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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From May 20 to June 25, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: colourless liquid
Details on test material:
- Name of test material (as cited in study report): Propylidynetrimethyl trimethacrylate
- Physical state: colourless liquid
- Storage condition of test material: in a sealed container, at room temperature in the dark

Method

Target gene:
Not applicable
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 102
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver post-mitochondrial fraction (S-9), obtained from Molecular Toxicology Incorporated, USA
Test concentrations with justification for top dose:
- Range-finder experiment and experiment 1: 1.6, 8, 40, 200, 1000 and 5000 µg/plate (with and without S-9)
- Experiment 2: 156.3, 312.5, 625, 1250, 2500 and 5000 µg/plate (with and without S-9)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: Solubility of propylidynetrimethyl trimethacrylate in DMSO = 100 mg/mL
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: See table 1
Details on test system and experimental conditions:
METHOD OF APPLICATION: Plate incorporation method; as the results of Experiment 1 were negative, treatments in the presence of S-9 in experiment 2 included a pre-incubation step (incubation for 1 hour at 37±1°C).

DURATION
For all assays, bacteria were cultured at 37±1°C for 10 hours in nutrient broth, containing ampicillin (TA98, TA100) or ampicillin and tetracycline (TA102) as appropriate. Incubation was carried out with shaking in an anhydric incubator. All treatments were completed within 6 hours of the end of the incubation period.
After plating with test substance or control, the plates were inverted and incubated at 37±1°C in the dark for 3 days.

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS:
Range-finding test: triplicate plates; negative (vehicle) and positive controls were included in quintuplicate and triplicate, respectively.
Main experiments: triplicate plates; negative (vehicle) controls were included in quintuplicate, and positive controls were included in triplicate.

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of revertants; thinning of background bacterial lawn.
Evaluation criteria:
For valid data, the test article was considered to be mutagenic if:
- Dunnett's test gave a significant response (p=< 0.01) which was concentration related
- The positive trend/effects described above were reproducible.
Negative: If all of the above criteria were not met
Statistics:
Dunnett's test was used to compare the counts at each concentration with the control. The presence or otherwise of a concentration response was checked by non-statistical analysis, up to limiting levels.

Results and discussion

Test resultsopen allclose all
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:
cytotoxicity
Remarks:
Marked reduction in revertant numbers was observed at 2500 µg/plate and above in strains TA1535 and TA1537 in the presence of S-9 and at 5000 µg/plate in strain TA1537 in the absence of S-9 in experiment 2
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
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:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Precipitation of the test article was observed on all test plates treated at 5000 µg/plate in the Range-Finder Experiment and Experiment 1 and at 2500 µg/plate and above in Experiment 2.
In the range-finder experiment and in experiment 1, no evidence of toxicity was observed as would normally manifest as a diminution of the background bacterial lawn or a marked reduction in revertant numbers.
In the second experiment, evidence of toxicity in the form of a marked reduction in revertant numbers was observed at 2500 µg/plate and above in strains TA1535 and TA1537 in the presence of S-9 and at 5000 µg/plate in strain TA1537 in the absence of S-9.

Applicant's summary and conclusion

Conclusions:
Under the test conditions, propylidynetrimethyl trimethacrylate is not considered as mutagenic in this bacterial system according to the criteria of the Annex VI to the Directive 67/548/EEC and CLP Regulation(EC) N° (1272-2008). 
Executive summary:

In a GLP study performed according to OECD guideline 471, propylidynetrimethyl trimethacrylate was tested for mutagenicity using Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 with the plate incorporation and preincubation methods in the presence and absence of metabolic activation system (S-9 mix). Due to absence of toxicity in the range-finder test, using TA100 tested at concentrations of 1.6, 8, 40, 200, 1000 and 5000 µg/plate with and without S-9, all strains in experiment 1 were tested with and without S-9 at these concentrations. In order to examine more closely those concentrations of propylidynetrimethyl trimethacrylate approaching the maximum test concentration, the following concentrations were tested in the second experiment (using the preincubation method (1h at 37°C) when S-9 was used): 156.3, 312.5, 625, 1250, 2500 and 5000 µg/plate, with and without S-9. Precipitation of the test article was observed on all test plates treated at 5000 µg/plate in the Range-Finder Experiment and Experiment 1 and at 2500 µg/plate and above in Experiment 2. In the range-finder experiment and in experiment 1, no evidence of toxicity was observed as would normally manifest as a diminution of the background bacterial lawn or a marked reduction in revertant numbers. In the second experiment, evidence of toxicity in the form of a marked reduction in revertant numbers was observed at 2500 µg/plate and above in strains TA1535 and TA1537 in the presence of S-9 and at 5000 µg/plate in strain TA1537 in the absence of S-9. The positive controls induced the appropriate responses in the corresponding strains. Propylidynetrimethyl trimethacrylate showed no substantial increases in revertant colony numbers over control count obtained with any of the tester strains at any concentrations in either presence or absence of S-9. Under the test conditions, propylidynetrimethyl trimethacrylate is not considered as mutagenic in this bacterial system according to the criteria of the Annex VI to the Directive 67/548/EEC and CLP Regulation(EC) N° (1272-2008).