Hexyl methacrylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

There are two in vitro mutagenicity studies concerning the mutagenic potential for n-hexyl methacrylate available where n-hexyl methacrylate was negative in vitro (Ames test, Zeiger et al., (1987)) and (Ames test, Waegenmachers et al, (1984)).

Bacterial gene mutation assay

The potential of n-hexyl methacrylate (n-HMA) to induce reverse mutation in Salmonella typhimurium (strains: TA 98, TA 100, TA 1535, TA 1537) was evaluated according to a protocol comparable to the OECD guidelines 471 (Zeiger et al., 1987). n-HMA was tested in single experiment, with and without a metabolic activation system, according to the preincubation method (20 min at 37 °C). Concentrations of n-HMA (0, 0.1, 0.33, 1, 3., 10, 33, 100, 333, 1000, 3333, and 10000 µg/plate.), overnight culture of S. typhimurium (0.05-0.10 ml) and S-9 mix or buffer were incubated without shaking for 20 minutes. The top agar was added and the contents of the tubes were mixed and poured onto the surfaces of Petri dishes. His+ (histidine dependent) colonies arising on plates were machine-counted after two days incubation at 37 °C. Testing was without metabolic activation, with 10% rat liver S-9, or with 10% hamster liver S-9. The positive control chemicals induced a significant increase of the revertant frequency in all tester strains, either with or without metabolic activation. n-HMA was negative, in the presence and absence of metabolic activation, in all tester strains.

Furthermore the potential of n-hexyl methacrylate (n-HMA) to induce reverse mutation in Salmonella typhimurium (strains: TA 98, TA 100, TA 1535, TA 1537, TA1538) was evaluated on triplicate plates according to a protocol comparable to the OECD guidelines 471 (standard Ames assay, Waegermakers et al., 1984). n-hexyl methacrylate was tested with and without a metabolic activation system, according to the standard procedure as described by Ames et al. (1975). At least 4 concentrations up to 2500 µg/plate were tested. n-HMA was diluted in DMSO prior to use. Solvent controls, positive controls and sterility controls for S9 mix were run with each experiment. His+ (histidine dependent) colonies arising on plates were manually-counted after 48 to 72 h after incubation in the dark at 37 °C. The positive control chemicals induced a significant increase of the revertant frequency in all tester strains, either with or without metabolic activation. n-HMA was negative, in the presence and absence of metabolic activation, in all tester strains.

Short description of key information:
There are two in vitro mutagenicity studies concerning the mutagenic potential for n-hexyl methacrylate available where n-hexyl methacrylate was negative in vitro (Ames test, Zeiger et al., (1987)) and (Ames test, Waegemaekers et al., (1984)). The results of genetic toxicity studies from the category Lower Alkyl (C1-C8) Methacrylates support the findings in n-hexyl methacrylate which are structurally closely related and are considered to be representative for the genetic toxicity of n-hexyl methacrylate.

So we expect n-hexyl methacrylate to be none mutagenic as well.

Endpoint Conclusion: No adverse effect observed (negative)

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:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Similar to OECD-guideline 471. Study well documented, meets generally accepted scientific principles, acceptable for assesment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Method: standard procedure as described by Ames et al. (1975) Ames test

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

his-

Species / strain / cell type:

S. typhimurium, other: TA1535, TA1537, TA1538, TA98, TA100

Metabolic activation:

with and without

Metabolic activation system:

AROCLOR 1254 or phenobarbitone induced rat liver S9 mix.

Test concentrations with justification for top dose:

40-25000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

benzo(a)pyrene

other: 2-Aminoantracene

Details on test system and experimental conditions:

Salmonella typhimurium reverse mutation assay, Ames test
To minimise evaporation treated plates were sealed in glass air-tight exposure jars.

