[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Under the conditions of this test N,N,N-trimethyl-3-{[(2E)-3-phenylprop-2- enoyl]amino}propan-1-aminium chloride 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:

27 January 2021 to 18 February 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Appearance/Physical state: Solid (deliquescent)
Batch: 209-653 (dried material from [Tradename] batch number 0001569403)
Purity: 94.5%
Expiry date: 03 December 2021
Storage conditions: Room temperature under nitrogen in the dark
Formulated concentrations were adjusted to allow for the stated impurity content (5.5%) of the test item.

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:

The Phenobarbitone / β-Naphthoflavone induced S9 Microsomal fractions (Sprague-Dawley) used in this study were purchased from Moltox; Lot No.’s 4272 (Experiment 1) and 4217 (Experiment 2) and the protein level was adjusted to 20 mg/mL. Copies of the S9 Quality Control and Production Certificates are presented in Appendix 3 of the attached report.

The S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test.
S9 fraction 5.0 mL; 1.65 M KCl/0.4 M MgCl2 1.0 mL; 0.1 M Glucose-6-phosphate 2.5 mL; 0.1 M NADP 2.0 mL; 0.2 M Sodium phosphate buffer (pH 7.4) 25.0 mL; Sterile distilled water 14.5 mL

Test concentrations with justification for top dose:

- Experiment 1: Plate Incorporation Method
1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate (the OECD TG 471 maximum recommended dose level)



– Experiment 2: Pre-Incubation Method
15, 50, 150, 500, 1500 and 5000 μg/plate (determined by the results of Experiment)

Six 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 the lack of 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.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:
DMSO

- Justification for choice of solvent/vehicle:
Test substance not soluble in water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-Aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: twice

METHOD OF TREATMENT/ EXPOSURE:
Experiment 1 - Plate Incorporation Method; Experiment 2 – Pre-Incubation Method

All of the plates were incubated at 37 ± 3 °C for between 48 and 72 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning of the background bacterial lawn (toxicity).

Evaluation criteria:

Any, one, or all of the following were used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537

Statistics:

It was not required

Key result

Species / strain:

other: 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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Experiment 2 (pre-incubation)
visible reduction in the growth of the bacterial background lawns, was observed with test item exposure to all tester strains in the absence of S9-mix at 5000 μg/plate. However, there was no visible reduction in the growth of the bacterial background lawn noted at any dose level in the presence of S9-mix.
No test item precipitate was observed on the plates at any of the doses tested either in the presence or in the absence of S9-mix.
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.

Conclusions:

Under the conditions of this test N,N,N-trimethyl-3-{[(2E)-3-phenylprop-2- enoyl]amino}propan-1-aminium chloride was considered to be non-mutagenic.

Executive summary:

Mutagenicity of the test substance was evaluated in Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA with Bacterial Reverse Mutation Test (OECD 471). 

 

Bacterial strains were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 (plate incorporation) was based on the guidance given in OECD TG 471 and was selected as 1.5 to 5000 μg/plate. As the overall result of the first experiment 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 modified following the results of Experiment 1 and was selected as 15 to 5000 μg/plate.

 

Under the conditions of this test N,N,N-trimethyl-3-{[(2E)-3-phenylprop-2- enoyl]amino}propan-1-aminium chloride was considered to be non-mutagenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

In an Ames test conducted according to OECD 471 Test Guideline, the test substance showed no mutagenic acitivity.