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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
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2012-11-28 to 2013-03-14
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP Guideline Study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

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
Details on test material:
Name: FAT 40224/H TE
Batch No.: TZ 2382-BB-503C81
Physical State: powder
Colour: red
Density: 1.46 g/cm3 (20°C)
pH: 6.5 to 7.5 conc.(% w/w): 1%
Melting Point: > 200°C
Purity / Active Components: sum of all coloured substances: 85.4%, main constituents: 50.6%
Date of Analysis: 23.07.2012
Storage Conditions: at room temperature
Expiry Date: 28.01.2017

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):
5, 10, 25, 50, 100, 250, 500, 1000, 2500 and 5000 µg/mL
Pre-experiment for experiment II (only without metabolic activation, 20 h long-term exposure assay):
50, 100, 250, 500, 1000, 2000, 3000 and 5000 µg/mL
Experiment I
without metabolic activation: 5, 10, 25, 50, 100, 250, 500, 1000, 2000 and 2500 µg/mL
and with metabolic activation: 5, 10, 25, 50, 100, 250, 500, 1000 and 2500 µg/mL
Experiment II
without metabolic activation: 10, 25, 50, 100, 200, 400, 600 and 800 µg/mL
and with metabolic activation: 100, 316, 1000, 1250, 1500, 2000, 2400, 2800 and 3000 µg/mL
Vehicle / solvent:
Vehicle (Solvent) used: cell culture medium (MEM + 0% FBS 4h treatment; MEM + 10% FBS 20h treatment). The test item was dissolved under stirring in cell culture medium and diluted prior to treatment
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation; 300 µg/mL
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with metabolic activation; 1 and 1.5 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: ≥ 500 μg/mL; experiment I with S9: ≥ 100 μg/mL; Experiment II without S9: ≥ 400 μg/mL; Experiment II with S9:≥ 1000 μg/mL
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 in vitro cell gene mutagenicity test under the experimental conditions reported, the test item FAT 40224/H TE 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 FAT 40224/H TE dissolved in cell culture medium at concentrations of

- 5, 10, 25, 50, 100, 250, 500, 1000, 2000 and 2500 µg/mL (without metabolic activation, Experiment I)

- 5, 10, 25, 50, 100, 250, 500, 1000 and 2500 µg/mL (with metabolic activation, Experiment I)

- 10, 25, 50, 100, 200, 400, 600 and 800 µg/mL (without metabolic activation, Experiment II)

- 100, 316, 1000, 1250, 1500, 2000, 2400, 2800 and 3000 µg/mL (with metabolic activation, Experiment II).

FAT 40224/H 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 16.5% for the highest concentration (2500 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 2500 µg/mL with a relative growth of 17.4%. In experiment II without metabolic activation the relative growth was 12.2% for the highest concentration (800 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 3000 µg/mL with a relative growth of 21.8%, which is slightly higher than the postulated 10-20% survival. Due to the fact that in both experiments with metabolic activation no hint at mutagenicity was found, this deficiency is considered to be not biologically relevant.

In experiment I without metabolic activation the highest mutation rate (compared to the negative control values) of 1.10 was found at a concentration of 1000 µg/mL with a relative growth of 45.6%.

In experiment I with metabolic activation the highest mutation rate (compared to the negative control values) of 1.30 was found at a concentration of 25 µg/mL with a relative growth of 82.1%.
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 100.2%.
In experiment II with metabolic activation the highest mutation rate (compared to the negative control values) of 1.21 was found at a concentration of 2000 µg/mL with a relative growth of 35.5%.

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 forin vitromutagenicity (mammalian forward gene mutation) data.