Key result

Species / strain:

S. typhimurium, other: TA1535, TA1537, TA1538, TA98, TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Interpretation of results (migrated information):
negative

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

Executive summary:

The potential of n-hexyl methacrylate (n-HMA) to induce reverse mutation in Salmonella typhimurium (strains: TA 98, TA 100, TA 1535, TA 1537, TA1538) was evaluated on triplicate plates according to a protocol comparable to the OECD guidelines 471 (standard Ames assay, Waegermakers et al., 1984). n-hexyl methacrylate was tested with and without a metabolic activation system, according to the standard procedure as described by Ames et al. (1975). At least 4 concentrations up to 2500 µg/plate were tested. n-HMA was once tested with phenobarbial-induced S9 mix, once with Aroclor-1254 -induced S9 mix and twice without any additional metabolizing system. n-HMA was diluted in DMSO prior to use. Solvent controls, positive controls and sterility controls for S9 mix were run with each experiment. His+ (histidine dependent) colonies arising on plates were manually-counted after 48 to 72 h after incubation in the dark at 37 °C. The positive control chemicals induced a significant increase of the revertant frequency in all tester strains, either with or without metabolic activation. n-HMA was negative, in the presence and absence of metabolic activation, in all tester strains.

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

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Similar to OECD-guideline 471. Study well documented, meets generally accepted scientific principles, acceptable for assessment.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Method: Ames test

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

his-

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from Aroclor 1254 induced Sprague Dawley rat liver and male Syrian Hamster liver (10 %).

Test concentrations with justification for top dose:

At least five doses tested in triplicate.
S. typhimurium TA 100: 3 - 333 µg/plate (without activation), 100 - 10000 µg/plate (with activation, hamster liver and rat liver S-9-mix)
S. typhimurium TA 1535: 3- 333 µg/plate (without activation), 100 - 10000 µg/plate (with activation, hamster liver and rat liver S-9-mix)
S. typhimurium TA 1537: 0.1 - 100 µg/plate (without activation), 100 - 10000 µg/plate (with activation, hamster liver and rat liver S-9-mix)
S. typhimurium TA 98: 3 - 333 µg/plate (without activation), 100 - 10000 µg/plate (with activation, hamster liver and rat liver S-9-mix

Vehicle / solvent:

DMSO Dimethyl sulfoxid

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: without S9: sodium azide, 9-aminoacridine and 4-nitro-o-phenylenediamine. With S9: 2-aminoanthracene

Details on test system and experimental conditions:

Salmonella typhimurium reverse mutation assay
Two preincubation assays were conducted.

Evaluation criteria:

An individual trial was judged (+) mutagenic if a dose related increase over the corresponding solvent control was seen, and it was judged weakly mutagenic (+W) if a low dose response was seen. A trial was considered questionable (?) if a dose related increase was judged insufficiently high to justify a call of "+W" if only a single dose was elevated over control or if a non-dose related increase was seen. A chemical was judged weakly mutagenic "+W' or mutagenic "+" if it produced a reproducible, dose related increase in his+ revertants over the corresponding solvent controls in replicate trials.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

S9 mix from Aroclor 1254 induced Sprague Dawley rat liver and male Syrian Hamster liver (10% in test concentration)

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

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

Executive summary:

The potential of n-hexyl methacrylate (n-HMA) to induce reverse mutation in Salmonella typhimurium (strains: TA 98, TA 100, TA 1535, TA 1537) was evaluated according to a protocol comparable to the OECD guidelines 471 (Zeiger et al., 1987). n-HMA was tested in single experiment, with and without a metabolic activation system, according to the preincubation method (20 min at 37 °C). Concentrations of n-HMA (0, 0.1, 0.33, 1, 3., 10, 33, 100, 333, 1000, 3333, and 10000 µg/plate.), overnight culture of S. typhimurium (0.05-0.10 ml) and S-9 mix or buffer were incubated without shaking for 20 minutes. The top agar was added and the contents of the tubes were mixed and poured onto the surfaces of Petri dishes. His+ (histidine dependent) colonies arising on plates were machine-counted after two days incubation at 37 °C. Testing was without metabolic activation, with 10% rat liver S-9, or with 10% hamster liver S-9. The positive control chemicals induced a significant increase of the revertant frequency in all tester strains, either with or without metabolic activation. n-HMA was negative, in the presence and absence of metabolic activation, in all tester strains.

