induces either a statistically significant dose-related increase in the number of micronucleated
polychromatic erythrocytes or a reproducible statistically significant positive response for at least one of the test points.
A test article producing neither a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes nor a
statistically significant and reproducible positive response at anyone of the test points is considered nonmutagenic in this system.
This can be confirmed by means of the nonparametric Mann-Whitney test.
However, both biological and statistical significance should be considered together.
mntire

Statistics:

Statistical methods (nonparametric Mann-Whitney test) will be used as an aid in evaluating the results. However, the primary point of consideration
is the biological relevance of the results.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY (see Remarks on results including tables and figures)

In a pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 5000 mg/kg b.w., Dodecylmethacrylate
suspended in CMC (1%). The volume administered was 10 ml/kg b.w..
All treated animals expressed toxic reactions: reduction of spontaneous activity, eyelid closure and apathy. One female expressed pilo erection.

Summary of results test group                     dose mg/kg bw              sampling              PCEs with              range                            PCE/NCE                                                                                time (hours)        micronuclei        ------------------------------------------------------------------------------------------------------------------------------ suspending agent                     0                            24                     0.09%                 0 - 2                           1000 / 404                        test article                            5000                         24                    0.06%                  0 - 3                           1000 / 511 cyclophosphamide                40                           24                    1.06%                 3 - 27                           1000 /935         suspending agent                     0                            48                     0.14%                  1 - 4                           1000 / 688                      test article                            5000                          48                     0.03%                  0 - 2                          1000 / 900     suspending agent                     0                            72                      0.09%                 0 - 3                           1000 / 676                      test article                            5000                          72                      0.07%               0 - 2                           1000 / 670 -------------------------------------------------------------------------------------------------------------------------------

Conclusions:

Interpretation of results: negative
During the study decribed and under the experimental conditions reported, Dodecylmethacrylate did not induce micronuclei. This was determined by the micronucleus test with bone marrow cells of the mouse (OECD guideline 474).
Therefore, Dodecylmethacrylate is considered to be non-mutagenic in this micronucleus assay.

Executive summary:

In a NMRI mouse bone marrow micronucleus assay, 5 animals/male/female/dose were treated orally with Dodecylmethacrylate (98%, stabilizied) at a dose of 0, 5000 mg/kg bw. The test article was suspended in Carboxymethylcellulose (1%). This suspending agent was used as negative control. The volume administered orally was 10 ml/kg b.w.. 24 h, 48 h and 72 h after a single application of the test article the bone marrow cells were collected for micronuclei analysis. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei.

To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 1000 PCE.

The following dose level of the test article was investigated:

24 h, 48 h, and 72 h preparation interval: 5000 mg/kg b.w..

In a pre-experiment this dose level was estimated to be the maximum attainable dose. The animals expressed toxic reactions. After treatment with the test article the ratio between PCEs and NCEs was not affected as compared to the corresponding negative controls thus indicating no cytotoxic effects.

In comparison with the corresponding negative controls there was no enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article.

An appropriate reference mutagen was used as positive control which showed a distinct increase of induced micronucleus frequency. 

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

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)

Additional information

Additional information from genetic toxicity in vivo:

For the assessment of dodecyl methacrylate there are peer reviewed data of a review available and data of the structurally related substance isodecyl methacrylate (three in vitro studies).

One reverse mutation assay in bacteria with isodecyl methacrylate, one chromosome aberration micronucleus test and one HPRT test with the structurally related substance isodecyl methacrylate are available.

In vivo there is one micronucleus study with dodecyl methacrylate available.

 

Bacterial reverse mutation assay

No study is available on dodecyl methacrylate but there is a study with the structurally related substance isodecyl methacrylate.

In a reverse gene mutation assay in bacteria according to OECD guideline 471 Salmonella typhimurium TA98, TA100, TA1535, TA1537 and TA102 strains were exposed to isodecyl methacrylate in THF at concentrations up to 5000 µg/plate in the presence and absence of mammalian metabolic activation. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

 

In vitro Chromosome aberration test in human lymphocytes

No study is available on dodecyl methacrylate but there is a study with the structurally related substance isodecyl methacrylate.

 

In a chromosome aberration test in human lymphocytes according to OECD guideline 473 (adopted May 26, 1983), human lymphocyte cultures were exposed to Isodecyl methacrylate (98.9%) in THF with and without metabolic activation (S9 mix).

