C16-18-(even numbered, C18-unsaturated)-alkylamines acetates

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The registration substance was not mutagenic in a guideline conform Salmonella typhimurium reverse mutation assay (Ames test) using the five tester strains TA 98, TA 100, TA 1535, TA 1537 and TA 102 with and without metabolic activation (S9-mix from induced rat liver). Likewise, the structural and functional closely related substances were also not mutagenic in guideline compliant mammalian cell gene (HPRT) mutation assays in V79 Chinese hamster cells and did not induce chromosome aberrations or clastogenic effects in guideline conform cytogenetic studies in V79 cells in vitro with and without metabolic activation.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-04-08 to 2014-05-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP 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)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of assay:

bacterial reverse mutation assay

Target gene:

His

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

rat S9 liver microsomal fraction

Test concentrations with justification for top dose:

Experiment I:
0.316, 1.00, 3.16, 10.0, 31.6, 100 and 316 µg/plate
for all tester strains except for TA 98 (with metabolic activation)

3.16, 10.0, 31.6, 100, 316, 1000 and 2500 µg/plate
TA 98 (with metabolic activation)
Experiment II:
0.16, 0.5, 1.6, 5, 16, 50, 90, 200 and 350 µg/plate

Vehicle / solvent:

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

Untreated negative controls:

yes

Remarks:

A. dest., BSL Lot No. 140327, 140408

Negative solvent / vehicle controls:

yes

Remarks:

ethanol, AppliChem Lot No. 3D008610

Positive controls:

yes

Positive control substance:

other:

Remarks:

Positive control without metabolic activation: sodium azide for tester strains TA100, TA1535; 4-nitro-o-phenylene-diamine for TA98, TA1537; methylmethanesulfonate for TA102. Positive control with metabolic activation: 2-aminoanthracene for all strains

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
-For the plate incorporation method the following materials were mixed in a test tube and poured over the surface of a minimal agar plate:
100 µL Test solution at each dose level, solvent control, negative control or reference mutagen solution (positive control),
500 µL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation),
100 µL Bacteria suspension (cf. Preparation of Bacteria, pre-culture of the strain),
2000 µL Overlay agar.
After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.

NUMBER OF REPLICATIONS: For each strain and dose level, including the controls, three plates were used.


DETERMINATION OF CYTOTOXICITY
- Method: clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control

Evaluation criteria:

The Mutation Factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher
than the reversion rate of the solvent control.

Statistics:

According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Additional information on results:

Precipitation was observed in the pre-experiment in tester strain TA 98 and TA 100 at concentrations of 1000 µg/plate and higher (with and without metabolic activation). Precipitation was observed in experiment I in tester strain TA 98 at concentrations of 1000 µg/plate and higher (with metabolic activation) and in all tester strains used in experiment II at a concentration of 350 µg/plate (with and without metabolic activation).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Toxic effects of the test item were noted in all tester strains evaluated in experiment I and II.

In experiment I toxic effects of the test item were observed in tester strain TA 98 at concentrations of 31.6 µg/plate and higher (withoutmetabolic activation) and at concentrations of 316 µg/plate (withmetabolic activation). In tester strains TA 100, TA 1535 and TA 1537 toxic effects of the test item were noted at concentrations of 10.0 µg/plate and higher (withoutmetabolic activation) and at concentrations of 100 µg/plate and higher (withmetabolic activation). In tester strain TA 102 toxic effects of the test item were observed at concentrations of 10.0 µg/plate and higher (withoutmetabolic activation) and at a concentration of 316 µg/plate (withmetabolic activation).

In experiment II toxic effects of the test item were noted in tester strains TA 98, TA 1535 and TA 1537 at concentrations of 16 µg/plate and higher (withoutmetabolic activation) and at concentrations of 200 µg/plate and higher (withmetabolic activation). In tester strain TA 100 toxic effects of the test item were noted at concentrations of 16 µg/plate and higher (withoutmetabolic activation) and at concentrations of 90 µg/plate and higher (withmetabolic activation). In tester strain TA 102 toxic effects of the test item were observed at concentrations of 50 µg/plate and higher (withoutmetabolic activation) and at concentrations of 200 µg/plate and higher (withmetabolic activation).

