Amines, C16-18-alkyl, 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 submission substance was also not mutagenic in a guideline compliant mammalian cell gene (HPRT) mutation assay in V79 Chinese hamster cells and did not induce chromosome aberrations or clastogenic effects in a guideline conform cytogenetic study in V79 cells in vitro with and without metabolic activation. This view is supported by a negative in vivo micronucleus assay with the analogous compound C16-18-(even numbered, C18-unsaturated)-alkylamines.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline Study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)

Type of assay:

mammalian cell gene mutation assay

Target gene:

hypoxanthine-guanine-phosphoribosyl-transferase (HPRT)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

-Type and identity of media: MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 of Wistar Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix

Test concentrations with justification for top dose:

Pre-experiment for experiment I (without metabolic activation):
0.039, 0.078, 0.156, 0.3125, 0.625, 1.25, 2.5, 5.0, 7.5, 10 mM
Pre-experiment for experiment I (with metabolic activation):
0.00066, 0.0010, 0.0033, 0.0066, 0.010, 0.039, 0.078, 0.156, 0.3125, 0.625, 1.25, 2.5, 5.0, 7.5, 10 mM
Pre-experiment for experiment II (only without metabolic activation, 20 h long-term exposure assay):
1.0, 2.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 µM
Experiment I
without metabolic activation:
0.033, 0.066, 0.10, 0.33, 0.66, 1.0, 3.3, 6.6 and 10 µM
and with metabolic activation:
2.5, 10, 40, 50, 60, 70, 80, 90 and 100 µM
Experiment II
without metabolic activation:
0.25, 0.5, 1, 2, 4, 6, 8, 10 and 11 µM
and with metabolic activation:
0.7, 2.2, 7, 22, 34, 46, 58, 70 and 82 µM

Vehicle / solvent:

Vehicle (Solvent) used: EtOH

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Migrated to IUCLID6: 300 µg/mL

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation

Migrated to IUCLID6: 1 µg/mL and 1.5 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: dissolved in EtOH
DURATION: 4 h (short-term exposure), 20 h (long-term exposure)
Expression time (cells in growth medium): 48-72 h
Selection time (if incubation with selection agent): about one week

SELECTION AGENT ( mutation assay) 11 µg/mL 6-thioguanine (TG)
NUMBER OF REPLICATIONS: two separate experiments (I+II) with single exposure; 5 individual flasks were seeded and evaluated
NUMBER OF CELLS EVALUATED: 400000 cells per flask
DETERMINATION OF CYTOTOXICITY: Method: relative growth

Evaluation criteria:

A test is considered to be negative if there is no biologically relevant increase in the number of mutants.
There are several criteria for determining a positive result:
-a reproducible three times higher mutation frequency than the solvent control for at least one of the concentrations;
-a concentration related increase of the mutation frequency; such an evaluation may be considered also in the case that a three-fold increase of
the mutant frequency is not observed;
-if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I without S9: ≥6.6 mM; experiment I with S9: ≥ 60 mM; Experiment II without S9: ≥ 8 mM; Experiment II with S9:≥ 58 mM

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item C16-18-(even numbered)-alkylamines acetates is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

Executive summary:

In a mammalian cell gene mutation assay (HPRT locus],V79 cells culturedin vitro were exposed to C16-18-(even numbered)-alkylamines acetates dissolved in EtOH at concentrations of

- 0.033, 0.066, 0.10, 0.33, 0.66, 1.0, 3.3, 6.6 and 10 µM (without metabolic activation, Experiment I)

- 2.5, 10, 40, 50, 60, 70, 80, 90 and 100 µM (with metabolic activation, Experiment I)

- 0.25, 0.5, 1, 2, 4, 6, 8, 10 and 11 µM (without metabolic activation, Experiment II)

- 0.7, 2.2, 7, 22, 34, 46, 58, 70 and 82 µM (with metabolic activation, Experiment II).

C16-18-(even numbered)-alkylamines acetates

was tested up to cytotoxic concentrations.

A biologically relevant growth inhibition (reduction of relative growth below 70%) was observed after the treatment with the test item in experiment I and II with and without metabolic activation. In experiment I without metabolic activation the relative growth was 16.3% for the highest concentration (10 µM) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 100 µM with a relative growth of 15.2%. In experiment II without metabolic activation the relative growth was 8.2% for the highest concentration (11 µM) evaluated. The highest concentration evaluated with metabolic activation was 82 µM with a relative growth of 5.0%.

In both experiments no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). No dose-response relationship was observed.

The positive controlsdidinduce the appropriate response. 

There was no evidence of a concentration related positive responseof induced mutant colonies over background.

This study is classified as acceptable.  This study satisfies the requirement for Test Guideline OPPTS 870.5300, OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

The registration substance 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.

The registration substance was tested for potential gene mutation in a guideline conform mammalian cell gene mutation assay (HPRT locus) according to OECD TG 476 in V79cells of the Chinese hamster. Independent experiments were performed using several test concentrations up to the limit of5000 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.

The submission substance was tested 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 submission substance is found 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 key studies covering bacterial point mutation testing, testing for gen mutations in mammalian cells as well as 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 the test item can most probably be excluded. Thus, the submission substance does not have to be classified for mutagenicity in accordance with the criteria laid down in the EU Dangerous Substances Directive (67/548/EEC) as well as in the EU Classification, Labellling and Packaging Regulation (1272/2008/EC).