2-octyldodecyl 5-oxo-L-prolinate

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:

From September 01, 2016 to October 06, 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

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Batch no.: CH 192955/01
Purity: 99.98%
Appearance: clear yellow liquid

Target gene:

Histidine and tryptophan

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

Metabolic activation system:

S9-mix (rat liver S9-mix induced Aroclor 1254)

Test concentrations with justification for top dose:

1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate were tested in triplicate (based on range finding dose study and cytotoxicity).
The highest concentration of the test susbtance used in the mutation assays was 5000 μg/plate or the level at which the test substance inhibited bacterial growth.

Vehicle / solvent:

Ethanol for the test substance (and DMSO or saline for positive controls)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

ethanol, DMSO or saline

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR191 (without metabolic activation) and 2-aminoanthracene (with metabolic activation)

Details on test system and experimental conditions:

- Following dose range findings studies, the test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay both in the absence and presence of S9-mix. An additional pre-incubation test was performed with the tester strains TA1535, TA1537 and TA98 in the absence of S9-mix.
- Exposure: 48 ± 4h (+ a pre-incubation of 30 min if needed)

Rationale for test conditions:

- Based on the most recent OECD and EC guidelines
- Dose range finding studies
- First mutation experiments

Evaluation criteria:

In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
- A test substance was considered negative (not mutagenic) in the test if: a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent control. b) The negative response should be reproducible in at least one follow up experiment.
- A test substance was considered positive (mutagenic) in the test if: a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three times the concurrent control. b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Statistics:

No formal hypothesis testing was done.
- Revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.

Key result

Species / strain:

other: Salmonella typhimurium TA1535, TA1537 and TA98

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:

other: Salmonella typhimurium TA1535, TA1537, TA98 and TA100 and Escherichia coli WP2uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not valid

Remarks:

for S. typhimurium TA1535 in the absence of S-9-mix

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

other: Salmonella typhimurium TA1535, TA1537, TA98 and TA100 and Escherichia coli WP2uvrA

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- In the dose range finding study, the test substance was initially tested up to concentrations of 5000 µg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The test substance precipitated on the plates at dose levels of 1600 and 5000 μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.
- In the first mutation experiment, the test substance was tested up to concentrations of 1600 µg/plate in the strains TA1535, TA1537 and TA98. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.
- In the second mutation experiment, the test substance was tested up to concentrations of 1600 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the preincubation assay. The test substance precipitated on the plates at dose levels of 512 and 1600 μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. In the second mutation test, in tester strains TA1535, TA1537 and TA98 in the absence of S9-mix outliers in the number of revertants were observed in the solvent control (TA1535) or in the lowest concentration tested (TA1537 and TA98), therefore an additional experiment was performed.
- In this third mutation experiment, the test substance was tested up to concentrations of 1600 µg/plate in the absence of S9-mix in the pre-incubation assay. The test substance precipitated on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.
- The negative control values were within the laboratory historical control data ranges, except the response for TA1535 in the absence of S9-mix, second experiment. However since the elevated number of revertants were caused by one outlier and this part of the mutation assay was repeated, the validity of the study was considered to be not affected. In this study, acceptable responses were obtained for the negative and strain-specific positive control substances indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
- The test substance did not induce a biologically relevant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation in any of the experiments. Based on the results of this study it was concluded that the test substance was not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Remarks on result:

other: first experiment

Conclusions:

Under the study conditions, the test substance was not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

A study was conducted to determine the in vitro genetic toxicity according to OECD Guideline 471 and EU Method B.13/14 (mutagenicity - reverse mutation test using bacteria), in compliance with GLP. Dose range finding tests as well as direct plate and pre-incubation assays both in the absence and presence of S9-mix were performed. Salmonella typhimurium strains TA1535, TA1537, TA100 and TA98 and Escherichia coli strain WP2uvrA were exposed to the test substance at concentration levels of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate, to negative or positive control substances for 48 ± 4h (plus a pre-incubation of 30 min if needed). In the dose range finding study, the test substance was initially tested up to concentrations of 5000 µg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The test substance precipitated on the plates at dose levels of 1600 and 5000 μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. In the first mutation experiment, the test substance was tested up to concentrations of 1600 µg/plate in the strains TA1535, TA1537 and TA98. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. In the second mutation experiment, the test substance was tested up to concentrations of 1600 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the preincubation assay. The test substance precipitated on the plates at dose levels of 512 and  1600 μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. In the second mutation test, in tester strains TA1535, TA1537 and TA98 in the absence of S9-mix outliers in the number of revertants were observed in the solvent control (TA1535) or in the lowest concentration tested (TA1537 and TA98), therefore an additional experiment was performed. In this third mutation experiment, the test substance was tested up to concentrations of 1600 µg/plate in the absence of S9-mix in the pre-incubation assay. The test substance precipitated on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. The negative control values were within the laboratory historical control data ranges, except the response for TA1535 in the absence of S9-mix, second experiment. However since the elevated number of revertants were caused by one outlier and this part of the mutation assay was repeated, the validity of the study was considered to be not affected. In this study, acceptable responses were obtained for the negative and strain-specific positive control substances indicating that the test conditions were adequate and that the metabolic activation system functioned properly. The test substance did not induce a biologically relevant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation in any of the experiments. Under the study conditions, the test substance was not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay (Verspeek-Rip, 2016).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Genetic toxicity, in vitro:

A study was conducted to determine the in vitro genetic toxicity according to OECD Guideline 471 and EU Method B.13/14 (mutagenicity - reverse mutation test using bacteria), in compliance with GLP. Dose range finding tests as well as direct plate and pre-incubation assays both in the absence and presence of S9-mix were performed. Salmonella typhimurium strains TA1535, TA1537, TA100 and TA98 and Escherichia coli strain WP2uvrA were exposed to the test substance at concentration levels of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate, to negative or positive control substances for 48 ± 4h (plus a pre-incubation of 30 min if needed). In the dose range finding study, the test substance was initially tested up to concentrations of 5000 µg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The test substance precipitated on the plates at dose levels of 1600 and 5000 μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. In the first mutation experiment, the test substance was tested up to concentrations of 1600 µg/plate in the strains TA1535, TA1537 and TA98. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. In the second mutation experiment, the test substance was tested up to concentrations of 1600 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the preincubation assay. The test substance precipitated on the plates at dose levels of 512 and  1600 μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. In the second mutation test, in tester strains TA1535, TA1537 and TA98 in the absence of S9-mix outliers in the number of revertants were observed in the solvent control (TA1535) or in the lowest concentration tested (TA1537 and TA98), therefore an additional experiment was performed. In this third mutation experiment, the test substance was tested up to concentrations of 1600 µg/plate in the absence of S9-mix in the pre-incubation assay. The test substance precipitated on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. The negative control values were within the laboratory historical control data ranges, except the response for TA1535 in the absence of S9-mix, second experiment. However since the elevated number of revertants were caused by one outlier and this part of the mutation assay was repeated, the validity of the study was considered to be not affected. In this study, acceptable responses were obtained for the negative and strain-specific positive control substances indicating that the test conditions were adequate and that the metabolic activation system functioned properly. The test substance did not induce a biologically relevant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation in any of the experiments. Under the study conditions, the test substance was not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay (Verspeek-Rip, 2016).