Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March - April (2009)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
GLP compliance:
yes (incl. certificate)
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 MATERIALBatch No.of test material: S10664-426Expiration date of the lot/batch: not available Purity test date: 99.3%

Method

Target gene:
Bacterial reverse mutation assays use amino acid requiring strains of Salmonella typhimurium to detect point mutations, which involve substitution, addition or deletion of one or a few DNA base pairs.The Salmonella typhimurium histidine (his) reversion system measures (his-) to (his+) reversion.These assay directly measure heritable DNA mutations of a type which is associated with adverse effects.
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Test concentrations with justification for top dose:
The test item concentrations to be applied in the main experiments were chosen according to the results of a pre-experiment.5.0 μL/plate was selected as the maximum concentration.Two indipendent experiments were performed with the following concentrations:Experiment I:0.0100, 0.0316, 0.100, 0.316,1.0, 2.5 and 5.0 μL/plateExperiment II:0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 μL/plate(TA 98, TA 1535, TA 1537 and TA 102)0.00100, 0.00316, 0.0100, 0.0316, 0.100, 0.316 and 1.0 μL/plate(TA 100)
Vehicle / solvent:
Vehicle/solvent used: DMSO Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Solvent controls, consisting of solvent or vehicle alone as well as untreated controls were treated in the same way as the treatment groups
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
S. typhimurium: TA 100, TA 1535
Untreated negative controls:
yes
Remarks:
Solvent controls, consisting of solvent or vehicle alone as well as untreated controls were treated in the same way as the treatment groups
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Remarks:
S. typhimurium: TA 98, TA 1537
Untreated negative controls:
yes
Remarks:
Solvent controls, consisting of solvent or vehicle alone as well as untreated controls were treated in the same way as the treatment groups
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
S. typhimurium: TA 102
Untreated negative controls:
yes
Remarks:
Solvent controls, consisting of solvent or vehicle alone as well as untreated controls were treated in the same way as the treatment groups
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: Vogel-Bonner Medium E agar plates with 2% glucoseDURATION- Preincubation period: 60 minutes at 37 °C (100 μL of the test item preparation was pre-icubated with the tester strains (100 μL) and sterile buffer or the metabolic activation system (500 μL))- Exposure duration: after solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark - Expression time (cells in growth medium):Evaluation of Cytotoxicity:Cytotoxicity can be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.Evaluation of Mutagenicity:The mutation Factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control (the exact and not the rounded values are used for calculation).According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.
Rationale for test conditions:
According to OECD guidelines
Evaluation criteria:
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessaryEvaluation of Cytotoxicity:Cytotoxicity can be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.Evaluation of Mutagenicity:The mutation Factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control (the exact and not the rounded values are used for calculation).According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Remarks:
(Experiment: I)
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
Key result
Species / strain:
S. typhimurium TA 100
Remarks:
(Experiment: I)
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
Key result
Species / strain:
S. typhimurium TA 1535
Remarks:
(Experiment: I)
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
Key result
Species / strain:
S. typhimurium TA 1537
Remarks:
(Experiment: I)
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
Key result
Species / strain:
S. typhimurium TA 102
Remarks:
(Experiment: I)
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
Key result
Species / strain:
S. typhimurium TA 98
Remarks:
(Experiment: II)
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
Key result
Species / strain:
S. typhimurium TA 100
Remarks:
(Experiment: II)
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
Key result
Species / strain:
S. typhimurium TA 1535
Remarks:
(Experiment: II)
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
Key result
Species / strain:
S. typhimurium TA 1537
Remarks:
(Experiment: II)
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
Key result
Species / strain:
S. typhimurium TA 102
Remarks:
(Experiment: II)
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

Applicant's summary and conclusion

Conclusions:
In conclusion, it can be started that during the described mutagenicity test and under the experimental conditions reported, 1-chloro-3-phenylpropane did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.Therefore, 1-chloro-3-phenylpropane is considered to be non-mutagenic in this bacterial reverse mutation assay.