Amines, C16-18-alkyl, acetates

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

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

Test material

Test material information

Constituent 1

Reference substance name:

C16-18-(even numbered)-alkylamines acetates

EC Number:

800-526-8

Cas Number:

1273322-45-4

Molecular formula:

R-NH2xHOOCCH3 R = alkyl, mainly C16-18-(even numbered)

IUPAC Name:

C16-18-(even numbered)-alkylamines acetates

Test material form:

other: solid wax

Method

Target gene:

hypoxanthine-guanine-phosphoribosyl-transferase (HPRT)

Species / strain

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

Controls

Controls 1

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

Controls 2

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.

Results and discussion

Test results

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'.

Applicant's summary and conclusion

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.