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Toxicological information

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:
27 Oct 2015 to 23 March 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Refer to the main study report
GLP compliance:
yes
Remarks:
Refer to the main study report
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Pale amber waxy solid (per Certificate of Analysis)
Details on test material:
- Name of test material (as cited in study report): EXP1505385
- Molecular weight (if other than submission substance): 368.6 g/mol
- Physical state: Pale amber waxy solid (per Certificate of Analysis)
- Analytical purity: 100%
- Composition of test material, percentage of components: >=90-<=100% Alkyl ester carboxylic acid
- Purity test date: 08 Oct 2015
- Lot/batch No.: E00974-38D&F(Batch/Lot/Notebook Ref No) E00350-288 (Label Lot)
- Expiration date of the lot/batch: 22 Jun 2018
- Storage condition of test material: Room temperature, protected from light

Method

Target gene:
The Salmonella strains contain mutations in the histidine operon, thereby imposing a requirement for histidine in the growth medium. These strains contain the deep rough (rfa) mutation, which deletes the polysaccharide side chain from the lipopolysaccharides of the bacterial cell surface. This increases cell permeability of larger substances. The other mutation is a deletion of the uvrB gene, which codes for a protein of the DNA nucleotide excision repair system, resulting in an increased sensitivity in detecting many mutagens. This deletion also includes the nitrate reductase (chi) and biotin (bio) genes (bacteria require biotin for growth). Tester strains TA98 and TA100 contain the R-factor plasmid, pKM101. These strains are reverted by a number of mutagens that are detected weakly or not at all with the non-R-factor parent strains. pKM101 increases chemical and spontaneous mutagenesis by enhancing an error-prone DNA repair system, which is normally present in these organisms. The tester strain Escherichia coli WP2 uvrA carries the defect in one of the genes for tryptophan biosynthesis. Tryptophan-independent mutants (revertants) can arise either by a base change at the site of the original alteration or by a base change elsewhere in the chromosome so that the original defect is suppressed. This second possibility can occur in several different ways so that the system seems capable of detecting all types of mutagens, which substitute one base for another. Additionally, the strain is deficient in the DNA nucleotide excision repair system.
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
Preliminary toxicity assay: 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 µg per plate for all test conditions
Mutagenicity assay: 33.3, 100, 333, 1000, 3333 and 5000 µg per plate (for TA98, TA1537 and WP2A uvrA) and 1.00, 3.33, 10.0, 33.3, 100, 333 and 1000 µg per plate (for TA100 and TA1535)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO (test substance, 2 aminoanthracene, 2-nitrofluorene, 9-aminoacridine, methyl methanesulfonate); water (sodium azide)
- Justification for choice of solvent/vehicle: DMSO was the vehicle of choice based on the solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in DMSO at a concentration of approximately 500 mg/mL with sonication at 35.8ºC for 10 minutes in the solubility test conducted at BioReliance.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 to 72 hours

NUMBER OF REPLICATIONS: 1 in the preliminary toxicity assay; 3 in the mutagenicity assay

NUMBER OF CELLS EVALUATED: >/= 0.3 * 10^8 cells/plate

DETERMINATION OF CYTOTOXICITY
- Method: other: Counting of revertant colony numbers and evaluation of the condition of the bacterial background lawn.
Evaluation criteria:
The revertant colony numbers were determined for each plate (counted either manually or by automatic colony counter). The mean and standard deviation of the number of revertants per plate were calculated and reported.

For the test article to be evaluated positive (mutagenic), it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article as specified below:
Strains TA1535 and TA1537
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean vehicle control value.
Strains TA98, TA100 and WP2 uvrA
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean vehicle control value.

An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response was evaluated as negative if it was neither positive nor equivocal.
Statistics:
According to the test guidelines, the biological relevance of the results is the criterion for the interpretation of the results, and a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Toxicity, either background or as a reduction in revertant count, was observed beginning at 100 or at 1000 µg per plate with tester strains TA100 and TA1535 in the presence and absence of S9 activation
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the conditions of this study, The Substance did not induce reverse mutations at selected loci of several tester strains in either the presence or absence of Aroclor induced rat liver S9. A confirmatory (independent repeat) assay was not required as the study was concluded to be unequivocally negative
Executive summary:

The test Substance was tested to evaluate its mutagenic potential by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonellatyphimuriumand at the tryptophan locus ofEscherichia colistrain WP2 uvrA in the presence and absence of an exogenous metabolic activation system. Dimethyl Sulfoxide (DMSO) was used as the vehicle. All criteria for a valid study were met as described in the protocol.

In the preliminary toxicity assay, the dose levels tested were 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 µg per plate. Precipitate of the test material was observed from3333-5000 µg per plate. Toxicity, either background or as a reduction in revertant count, was observed beginning at 33.3, 66.7, 100 or 333 µg per plate with tester strains TA100 and TA1535 in the presence and absence of S9 activation. No toxicity was observed in other tester strains treated. Based upon these results, the maximum dose tested in the mutagenicity assay was 5000 µg per plate (for TA98, TA1537 and WP2AuvrA) and 1000 µg per plate (for TA100 and TA1535).

In the mutagenicity assay, the dose levels tested were 33.3, 100, 333, 1000, 3333 and 5000 µg per plate (for TA98, TA1537 and WP2AuvrA) and 1.00, 3.33, 10.0, 33.3, 100, 333 and 1000 µg per plate (for TA100 and TA1535). Precipitate was observed from 3333-5000 µg per plate. Toxicity, either background or as a reduction in revertant count, was observed beginning at 100 or at 1000 µg per plate with test strains TA100 and TA1535 in the presence and absence of S9 activation. No toxicity was observed in other tester strains treated. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

All criteria for a valid study were met as described in the protocol. Under the conditions of this study, test Substance was negative for the ability to induce reverse mutations at selected loci of several strains ofSalmonellatyphimurium and at the tryptophan locus of Escherichia colistrain WP2 uvrA in the presence and absence of an exogenous metabolic activation system.