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with C16-18-(even numbered, C18-unsaturated)-alkylamines acetates at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.

The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiments.

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, C16-18-(even numbered, C18-unsaturated)-alkylamines acetates did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, C16-18-(even numbered, C18-unsaturated)-alkylamines acetates is considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

In a reverse gene mutation assay in bacteria, strains TA98, TA100, TA1535, TA1537 and TA102 of S. typhimurium were exposed to  C16-18-(even numbered, C18-unsaturated)-alkylamines acetates at concentrations of 0.316, 1.00, 3.16, 10.0, 31.6, 100 and 316 µg/plate for all tester strains except for TA 98 (withmetabolic activation) and 3.16, 10.0, 31.6, 100, 316, 1000 and 2500 µg/plate

TA 98 (withmetabolic activation) (experiment I) and 0.16, 0.5, 1.6, 5, 16, 50, 90, 200 and 350 µg/plate (experiment II), in the presence and absence of mammalian metabolic activation according to the plate incorporation method (experiment I and II).

The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5100; OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

The registration substance C16 -18 -(even numbered, C18 -unsaturated)-alkylamines acetates was tested for potential point mutation in a guideline conform bacterial reverse mutation assay (Ames test) according to OECD TG 471 with and without metabolic activation. Independent experiments using several test concentrations up to the limit dose of 5000µg/plate did not cause gene mutations by base pair changes or frameshifts in the genome of any of the tester strains used. Therefore, the submission substance is considered to be non-mutagenic in this bacterial reverse mutation assay. This study was selected as key study.

Additional mutagenicity assays with the registration substance have not been carried out. However, the structural and functional analogue C16 -18 -(even numbered)-alkylamines acetates can be used as surrogate substance for assessment purposes. This compound was tested for potential gene mutation in a guideline conform mammalian cell gene mutation assay (HPRT locus) according to OECD TG 476 in V79 cells of the Chinese hamster. Independent experiments were performed using several test concentrations up to the limit of 5000 µg/mL (with and without metabolic activation). No biologically relevant increase of mutants was found after treatment with the test item, neither with nor without metabolic activation. No dose-response relationship was observed. Therefore, the submission substance is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese hamster. This study was selected as key study.

C16 -18 -(even numbered)alkylamines acetates was tested as surrgate substance also for the potential to induce structural chromosome aberrations in an guideline conform in vitro cytogenetic assay in Chinese hamster V79 cells according to OECD TG 473. Two independent experiments with and without metabolic activation were carried out using test item concentrations up to the limit of 5000µg/mL. The aberration rates of all dose groups treated with the test item were within the historical control data of the negative controls. No biologically relevant increase of the aberration rates and no biologically relevant increase in the frequencies of polyploid cells were noted after treatment with the test item as compared to the controls. EMS and CPA were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations. The test item did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line and is thus considered to be non-clastogenic in this test. This study was selected as key study.

In conclusion, the registration substance C16 -18 -(even numbered, C18 -unsaturated)-alkylamines acetates subs can be considered to be not mutagenic in the bacterial reverse mutation assay, the mammalian (HPRT) mutation test in V79 cells and in the in vitro chromosome aberration test in V 79 cells.

Justification for selection of genetic toxicity endpoint
There are several mutagenicity studies available for C16-18-(even numbered, C18-unsaturated)-alkylamines acetates, the structurally closely related C16-18-(even numbered)-alkylamines acetates as well as structural and functional similar C12-18-(even numbered)-alkylamines which cover point mutation testing in bacteria, testing for gen mutations in mammlian cells and chromosome mutations in mammalian cell systems. All studies are guideline conform tests according to GLP with a Klimisch rating of 1.

Justification for classification or non-classification

Based on the available data from three independent mutagenicity assays, a respective mutagenic potential of C16 -18 -(even numbered, C18 -unsaturated)-alkylamines acetates can most probably be excluded. Thus, the submission substance does not have to be classified for mutagenicity in accordance with the criteria of CLP/GHS laid down in the EU Classification, Labellling and Packaging Regulation (1272/2008/EC).