2-propen-1-amine, N-2-propen-1-yl-, hydrochloride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The in vitro genotoxicity in bacteria of the test substance 2-propen-1-amine, N-2-propen-1-yl, hydrochloride was determined during a Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli according to the OECD TG 471. The Ames preincubation method was performed both with and without the addition of a rat liver homogenate metabolizing system (S9 -mix). Sensitivity of the assay and the efficacy of the S9-mix were validated by positive, negative and vehicle controls. The dose range was determined during a preliminary toxicity assay and ranged between 1.5 and 5000 μg/plate. As a result of this experiment, 5000 μg/plate was selected as the highest dose to be tested during the main experiment performed according to the preincubation method.

The test item 2-propen-1-amine, N-2-propen-1-yl, hydrochloride 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. Under the conditions of this test 2-propen-1-amine, N-2-propen-1-yl, hydrochloride 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:

2020

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 (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

66% purity
Aqueous solution

Target gene:

Histidine (S. typhimurium) and Tryptophan (E. coli).

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:

Type and composition of metabolic activation system:
- source of S9 : Sprague Dawley Rat Liver
- method of preparation of S9 mix : prepared from rats pre-treated with a mixture known to induce an elevated level of these enzymes
- Volume of S9 mix in the final culture medium: 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer
- quality controls of S9 sterility: A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine or tryptophan 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.
S9 enzymatic activity was validated.

Test concentrations with justification for top dose:

Experiment 1 (using the direct plate incorporation method): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
Experiment 2 (using the pre-incubation method): 15, 50, 150, 500, 1500 and 5000 μg/plate, determined by the results of Experiment 1

Vehicle / solvent:

- Vehicle used: sterile distilled water
- Justification for choice of vehicle: The test item was supplied ‘neat’ at a maximum concentration of 660 mg/mL, therefore it was diluted in sterile distilled water to achieve a maximum recommended concentration of 50 mg/mL (equivalent to the test guideline maximum recommended dose level (5000 μg/plate) when plated out). Sterile distilled water was therefore selected as the vehicle.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-Aminoanthracene (2AA)

Details on test system and experimental conditions:

The test item was supplied ‘neat’ at a maximum concentration of 660 mg/mL, therefore it was diluted in sterile distilled water to achieve a maximum recommended concentration of 50 mg/mL (equivalent to the test guideline maximum recommended dose level (5000 μg/plate) when plated out). Sterile distilled water was therefore selected as the vehicle.
The test item was accurately measured and, on the day of each experiment, approximate half-log dilutions prepared in sterile distilled water. All test item preparation and dosing was performed under yellow safety lighting.
All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Rationale for test conditions:

The maximum concentration was 5000 μg/plate (the OECD TG 471 maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method

Evaluation criteria:

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. Biological relevance against in-house historical control ranges.
4. 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 (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
5. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).

Statistics:

Statistical significance was confirmed by using Dunnett’s Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Key result

Species / strain:

S. typhimurium TA 100

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

Key result

Species / strain:

S. typhimurium TA 1535

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

Key result

Species / strain:

S. typhimurium TA 1537

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

Key result

Species / strain:

S. typhimurium TA 98

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

Key result

Species / strain:

E. coli WP2 uvr A

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

Experiment 1 (plate incorporation) – Table 2 and Table 3 (see attachement in background material)

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. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix). No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix).

There were no significant 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 metabolic activation (S9-mix).

Experiment 2 (pre-incubation) – Table 4 and Table 5 (see attachement in background material)

The maximum dose level of the test item in the second experiment was the same as for Experiment 1 (5000 μg/plate). Toxicity, evaluated as a visible reduction in the growth of the bacterial background lawns and/or a reduction in revertant counts, was observed with test item exposure to all tester strains dosed in the absence of exogenous metabolic activation (S9-mix) from 1500 μg/plate. There was however, no visible reduction in the growth of the bacterial background lawn at any dose level, in the presence of exogenous metabolic activation (S9-mix). No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix). There were no significant 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 metabolic activation (S9-mix).

Conclusions:

The test item is considered to be non-mutagenic under the conditions of the test, for both methods, with and without metabolic activation.

Executive summary:

The in vitro genotoxicity in bacteria of the test substance 2-propen-1-amine, N-2-propen-1-yl, hydrochloride was determined during a Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli according to the OECD TG 471. The Ames preincubation method was performed both with and without the addition of a rat liver homogenate metabolizing system (S9 -mix). Sensitivity of the assay and the efficacy of the S9-mix were validated by positive, negative and vehicle controls. The dose range was determined during a preliminary toxicity assay and ranged between 1.5 and 5000 μg/plate. As a result of this experiment, 5000 μg/plate was

selected as the highest dose to be tested during the main experiment performed according to the preincubation

method

The test item 2-propen-1-amine, N-2-propen-1-yl, hydrochloride 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. Under the conditions of this test 2-propen-1-amine, N-2-propen-1-yl, hydrochloride was considered to be non-mutagenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

The test item 2-propen-1-amine, N-2-propen-1-yl, hydrochloride 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. Under the conditions of this test 2-propen-1-amine, N-2-propen-1-yl, hydrochloride was considered to be non-mutagenic.