Reaction mass of dodecyl methacrylate and tridecyl methacrylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

consistenty negative in a bacteria reverse mutation assay (OECD TG 471; RL1, GLP), mammalian cell gene mutation assay (OECD TG 476, RL1, GLP) and chromosome aberration assay (OECD TG 473, RL1, GLP); read-across from Isodecyl methacrylate

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar physicochemical, ecotoxicological and toxicological properties because
• they are manufactured from similar or identical precursors under similar conditions
• they share structural similarities with common functional groups: methacrylate esters
• the metabolism pathway leads to comparable products (methacrylic acid and medium chain alcohol).

Therefore, read-across from the existing physicochemical, ecotoxicity and toxicity studies on the source substances is considered as an appropriate adaptation to the standard information requirements of REACH regulation

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see “Justification for read-across” attached to IUCLID section 13

3. ANALOGUE APPROACH JUSTIFICATION
see “Justification for read-across” attached to IUCLID section 13

4. DATA MATRIX
see “Justification for read-across” attached to IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Type of assay:

bacterial reverse mutation assay

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)

Species / strain:

other: Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and 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

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results: negative

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar physicochemical, ecotoxicological and toxicological properties because
• they are manufactured from similar or identical precursors under similar conditions
• they share structural similarities with common functional groups: methacrylate esters
• the metabolism pathway leads to comparable products (methacrylic acid and medium chain alcohol).

Therefore, read-across from the existing physicochemical, ecotoxicity and toxicity studies on the source substances is considered as an appropriate adaptation to the standard information requirements of REACH regulation

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see “Justification for read-across” attached to IUCLID section 13

3. ANALOGUE APPROACH JUSTIFICATION
see “Justification for read-across” attached to IUCLID section 13

4. DATA MATRIX
see “Justification for read-across” attached to IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Type of assay:

in vitro mammalian chromosome aberration test

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

Remarks on result:

other: strain/cell type: Human Lymphocytes

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results: negative

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar physicochemical, ecotoxicological and toxicological properties because
• they are manufactured from similar or identical precursors under similar conditions
• they share structural similarities with common functional groups: methacrylate esters
• the metabolism pathway leads to comparable products (methacrylic acid and medium chain alcohol).

Therefore, read-across from the existing physicochemical, ecotoxicity and toxicity studies on the source substances is considered as an appropriate adaptation to the standard information requirements of REACH regulation

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see “Justification for read-across” attached to IUCLID section 13

3. ANALOGUE APPROACH JUSTIFICATION
see “Justification for read-across” attached to IUCLID section 13

4. DATA MATRIX
see “Justification for read-across” attached to IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Type of assay:

mammalian cell gene mutation assay

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

Remarks on result:

other: strain/cell type: V79

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results: negative

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 February 2018 to 14 March 2018 (Study initiation to experimental completion)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Kyoeisha Chemical Co., Ltd-
- Lot No. of test material: 7061301
- Expiration date of the lot: 13 June 2019
- Purity test date: 3 October 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In original container as supplied by the Sponsor, at room temperature (Ambient). container kept tightly closed and away from heat or sunlight
- Stability under test conditions: Assumed stable for the duration of the test
- Solubility and stability of the test substance in the solvent/vehicle: Soluble in diimethyl sulphoxide (DMSO) at 50 µL/mL
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: None

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Solublised in DMSO at 50 µL/mL

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Initial Toxicity-Mutation Assay: 0.0015, 0.005, 0.015, 0.05, 0.15, 0.5, 1.5, 5 µl/plate
Confirmatory Mutation Assay: 0.16, 0.31, 0.63, 1.25, 2.5, 5 µl/plate
In accordence with OECD 471, the recommended maximum test concentration for soluble non-cytotoxic substances is 5 µl/plate. This was the highest selected concentration for testing in the absence and presence of metabolic activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Soluble in DMSO at 50 µL/mL to permit the recommended maximum test concentration of 5 µl/plate.

Untreated negative controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Aminoanthracene (+S9) to verify the efficiency of the S9 fraction

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar using the plate incorporation method
- Cell density at seeding (if applicable): Cell densities (OD at 660 nm) of all tester strains were within the required range to produce cultures with approximately 1 - 2 x 109 bacteria/mL, demonstrating appropriate numbers of bacteria were plated.

DURATION
- Exposure duration: Petri plates were incubated at 37 ± 1 °C for 48 hours

NUMBER OF REPLICATIONS: Duplicate plates (Initial Toxicity Mutation Assay) and Triplicate plates (Confirmatory Mutation Test)


DETERMINATION OF CYTOTOXICITY
- Method: other: In the confirmatory mutation assay, six analysable doses were available to evaluate assay data. Cytotoxicity is detectable as decrease in the number of revertant colonies per plate and/or by a thinning of the bacterial background lawn.
- Any supplementary information relevant to cytotoxicity: Cytotoxicity was characterised by inhibition of the background bacterial lawn and/or reduction in the number of revertant colonies. In the initial Toxicity Mutation Assay, a normal bacterial background lawn and no increase in the number of revertant colonies was observed up to 5 µL/plate both in the absence and presence of metabolic activation.

Rationale for test conditions:

Standard test conditions, ie for the solvent/vehicle, exposure concentrations and controls, were performed in accordence with OECD 471 guidence.

Evaluation criteria:

Once the criteria for a valid assay had been met, responses observed in the assay were evaluated. The conditions necessary for determining a positive result were: there should be a dose-related increase in the mean revertants per plate in at least one tester strain over the range tested and/or at one or more doses of the test item either in the absence or presence of the metabolic activation system. The biological relevance of the results was considered:

Strains TA1535 and TA1537 and Escherichia coli WP2uvrA:
Data sets were judged positive, when the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0 times the mean negative control value.