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Methacrylates behave as a chemical family when studied for genotoxicity potential and, with few exceptions based on alerting chemical structures, new compounds in this family may be considered for waiver from acutal testing based on structure-activity relationships. Therefore the genotoxic behaviour of a similar chemical can be predicted with confidence by inclusion within this chemical class, thus avoiding unnessary testing. (Johannsen FR et al.(2008) Regulatory Toxicology and Pharmacology 50: 322 - 335)

Data availability

For n-hexyl methacrylate gene mutation data in bacteria are available in vitro. Data from the category Lower Alkyl (C1-C8) Methacrylates support the findings in n-hexyl methacrylate. In vivo data for n-hexyl methacrylate are not available.

Data from analogous substances and metabolites

Analogous substances

Reliable in vitro gene mutation assays in mammalian cells, test method OECD Guideline 476 (In vitro Mammalian Cell Gene Mutation Test), are available for methyl methacrylate (CAS 80-62-6); ethyl methacrylate (CAS 97-63-2) and 2-ethyl-hexyl methacrylate (CAS 688-84-6). In general, methyl methacrylate and ethyl methacrylate were positive in high and mainly toxic concentrations in several mouse lymphoma assays (small colony mutants, indicating that the genetic effect was derived from clastogenicity and not from gene mutations). 2-ethylhexyl methacrylate did not induce gene mutations at the HPRT locus in V79 cells (this study is attached as read-across information).

Further support for the absence of genotoxic potential in vivo can be gained by read-across from MMA (CAS 80-62-6), referring to a dominant lethal test in CD-1 mice (Anderson and Hodge, 1976). In this study groups of 20 male CD-1 mice were exposed via inhalation to MMA at 100, 1000, or 9000 ppm (416, 4160 and 37440 mg/m³) for 6 h/day for 5 days. Each male was subsequently mated with 2 different unexposed female mice weekly over a period of 8 weeks. MMA did not induce dominant lethal mutations as indicated by no adverse effect on total implants and early or late post-implantation death in the offspring of treated males compared to controls (this study is attached as read-across information).

 

Metabolites

It has been established that the lower alkyl methacrylates have a common mode of chemical reactivity via the C=C double bond and Michael addition and as such this lends them the potential to be chemically reactive towards macromolecules such as protein and DNA though a mechanism of electrophilic attack, albeit with a low reactivity (see ch. 5.1.3 toxicokinetics; Schwöbel et al., 2010, Cronin, 2012, 2015). It has also been established that the primary metabolic pathway of the esters is fast hydrolysis by ubiquitous carboxylesterases in the human body with a half-life in the order of minutes (Jones 2002). The resultant acid (methacrylic acid) and alcohol metabolites are non genotoxic.

The EU ESR on MAA (methacrylic acid, CAS 79-41-4) concluded: “Methacrylic acid is negative in a bacterial gene mutation test. 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.”

None of the lower alkyl alcohol metabolites are regarded as mutagenic.

 

Summary                                  

Overall, n-hexyl methacrylate is negative in gene mutation tests in in bacteria. This, also, is supported by analogous substances from the lower alkyl methacrylates category. In conclusion, n-hexyl methacrylate is regarded as non-genotoxic.

Short description of key information:
Gene mutation in bacteria
Negative S. typhimurium TA 1535, TA 1537, TA 98, and TA 100, with and without metabolic activation (similar to OECD 471) (Zeiger, 1987)

Negative S. typhimurium TA 1535, TA 1537, TA1538, TA 98, and TA 100, with and without metabolic activation (similar to OECD 471) (Waegemaekers, 1984)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

According to the available data and the CLP criteria for classification as germ cell mutagens, no classification is warranted for n-hexyl methacrylate.