N,2-dimethyl-N-phenylbutyramide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a bacterial reverse mutation assay, conducted to OECD guidelines (TG 417) and to GLP, Gardamide was not mutagenic, with or without metabolic activation.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Identification: Gardamide
Chemical Name: N-Methyl-N-Phenyl-2-Methylbutyramide
Purity: 99.7%
CAS No.: 84434-18-4
Stability: Stable under normal temperatures and pressure

Target gene:

Histidine [S. typhimurium] and tryptophan [E. coli]

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix, prepared from male Sprague-Dawley rats that had been injected with phenobarbitone/β-naphthoflavone

Test concentrations with justification for top dose:

Test material dose rang in range finding and main test: 50, 150, 500, 1500, 5000 μg/plate
Confirmatory test dose range: 2000, 3000, 4000 and 5000 μg/plate.

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Remarks:

2-Aminoacridine and Benzo(a)pyrene

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Details on test system and experimental conditions:

Salmonella typhimurium strains TA 1525, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA' were treated with test material using Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S( in standard cofactors). The dose range for the range finding test was determined in a preliminary toxicity assay and was 50 to 5000 μg/plate. The experiment was repeated on a separate day using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test meaterial formulations.
A third experiment was performed to confirm whether a mutagenic response noted in the main test was real or spurious. The experiment was carried out using bacterial strain TA100 in the absence of S9 only and employed a narrowed test material dose range of 2000, 3000, 4000 and 5000 μg/plate.

Evaluation criteria:

There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS (5) can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive result.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was therefore test up to the maximum recommended dose level of 5000 μg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. Small. statistically significant increases in revertant colony frequency were observed for tester strain TA100, (absence of S9 only) at 5000 μg/plate in the range finding test and 1500 and 5000 μg/plate in the main test. These increases were considered to be of no biological relevance becuase there was no clear evidence of a dose-response relationship or reproducibility over four separate experiments, one of which, employed quintuplicate dosing and a narrowed test material dose range. Furthermore, indicidual revertant counts were within the in-house historical control maxima for the bacterial strain 5000 and 1500 μg/plate in the range-finding and main testss respectively.
All of the positive control chemical sused in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9 mix and the sensitivity of the bacterial strains.

Conclusions:

In a guideline study, conducted to GLP, Gardamide was found to be not mutagenic in the bacterial reverse mutation assay, tested in four strains of Salmonella typhimurium and one strain of Escherichia coli in the presence and absence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Additional information from genetic toxicity in vitro:

Justification for selection of genetic toxicity endpoint
Reliable OECD guideline study, to GLP.

Justification for classification or non-classification

Based on negative results in a reliable bacterial reverse mutation assay in four strains of Salmonella typhimurium and one of Escherichia coli, classification under the EU DSD or CLP regulations as a mutagen is not required.