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Genetic toxicity: in vitro

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Administrative data

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:
06 Oct to 13 Nov 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-study OECD 471, GLP. Study according to relevant guideline.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted July 21, 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
dayted May 30, 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Principles of method if other than guideline:
The bacterial reverse mutation test is able to identify substances that cause point mutations, by substitution, addition or deletion of one or a few
DNA base-pairs. Mutagenic substances can induce reversion in histidine-(for Salmonella typhimurium) or tryptophan-(for Escherichia coli)
deficient strains which are then able to grow and form colonies in a histidine- or tryptophan limited medium, while non-reverted strains can not.
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Ethyltriglycol methacrylate
- Substance type: organic
- Physical state at room temperature: liquid

Method

Species / strain
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 phenobarbital and beta-naphthoflavone -induced, male Wistar rats
Test concentrations with justification for top dose:
Main experiment:
+/-S9 mix; 0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (experiment I and experiment II)

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. 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: distilled water
- Justification for choice of solvent/vehicle: The solvent was chosen because of its relative nontoxicity for the bacteria.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Concurrent untreated and solvent control: DMSO were performed
Negative solvent / vehicle controls:
yes
Remarks:
vehicle: DMSO
True negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
for TA 1535, TA 100
Positive control substance:
sodium azide
Remarks:
sodium azide (supplier: Sigma, D-82041 Deisenhofen, Germany) dissolved in aqua dest.; concentration: 10 µg/plate

Migrated to IUCLID6: without metabolic activation
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 (supplier: Fluka dissolved in DMSO; concentration: 10 µg/plate for TA98, 40 µL/plate for TA 1537
Positive controls:
yes
Remarks:
for TA 102
Positive control substance:
methylmethanesulfonate
Remarks:
methylmethanesulfonate (supplier: Sigma, D-82041 Deisenhofen, 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 (supplier: Sigma, D-82041 Deisenhofen, Germany) dissolved in DMSO; concentration: 2.5 µg/plate (10 µg/plate for TA 102)
Details on test system and experimental conditions:
METHOD OF APPLICATION: Plate-incorporation test (experiment I) and pre-incubation test (experiment II)

- Salmonella typhimurium strains were obtained from MOLTOX Inc., NC 28607, USA
-Metabolic activation system: S9 from rat liver, induced with phenobarbital and beta-naphthoflavone.
Administration:
-Number of replicates: 2
-Plate per test: 3
Evaluation criteria:
EVALUATION OF RESULTS
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 TA98, TA 100 and TA 102 the number of reversions is at least twice as high and in strains TA 1535,
TA 1537 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.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: Toxicity was not observed up to 5000 µg/plate
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: other: Salmonella typhimurium TA100, TA1535, TA98 and TA1537, and TA102
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion, it can be stated that during the decribed mutagenicity test and under the experimental conditions reported, the test article did not
induce gene mutations by base pair changes or frame shifts in the genome of the strains tested.
Therefore, Ethyltriglycol methacrylate has to be judged as nonmutagenic up to 5000 µg/plate 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 Ethyltriglycol methacrylate ( 98.95 % stabilised with 103 ppm HQME) at concentrations of 31.6, 100, 316, 1000, 2500 and 5000 µg/plate in the presence and absence of mammalian metabolic activation S9 -mix. In two independent experiments Ethyltriglycol methacrylate was investigated for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II).

No precipitation of Ethyltriglycol methacrylate was observed in any tester strain used in experiment I and II (+/- S9 metabolic activation).

 

No toxic effects occurred in the test groups with and without metabolic activation in both independent experiments. The reduction in the number of revertants down to a mutation factor of 0.5 found in experiment I in tester strain TA 1537 at a concentration of 2500 µg/plate (without metabolic activation) was regarded as not biologically relevant due to lack of a dose-reponse relationship.

The plates incubated with the test article showed normal background growth up to 5000 µg/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 Ethyltriglycol methacrylate 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 significance.

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.

 

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

Conclusion:

Under the conditions of this study according to OECD 471, the test substance, Ethyltriglycol methacrylate

(ETMA; CAS: 39670 -09 -2), did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

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