N,N-Dimethyl-9-dodecenamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation study in bacteria

In a Reverse Mutation Assay ‘Ames Test’ using Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA

(OECD TG 471) the test item N,N-dimethyl dodec-9 -enamide did not induce an increase in the frequency of revertant colonies that met the criteria for a positive result, either with or without metabolic activation (S9-mix). Under the conditions of this test N,N-dimethyl dodec-9-enamide was considered to be non-mutagenic.

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:

28 January - 21 February 2022

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 (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

S. typhimurium: histidine reversion system
E. coli: tryptophan reversion system

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:

Type and composition of metabolic activation system:
The metabolic activation system was lyophilized phenobarbital/beta-naphthoflavone induced rat liver S9 and cofactors mix (MutazymeTM) reconstituted with cold, sterile water to provide a 10% phenobarbital/beta-naphthoflavone induced rat liver S9 and cofactors mix. Lot No. 4502 was used in this study. A copy of the S9 Quality Control and Production Certificate is presented in Appendix 2 (attached).
S9-Mix and Agar
The S9-mix was prepared by adding 20 mL of cold, sterile water to each vial.
Final concentrations of S9-mix of approximately 10% (v/v), MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium phosphate buffer (100 mM, pH 7.4) and was maintained on ice for the duration of the test.
A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine (for S. typhimurium strains) or tryptophan (for E.coli strain) supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.

Test concentrations with justification for top dose:

Experiment 1 – Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2 – Pre-Incubation Method: 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150 and 500 µg/plate
The maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 µg/plate as recommended for a soluble and non-toxic substance.
The dose range used for Experiment 2 was determined by the results of Experiment 1

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide (DMSO) at the same concentration in solubility checks performed in house. DMSO was therefore selected as the solvent.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : Experiment 1 – Plate Incorporation Method; Experiment 2 – Pre-Incubation Method

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
- Test substance added in medium (Expt 1 - plate incorporation); Expt 2 - preincubation; in suspension

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: Experiment 2 - 20 mins
- Exposure duration/duration of treatment: Experiment 1 & 2 - All of the plates were incubated at 37 ± 3 °C for between 48 and 72 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition
- Any supplementary information relevant to cytotoxicity: The plates were viewed microscopically for evidence of thinning of the background bacterial lawn (toxicity).

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Plates were scored for the presence of revertant colonies using an automated colony counting system.

OTHER
- Acceptability Criteria
The reverse mutation assay may be considered valid if the following criteria are met:
1. The vehicle control counts should be consistent with the laboratory’s historical control ranges
2. The positive control chemicals should induce increases in mean revertant numbers of ≥2-fold (in strains TA98, TA100 and WP2 uvrA) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control values, confirming discrimination between different strains, and an active S-9 preparation.
3. Contamination or some other confounding effect should not adversely impact the strain or assay functioning or prevent endpoint determination.

Evaluation criteria:

A test article is considered to have provided a mutagenic response if the assay data are valid, and:
1. Treatments with the test article provide a concentration-related increase in revertant numbers at one or more concentrations in at least one strain with or without metabolic activation system
2. An increase in mean revertant colony numbers per plate is observed which is ≥2-fold (in strains TA98, TA100 and WP2 uvrA) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control values
3. Any increase in revertant numbers is reproducible, where applicable.

Statistics:

Statistical significance was not included as part of the result evaluation.

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:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

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:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

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:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

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:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Results for the untreated controls (spontaneous mutation rates) and viability are presented in Table 1 (attached) and were considered to be acceptable.
Two counts for WP2uvrA after the first and second experiments were marginally below the minimum level of the in-house historical untreated/solvent control minima for the tester strain. These counts were considered acceptable as the other solvent and untreated control counts were within the expected range and the tester strain responded very well to the respective positive controls in both the presence and absence of S9 mix. These data are for concurrent untreated control plates dosed in the absence of S9 performed on the same day as the Mutation Test.
The number of revertant counts for the solvent (DMSO) control plates were generally consistent with the laboratory’s historical control ranges. A single count for TA98 (dosed in the presence of S9-mix after the first experiment) was marginally below the minimum level of the in-house historical untreated/solvent control minima for the tester strain. This count was considered acceptable as the other solvent and untreated control counts were within the expected range and the tester strain responded very well to the respective positive controls in both the presence and absence of S9 mix. The positive control chemicals induced increases in mean revertant numbers of >2-fold in strains TA98, TA100, and WP2uvrA, and >3-fold in strains TA1535 and TA1537 the concurrent vehicle control values, confirming discrimination between different strains and an active S9 preparation.
The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, positive and solvent controls, both with and without S9-mix, are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and Table 5 for Experiment 2 (attached). A repeat of a section of the second experiment was required (WP2uvrA dosed in the absence of S9-mix) as the average solvent revertant colony counts were below the in-house historical control minima for the strain.
A history profile of solvent, untreated and positive control values (reference items) is presented in Appendix 1 (attached).
Experiment 1 (plate incorporation) – Table 2 and Table 3
The maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 µg/plate as recommended for a soluble and non-toxic substance.
The test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains in both the presence and absence of S9-mix from 150 µg/plate.
A test item precipitate was noted in both the presence and absence of S9-mix at 5000 g/plate. The precipitate did not prevent the scoring of revertant colonies.
There were no biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix.
Experiment 2 (pre-incubation) – Table 4 and Table 5
The toxic limit of the test item was selected as the maximum dose level in the second experiment.
The test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains in the absence of S9-mix from 150 µg/plate and from 500 µg/plate in the presence of S9-mix.
There was no evidence of a precipitate noted in the second experiment.
There were no biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix.

