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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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
publication
Title:
Non-mutagenicity of 27 aliphatic acrylate esters in the Salmonella-microsome test
Author:
T.H.J.M. Waegemaekers, and M.P.M Bensink
Year:
1984
Bibliographic source:
Mutation Research Vol. 137:95-102

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
- Principle of test: The mutagenicity of n-hexyl acrylate was tested in the Salmonella-microsome assay according to the method of Ames et al. (1975).
- Short description of test conditions: N-hexyl acrylate was tested in triplicate in S. typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100 with and without microbial activation (phenobarbital and aroclor 1254 induced rat liver S9 fractions). N-hexyl acrylate was tested up to cytotoxic concentrations.
- Parameters analysed / observed: Revertant colonies were counted manually after incubation in the dark at 37C for 48-72 hours.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Polyscience Inc.
- Expiration date of the lot/batch: not specified
- Purity test date:not specified

Method

Target gene:
Each S. typhimurium tester strain contains, in addition to a mutation in the histidine operon, additional mutations that enhance sensitivity to some mutagens. The rfa mutation results in a cell wall deficiency that increases the permeability of the cell to certain classes of chemicals such as those containing large ring systems that would otherwise be excluded. The deletion in the uvrB gene results in a deficient DNA excision-repair system. Tester strains TA98, and TA100 also contain the pKM101 plasmid (carrying the R-factor). It has been suggested that the plasmid increases sensitivity to mutagens by modifying an existing bacterial DNA repair polymerase complex involved with the mismatch-repair process.

TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. TA100 is reverted by both frameshift and base substitution mutagens and TA1535 are reverted only by mutagens that cause base substitutions.
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535
Species / strain / cell type:
S. typhimurium TA 1537
Species / strain / cell type:
S. typhimurium TA 1538
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital and Aroclor 1254 induced S9 mix
Test concentrations with justification for top dose:
Main assay: 25, 100, 400 and 1600 ug/plate
The top dose was chosen based on the presence of cytotoxicity as indicated by a cleared background lawn
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: not specified
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
historical average of spontaneous revertants for TA1535
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
other: 2-Aminoanthracene
Untreated negative controls:
yes
Remarks:
historical average of spontaneous revertants for TA 1537
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
other: 2-aminoanthracene Strain TA1537 Phenobarbital induced S9
Untreated negative controls:
yes
Remarks:
historical average of spontaneous revertants for TA 1538
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 4-Nitro-o-phenylenediamine Strain TA1538 without S9; 2 Aminoathracene Strain TA1538 Phenobarbital induced S9
Untreated negative controls:
yes
Remarks:
historical average of spontaneous revertants for TA98
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 4-Nitro-o-phenylenediamine Strain TA98 without S9; 2 Aminoanthracene Strain TA98 Phenobarbital induced S9
Untreated negative controls:
yes
Remarks:
historical average of spontaneous revertants for TA100
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
other: 2-Aminoanthracene Strain TA100 Phenobarbital induced S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): 0.5 - 1.0 x 10^9 bacteria/mL

DURATION
- Exposure duration: 48 - 72 hours

NUMBER OF REPLICATIONS: 3 replicate plates per dose

DETERMINATION OF CYTOTOXICITY
- Method: cleared background lawn
Rationale for test conditions:
The tester strains were exposed to the test article via the plate incorporation methodology originally described by Ames et al (1975). This methodology has been shown to detect a wide range of classes of chemical mutagens.
Evaluation criteria:
For tester strains TA98, TA1535, and TA1537, and TA100, an individual dose was considered positive if the mean revertant colony count on the test plates was equal to or greater than two times the mean number of spontaneous revertants on the vehicle control plates. A positive result for the assay was defined as a dose-related increase in the mean number of revertant colonies over at least three concentrations of test substance, including at least one positive dose.
Statistics:
The mean revertant colony count and standard deviation were determined for each dose point.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
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
Key result
Species / strain:
S. typhimurium TA 1537
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
Key result
Species / strain:
S. typhimurium TA 98
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
Key result
Species / strain:
S. typhimurium TA 1538
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
Key result
Species / strain:
S. typhimurium TA 100
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

Applicant's summary and conclusion

Conclusions:
N-hexyl acrylate was not mutagenic in five strains of Salmonella typhimurium bacteria in the presence or absence of metabolic activation.
Executive summary:

The substance was tested for its mutagenic potential based on the ability to induce back mutations in selected loci in Salmonella typhimurium strains TA 1535, TA 100, TA 1537, TA 98, and TA1538 at concentrations ranging from 25 to 1600 µg/plate according to the methods described in Ames et al. (1975) (equivalent or similar to OECD 471). The assay was performed using the Standard plate test with and without metabolic activation (phenobarbital/aroclor 1254 induced rat liver microsomes), respectively. The test material caused visible reduction in the growth of the bacterial background lawn at at a concentration of 1600 µg/plate and was used as the top dose level in the main assay. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.