3-sec-[C15-18-(branched and linear)-alk-2-enyl]pyrrolidine-2,5-dione

Genetic toxicity: in vitro

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:

29 November 2018 to 08 March 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-study according to OECD Test Guideline 471

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Remarks:

Refer to main study report

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3-sec-[C15-18-(branched and linear)-alk-2-enyl]pyrrolidine-2,5-dione

EC Number:

701-350-3

Molecular formula:

Not possible to assign, UVCB

IUPAC Name:

3-sec-[C15-18-(branched and linear)-alk-2-enyl]pyrrolidine-2,5-dione

Test material form:

liquid

Details on test material:

- Appearance: brown liquid
- Storage: room temperature, in the dark

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL- Lot/batch No.of test material: R17 2934STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL- Storage condition of test material: Room temperature, protected from light- Solubility of the test substance in the solvent/vehicle: The test article formed a clear solution in DMSO at a concentration of approximately 500 mg/mL in the solubility test conducted at BioReliance.TREATMENT OF TEST MATERIAL PRIOR TO TESTING- Treatment of test material prior to testing: To achieve a solution, the most concentrated dilution was vortexed for 2 minutes in the confirmatory mutagenicity assay.

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:

In the initial toxicity-mutation assay, the dose levels tested were 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate. The top dose was based on the guideline. Based upon initial toxicity-mutation assay results, the maximum dose tested in the confirmatory mutagenicity assay was 5000 μg per plate. In the confirmatory mutagenicity assay, the dose levels tested were 33.3, 100, 333, 1000, 3333 and 5000 μg per plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:DMSO for the test article and all positive controls were diluted in dimethyl sulfoxide (DMSO) except for sodium azide, which was diluted in sterile water - Justification for choice of solvent/vehicle: DMSO was the vehicle of choice based on the solubility of the test article and compatibility with the target cells

Controls

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 hoursNUMBER OF REPLICATIONS: 2 in the initial-toxicity mutation assay; 3 in the confirmatory mutagenicity assayNUMBER OF CELLS EVALUATED: >/= 0.3 x 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 each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.For the test article to be evaluated positive, 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 TA1537Data 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 and above the corresponding acceptable vehicle control range.Strains TA98, TA100 and WP2 uvrAData 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 and above the corresponding acceptable vehicle control range.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

Key result

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:

No toxicity in the initial toxicity mutation assay. Toxicity as reduction in revertant count was observed at 5000 μg per plate with tester strain TA1537 in the absence of S9 activation in the confirmatory mutagenicity assay

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:

All criteria for a valid study were met as described in the protocol. The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, X-16151 did not cause a positive mutagenic response with any of the tester strains in either the presence or absence of Aroclor-induced rat liver S9.

Executive summary:

The test article, X-16151, was tested to evaluate its mutagenic potential by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system. Dimethyl sulfoxide (DMSO) was used as the vehicle.

In the initial toxicity-mutation assay, the dose levels tested were 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate. No toxicity was observed. Precipitate was observed beginning at 1500 μg per plate with all conditions. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Based upon these results, the maximum dose tested in the confirmatory mutagenicity assay was 5000 μg per plate.

In the confirmatory mutagenicity assay, the dose levels tested were 33.3,100, 333, 1000, 3333 and 5000 μg per plate. Precipitate was observed beginning at 3333 μg per plate with all conditions. Toxicity as reduction in revertant count was observed at 5000 μg per plate with tester strain TA1537 in the absence of S9 activation. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

These results indicate X-16151 was negative for the ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system.