Strain TA98, TA100:
Data sets were judged positive, when the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0 times the mean negative control value.
Statistical analysis was used as an aid in the evaluation of a dose response.
A response that did not meet all three of the above criteria (magnitude, concentration responsiveness, reproducibility) was not evaluated as positive.
Negative results obtained in the initial toxicity-mutation assay were confirmed by a confirmatory mutation assay, using the same method as specified above, with an alteration in concentration spacing.

Statistics:

Simple linear regression analysis was performed for tester strains of Salmonella typhimurium viz., TA1537, TA1535, TA98, TA100 and Escherichia coli WP2uvrA, separately, to assess the dose dependent nature of any increase in revertant colonies.

In the Confirmatory Mutation Assay, statistical analysis did not reveal any significant effect.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

not applicable

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

not applicable

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

not applicable

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: None specified
- Effects of osmolality: None specified
- Precipitation: No precipitation observed at the limit test concentration of 5 µL/plate.
- Other confounding effects: None

HISTORICAL CONTROL DATA (with ranges, means and standard deviation)
- Positive historical control data: yes
- Negative (solvent/vehicle) historical control data: yes

Conclusions:

Under the specified experimental conditions, C12-13 alkyl methacrylate is concluded to be non-mutagenic in the Bacterial Reverse Mutation Assay using tester strains of Salmonella typhimurium viz., TA1537, TA1535, TA98, TA100 and Escherichia coli WP2uvrA.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A bacterial reverse mutation assay is available for the assessment of genetic toxicity of Reaction mass of dodecyl methacrylate and tridecyl methacrylate. Further studies (mammalian cell gene mutation assay and chromosome aberration test) are available for the source substance Isodecyl methacrylate. A detailed justification for read-across is attached to IUCLID section 13.

 

The absence of a mutagenic potential was demonstrated for gene mutations as well as chromosome mutations for Isodecyl methacrylate. These results are representative for the target substance, as Isodecyl methacrylate represents a worst-case in terms of bioavailability due to its molecular size, lipophilicity and water solubility.

 

Hypothesis for the analogue approach

The read-across hypothesis relies on the close structural similarity between the source substance Isodecyl methacrylate and the target substance Reaction mass of dodecyl methacrylate and tridecyl methacrylate. This read-across hypothesis corresponds to scenario 2 - different compounds have qualitatively similar properties - of the read-across assessment framework i.e. properties of the target substance are predicted to be quantitatively equal to those of the source substance. Namely, the structurally similar source substance Isodecyl methacrylate predicts the toxicological properties of the target substance Reaction mass of dodecyl methacrylate and tridecyl methacrylate.

 

Toxicological data are summarised in the data matrix; robust study summaries are included in the Technical Dossier in the respective sections.

 

Therefore, read-across from the existing toxicity studies conducted with the source substance is considered as an appropriate adaptation to the standard information requirements of the REACH Regulation for the target substance, in accordance with the provisions of Annex XI, 1.5 of the REACH Regulation.

A detailed justification for the proposed read-across approach is attached to Iuclid section 13.

 

1. Identity and characterisation of the source substance

 

There is close structural similarity between the source and the target substances and the identity and characterisation of these substances is unambiguous thereby giving a high level of confidence in the validity of the read across.

The target and source substances are manufactured from similar compounds by esterification of methacrylic acid with the corresponding fatty alcohol. Typical trace impurities are water and the corresponding alcohols as well as < 1 % methacrylic acid, which are not of toxicological concern.

The carbon chain length distribution of the resulting mix of long-chain aliphatic methacrylate esters mirrors the chain length distribution of the alcohol(s) used.

 

2. Link of structural similarities and differences with the proposed prediction

Structural similarities:

The target substance Reaction mass of dodecyl methacrylate and tridecyl methacrylate is an ester of Methacrylic acid and mainly linear C12 / C13 alcohols.

 

Structural differences:

There are differences in alkyl chain length between the target and the source substances. Additionally, the source substance contains branched alkyl chains, whereas the target substance mainly contains linear C-chains.

The source substance Isodecyl methacrylate can be expected to have a slightly higher bioavailability due to lower molecular weight compared to the target substance.

The physicochemical properties (low water solubility and high log Kow) are, however, quite similar. Thus, no large differences in bioavailability are expected. No differences in reactivity are expected based on these minor structural differences.

 

Reliability and adequacy of the source data

All available studies have been conducted according to OECD guidelines and have been assigned a reliability of 1 or 2 as documented in the data matrix (see detailed justification for read-across attached to Iuclid section 13).

 

Overall, the study design of the respective source studies is adequate and reliable for the purpose of this read-across. The results of the selected key studies are adequate for classification and labelling and for risk assessment purposes.

 

 

Data availability

Reverse gene mutation assay in bacteria

Reaction mass of dodecyl methacrylate and tridecyl methacrylate was non-mutagenic in the Bacterial Reverse Mutation Assay using tester strains of Salmonella typhimurium viz., TA1537, TA1535, TA98, TA100 and Escherichia coli WP2uvrA when tested up to the limit concentration of 5 µL/plate.

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.

 

Chromosome aberration test

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.

 

in vitro gene mutation study in mammalian cells

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.

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.

 

 

There are no data gaps for the endpoint genetic toxicity. There is no reason to believe that the results would not be relevant to humans. 

 

Justification for classification or non-classification

Based on the available data, Reaction mass of dodecyl methacrylate and tridecyl methacrylate does not need to be classified for genetic toxicity according to the criteria given in regulation (EC) 1272/2008. Thus, no labelling is required.