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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-02-05 to 2014-03-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid

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
Additional strain / cell type characteristics:
not specified
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 (with and without metabolic activation):
5, 10, 25, 50, 100, 250, 500, 1000, 2500, 5000 µg/mL
Pre-experiment for experiment II (only without metabolic activation, 20 h long-term exposure assay):
5, 10, 25, 50, 100, 200, 350, 500, 750, 1000 µg/mL
Experiment I
without metabolic activation: 25, 50, 75, 100, 125, 150, 200, 225 and 250 µg/mL
and with metabolic activation: 5, 10, 25, 50, 100, 250, 500 and 1000 µg/mL

Experiment II
without metabolic activation: 10, 20, 50, 100, 250, 500, 750, 1000, 1250 and 1500 µg/mL
and with metabolic activation: 70, 150, 300, 400, 500, 600, 700, 800 and 900 µg/mL
Vehicle / solvent:
Vehicle (Solvent) used: cell culture medium (MEM + 0% FBS 4 h treatment; MEM + 10% FBS 20 h treatment).
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation; 300 µg/mL
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with metabolic activation; 0.8 and 1.0 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: dissolved in medium
DURATION: 4 h (short-term exposure), 20 h (long-term exposure)
Expression time (cells in growth medium): 5 days
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: ≥ 50 μg/mL; experiment I with S9: ≥ 500 μg/mL; Experiment II without S9: ≥ 250 μg/mL; Experiment II with S9:≥ 400 μg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid

Any other information on results incl. tables

Precipitation:

No precipitation of the test item was noted in any of the experiments.

Toxicity:

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 11.5% for the highest concentration (250 μg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 1000 μg/mL with a relative growth of 10.6%.

In experiment II without metabolic activation the relative growth was 12.6% for the highest concentration (1500 μg/mL) evaluated. The highest concentration evaluated with metabolic activation was 900 μg/mL with a relative growth of 11.5%.

Mutagenicity:

In experiment I without metabolic activation all mutant values of the negative controls and test item concentrations found were within the historical control data of the test facility BSL BIOSERVICE (about 5-43 mutants per 106 cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the negative controls.

Mutation frequencies with the negative control were found to be 18.35 and 20.81 mutants/106 cells and in the range of 4.71 to 29.49 mutants/106 cells with the test item, respectively. The highest mutation rate (compared to the negative control values) of 1.51 was found at a concentration of 225 μg/mL with a relative growth of 11.1%.

With metabolic activation all mutant values of the negative controls and test item concentrations found were within the historical control data of the test facility BSL BIOSERVICE (about 5-44 mutants per 106 cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the negative controls.

Mutation frequencies with the negative control were found to be 16.30 and 11.84 mutants/106 cells and in the range of 6.29 to 18.71 mutants/106 cells with the test item, respectively. The highest mutation rate (compared to the negative control values) of 1.33 was found at a concentration of 10 μg/mL with a relative growth of 113.8%.

In experiment II without metabolic activation all mutant values of the negative controls and test item concentrations found were within the historical control data of the test facility BSL BIOSERVICE (about 5-43 mutants per 106 cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the negative controls.

Mutation frequencies with the negative control were found to be 26.82 and 29.28 mutants/106 cells and in the range of 12.74 to 40.26 mutants/106 cells with the test item, respectively. The highest mutation rate (compared to the negative control values) of 1.44 was found at a concentration of 50 μg/mL with a relative growth of 90.4%.

In experiment II with metabolic activation all mutant values of the negative controls and test item concentrations found were within the historical control data of the test facility BSL BIOSERVICE (about 5-44 mutants per 106 cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the negative controls.

Mutation frequencies with the negative control were found to be 23.10 and 13.15 mutants/106 cells and in the range of 13.89 to 42.28 mutants/106 cells with the test item, respectively. The highest mutation rate (compared to the negative control values) of 2.33 was found at a concentration of 300 μg/mL with a relative growth of 77.4%.

DMBA (0.8 and 1.0 μg/mL) and EMS (300 μg/mL) were used as positive controls and showed distinct and biologically relevant effects in mutation frequency.

Applicant's summary and conclusion

Conclusions:
FAT 40045/Z TE is considered to be not mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.
Executive summary:

In this mammalian cell gene mutation assay (HPRT locus), V79 cells cultured in vitro were exposed to FAT 40045/Z TE at concentrations of

- 25, 50, 75, 100, 125, 150, 200, 225 and 250 µg/mL (without metabolic activation, Experiment I)

- 5, 10, 25, 50, 100, 250, 500 and 1000 µg/mL (with metabolic activation, Experiment I)

- 10, 20, 50, 100, 250, 500, 750, 1000, 1250 and 1500 µg/mL (without metabolic activation, Experiment II)

- 70, 150, 300, 400, 500, 600, 700, 800 and 900 µg/mL (with metabolic activation, Experiment II).

FAT 40045/Z TE was tested up to cytotoxic concentrations.

Biologically relevant growth inhibition was observed in experiment I and II with and without metabolic activation. In experiment I without metabolic activation the relative growth was 11.5 % for the highest concentration (250 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 1000 µg/mL with a relative growth of 10.6 %. In experiment II without metabolic activation the relative growth was 12.6 % for the highest concentration (1500 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 900 µg/mL with a relative growth of 11.5 %.

In experiment I without metabolic activation the highest mutation rate (compared to the negative control values) of 1.51 was found at a concentration of 225 µg/mL with a relative growth of 11.1 %.

In experiment I with metabolic activation the highest mutation rate (compared to the negative control values) of 1.33 was found at a concentration of 10 µg/mL with a relative growth of 113.8 %. In experiment II without metabolic activation the highest mutation rate (compared to the negative control values) of 1.44 was found at a concentration of 50 µg/mL with a relative growth of 90.4 %. In experiment II with metabolic activation the highest mutation rate (compared to the negative control values) of 2.33 was found at concentrations of 300 µg/mL with a relative growth of 77.4 %.

The positive controls did induce the appropriate response. 

There was no evidence of a concentration related positive response of induced mutant colonies over background. Hence FAT 40045/Z TE is considered to be not mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.