Schiff bases, benzylidene, C16-18 (even numbered) and C18-unsaturated alkyl

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2nd February 2016 -5th May 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine requirement in Salmonela typhimurium Stains
Tryptophan requirement in Eschericha coli strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Test concentrations with justification for top dose:

Dose levels tested were 2.50, 7.50, 25.0, 75.0, 250 and 750 μg per plate with S9 activation and 0.250, 0.750, 2.50, 7.50, 25.0 and 75.0 μg per plate without S9 activation.

Justification: - The above doses were used based on the results obtained for the prelimary studies. 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.P recipitate was observed beginning at 333 or 667 μg per plate. Toxicity was observed beginning at 33.3 to 667 μg per plate.

Vehicle / solvent:

Ethanol

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Details on test system and experimental conditions:

Overnight cultures were prepared by inoculating from the appropriate frozen permanent stock into a vessel, containing 30 to 50 mL of culture medium. To assure that cultures were harvested in late log phase, the length of incubation was controlled and monitored. Following inoculation, each flask was placed in a shaker/incubator programmed to begin shaking at 125 to 175 rpm and incubating at 37±2°C for approximately 12 hours before the anticipated time of harvest. Each culture was monitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titer of greater than or equal to 0.3x109 cells per milliliter. The actual titers were determined by viable count assays on nutrient agar plates. Each plate was identified by the BioReliance study number and a code system to designate the treatment condition, dose level and test phase, as described in detail in BioReliance's Standard Operating Procedures. Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats that were injected intraperitoneally with Aroclor™ 1254 (200 mg/mL in corn oil) at a dose of 500 mg/kg, five days before sacrifice. The S9 was stored at -60°C or colder until used. Each bulk preparation of S9 was assayed for its ability to metabolize benzo(a)pyrene and 2-aminoanthracene to forms mutagenic to Salmonella typhimurium TA100.

Evaluation criteria:

All Salmonella tester strain cultures must demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene. Cultures of tester strains TA98 and TA100 must demonstrate the presence of the pKM101 plasmid R-factor. All WP2 uvrA cultures must demonstrate the deletion in the uvrA gene.
All cultures must demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls as follows (inclusive): TA98, 10 - 50; TA100, 80 - 240; TA1535, 5 - 45; TA1537, 3 - 21; WP2 uvrA, 10 - 60.

To ensure that appropriate numbers of bacteria are plated, tester strain culture titers must be greater than or equal to 0.3x109 cells/mL.
The mean of each positive control must exhibit at least a 3.0-fold increase in the number of revertants over the mean value of the respective vehicle control.
A minimum of three non-toxic dose levels is required to evaluate assay data. A dose level is considered toxic if one or both of the following criteria are met: A >50 % reduction in the mean number of revertants per plate as compared to the mean vehicle control value. This reduction must be accompanied by an abrupt dose-dependent drop in the revertant count. At least a moderate reduction in the background lawn.

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:

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

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:

not applicable

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:

not applicable

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:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

2.0-fold, non-dose responsive increase was observed

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, the test substance was concluded equivocal in the presence of Aroclor-induced rat liver S9 with tester strain TA98.

Executive summary:

No contaminant colonies were observed on the sterility plates for the vehicle control, the test article dilutions or the S9 and Sham mixes.

Preliminary Toxicity Assay

The results of the preliminary toxicity assay conducted at dose levels of 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 μg per plate in ethanol. The maximum dose of 5000 μg per plate was achieved using a concentration of 100 mg/mL and a 50.0 μL plating aliquot. Precipitate was observed beginning at 333 or 667 μg per plate. Toxicity was observed beginning at 33.3 to 667 μg per plate.

Mutagenicity Assay

Based upon the results of the preliminary toxicity assay, the dose levels selected for the mutagenicity assay were 2.50, 7.50, 25.0, 75.0, 250 and 750 μg per plate with S9 activation and 0.250, 0.750, 2.50, 7.50, 25.0 and 75.0 μg per plate without S9 activation.

Precipitate was observed at 750 μg per plate in the presence of S9 activation. Toxicity was observed beginning at 25.0 to 750 μg per plate. A 1.5-fold, non-dose responsive increase was observed with tester strain TA98 in the presence of S9 activation. Two replicates at the highest nontoxic dose levels were outside the 95% vehicle control limits and one value and the mean were within 95% vehicle control limits.

Based upon these results, the dose levels selected for the retest of mutagenicity assay were 7.50, 25.0, 75.0, 150, 250, 300, 500 and 750 μg per plate for tester strain TA98 with S9 activation. Precipitate was observed beginning at 300 μg per plate. Toxicity was observed beginning at 500 μg per plate. A 2.0-fold, non-dose responsive increase was observed. Two replicates at the highest nontoxic dose levels were outside the 95% vehicle control limits without a clear dose response. Due to lack of a dose response the conclusion was equivocal.

Based on the study report, the results indicate the test substance is non mutagenic. A negative result was observed in 4 strains of bacteria but an equivocal result was seen in one strain.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Justification for classification or non-classification

Based on the study report, the results indicate the test substance is non mutagenic - Negative result was observed in 4 strains of bacteria but equivocal result was seen in one strain.