Executive summary:

 Introduction

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published the OECD TG No. 471 “Bacterial Reverse Mutation Test”, 21 July 1997 as updated in 2020.

Methods

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to nine dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors (S9-mix)). The dose range for Experiment 1 (plate incorporation) was based on the guidance given inOECD TG 471and was selected as 1.5 to 5000 mg/plate. As the overall result of Experiment 1 was Negative, Experiment 2 was performed using the pre-incubation method with fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended slightly, based on the results of Experiment 1 and was 0.05 to 500 µg/plate. Nine test item concentrations were selected in Experiment 2 in order to ensure the study achieved at least four non‑toxic dose levels as required by the test guideline, and were selected based on cytotoxicity noted in Experiment 1, and the potential for a change in the cytotoxicity of the test item following the change in test methodology from plate incorporation to pre-incubation.

Results

The number of revertant counts for the solvent (dimethyl sulphoxide (DMSO)) control plates were consistent with the laboratory’s historical control ranges. The positive control chemicals induced increases in mean revertant numbers of >2-fold in strains TA98, TA100, and WP2uvrA, and >3-fold in strains TA1535 and TA1537 the concurrent vehicle control values, confirming discrimination between different strains and an active S9 preparation.

The dose level of the test item in the first experiment was selected as the OECD TG 471 recommended maximum dose level of 5000 µg/plate. The test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains in both the presence and absence of metabolic activation (S9-mix) from 150 µg/plate in the first mutation test (plate incorporation method). 

Based on the results of Experiment 1, the toxic limit of the test item was employed as the maximum concentration in the second mutation test. The test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains in the absence of S9-mix from 150 µg/plate and from 500 µg/plate in the presence of S9-mix. 

A test item precipitate was noted in both the presence and absence of S9-mix at 5000 mg/plate in Experiment 1. The precipitate did not prevent the scoring of revertant colonies.

There were no biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix employing the plate incorporation method. 

Similarly, no biologically relevant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix employing the pre‑incubation method. 

Conclusion

In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium andEscherichia coli (OECD TG 471) the test item N,N-dimethyl dodec-9-enamide did not induce an increase in the frequency of revertant colonies that met the criteria for a positive result, either with or without metabolic activation (S9-mix). Under the conditions of this test N,N-dimethyl dodec-9-enamide was considered to be non-mutagenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro gene mutation study in bacteria

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published the OECD TG No. 471 “Bacterial Reverse Mutation Test”, 21 July 1997 as updated in 2020.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to nine dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors (S9-mix)). The dose range for Experiment 1 (plate incorporation) was based on the guidance given inOECD TG 471and was selected as 1.5 to 5000 mg/plate. As the overall result of Experiment 1 was Negative, Experiment 2 was performed using the pre-incubation method with fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended slightly, based on the results of Experiment 1 and was 0.05 to 500 µg/plate. Nine test item concentrations were selected in Experiment 2 in order to ensure the study achieved at least four non‑toxic dose levels as required by the test guideline, and were selected based on cytotoxicity noted in Experiment 1, and the potential for a change in the cytotoxicity of the test item following the change in test methodology from plate incorporation to pre-incubation.

The number of revertant counts for the solvent (dimethyl sulphoxide (DMSO)) control plates were consistent with the laboratory’s historical control ranges. The positive control chemicals induced increases in mean revertant numbers of >2-fold in strains TA98, TA100, and WP2uvrA, and >3-fold in strains TA1535 and TA1537 the concurrent vehicle control values, confirming discrimination between different strains and an active S9 preparation.

The dose level of the test item in the first experiment was selected as theOECD TG 471 recommended maximum dose level of 5000 µg/plate. The test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains in both the presence and absence of metabolic activation (S9-mix) from 150 µg/plate in the first mutation test (plate incorporation method). 

Based on the results of Experiment 1, the toxic limit of the test item was employed as the maximum concentration in the second mutation test. The test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains in the absence of S9-mix from 150 µg/plate and from 500 µg/plate in the presence of S9-mix. 

A test item precipitate was noted in both the presence and absence of S9-mix at 5000 mg/plate in Experiment 1. The precipitate did not prevent the scoring of revertant colonies.

There were no biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix employing the plate incorporation method. 

Similarly, no biologically relevant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix employing the pre‑incubation method. 

Under the conditions of this test N,N-dimethyl dodec-9-enamide was considered to be non-mutagenic.

Justification for classification or non-classification

The substance was found to be non-mutagenic in a bacterial reverse mutation assay. Classification is therefore not required in the absence of further information.