The following concentrations were evaluated:

Experiment I:

22 hrs prep. interval, 4 h treatment without metabolic activation: 14.5, 25.4, 44.4, 77.7, 136.0, 238.1, 416.7, 729.1, 1276.0, 2233 µg/ml

22 hrs prep. interval, 4 h treatment with metabolic activation: 14.5, 25.4, 44.4, 77.7, 136.0, 238.1, 416.7, 729.1, 1276.0, 2233 µg/ml

Experiment II:

22 hrs prep. interval, 22 h treatment without metabolic activation: 44.4, 77.7, 136.0, 238.1, 416.7, 729.1, 1276.0, 2233 µg/ml

22 hrs prep. interval, 4 h treatment with metabolic activation: 44.4, 77.7, 136.0, 238.1, 416.7, 729.1, 1276.0, 2233 µg/ml

In Experiment I, visible precipitation of the test item in the culture medium was observed at 136.0 µg/ml and above in the absence and presence of S9 mix. In addition, precipitation occurred in Experiment II, in the absence of S9 mix, at 136.0 µg/ml and above and in the presence of S9 mix at 416.7 µg/ml and above. No relevant increase in the osmolarity or pH value was observed. In Experiment I, in the absence and presence of S9 mix, and in Experiment II, in the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In Experiment II, in the absence of S9 mix, a single clearly reduced mitotic index was observed at the highest dose evaluated for cytogenetic damage.

Isodecyl methacrylate was tested up to cytotoxic concentration.

 

In the absence and presence of S9 mix neither a statistically nor a biologically relevant increase in the number of cells carrying structural chromosome aberrations were observed after treatment with the test item.

HPRT-test in mammalian cells

No study is available on dodecyl methacrylate but there is a study with the structurally related substance isodecyl methacrylate.

Isodecyl methacrylate was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster according to OECD 476.

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

The cell cultures were evaluated at the following concentrations:

Experiment I:

without S9 mix:  0.1; 0.3; 0.5; 1.0; and 2.0 µg/ml

with S9 mix: 37.5; 75; 150; 300; and 1200 µg/ml

 

Experiment II:

without S9 mix: 18.8 ;37.5; 75.0; 150; and 600 µg/ml

with S9 mix: 37.5; 75.0; 150; 300; and 600 µg/ml

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 5.7 up to 24.0 mutants per 10⁶cells; the range of the groups treated with the test item was from 3.3 up to 34.1 mutants per 10⁶cells.

 

EMS(150 µg/mL in experiment I and 75 µg/mL in experiment II) and DMBA (2.0 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies. This showed the sensitivity of the test system and the activity of the S9 mix.

 

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

 

In vivo Micronucleus test

In a NMRI mouse bone marrow micronucleus assay, 5 animals/male/female/dose were treated orally with dodecyl methacrylate (98%, stabilizied) at a dose of 0, 5000 mg/kg bw according to OECD 474. The test article was suspended in carboxymethyl cellulose (1%). This suspending agent was used as negative control. The volume administered orally was 10 ml/kg b.w.. 24 h, 48 h and 72 h after a single application of the test article the bone marrow cells were collected for micronuclei analysis. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei.

To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 1000 PCE.

The following dose level of the test article was investigated:

24 h, 48 h, and 72 h preparation interval: 5000 mg/kg b.w..

In a pre-experiment this dose level was estimated to be the maximum attainable dose. The animals expressed toxic reactions. After treatment with the test article the ratio between PCEs and NCEs was not affected as compared to the corresponding negative controls thus indicating no cytotoxic effects.

In comparison with the corresponding negative controls there was no enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article.

An appropriate reference mutagen was used as positive control which showed a distinct increase of induced micronucleus frequency. 

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

Therefore, dodecylmethacrylate is considered to be non-mutagenic in this micronucleus assay.

Conclusion:

The absence of a mutagenic potential was demonstrated for gene mutations as well as chromosome mutations for isodecyl methacrylate.

These results are representative for Dodecyl methacrylate as isodecyl methacrylate represents a worst-case in terms of bioavailability due to its molecular size, lipophilicity and water solubility.

A negative mouse micronucleus tests with Dodecyl methacrylate supports the absence of a mutagenic potential in vivo.

Justification for selection of genetic toxicity endpoint
There is one reliable (Klimisch score: 1, GLP) in vivo study with Dodecyl methacrylate.
In three different reliable (Klimisch score: 1, with GLP) in vitro genetic toxicity studies Isodecyl methacrylate (structurally related substance of Dodecyl methacrylate) received negative results.

Justification for classification or non-classification

Based on the reliable available data on Dodecyl methacrylate and Isodecyl methacrylate (structurally related substance of Dodecyl methacrylate), Dodecyl methacrylate does not need to be classified for mutagenicity according to the criteria given in regulation (EC) 1272/2008 (CLP (EU-GHS) or the former European directive on classification and labelling 67/548/EEC. Thus, no labelling is required.