Registration Dossier

Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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:
2014-06-23 to 2014-09-25
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
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

Test material

Constituent 1
Chemical structure
Reference substance name:
2-[(1-amino-9,10-dihydro-4-hydroxy-9,10-dioxo-2-anthryl)oxy]ethyl phenyl carbonate
EC Number:
248-882-8
EC Name:
2-[(1-amino-9,10-dihydro-4-hydroxy-9,10-dioxo-2-anthryl)oxy]ethyl phenyl carbonate
Cas Number:
28173-59-3
Molecular formula:
C23H17NO7
IUPAC Name:
2-[(1-amino-4-hydroxy-9,10-dioxo-9,10-dihydroanthracen-2-yl)oxy]ethyl phenyl carbonate
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

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 (with and without metabolic activation):
0.0025, 0.0050, 0.010, 0.020, 0.030, 0.040, 0.050, 0.060, 0.070, 0.080, 0.090, 0.100, 0.125, 0.15 mM
Experiment I
with and without metabolic activation: 0.0010, 0.0025, 0.0050, 0.010, 0.020, 0.040, 0.060, 0.080, 0.100, 0.125 mM
Experiment II
without metabolic activation: 0.0010, 0.0025, 0.0050, 0.010, 0.020, 0.040, 0.060, 0.080, 0.100, 0.125 mM
and with metabolic activation: 0.0030, 0.0075, 0.015, 0.030, 0.045, 0.060, 0.075, 0.090, 0.011, 0.125 mM
Experiment III:
without metabolic activation: 0.07, 0.08, 0.09 mM
Vehicle / solvent:
Vehicle (Solvent) used: The test item was dissolved in DMSO and diluted in cell culture medium (MEM + 0% FBS 4h treatment; MEM + 10 % FBS 20h treatment) prior to 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
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 DMSO
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
Key result
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: 0.010, 0.040 and ≥0.080 mM; Experiment II without S9: ≥0.040 mM; Experiment III without S9: ≥0.07 mM
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
FAT 93504/B 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 cultured in vitro were exposed to FAT 93504/B at concentrations of


- 0.0010, 0.0025, 0.0050, 0.010, 0.020, 0.040, 0.060, 0.080, 0.100, 0.125 mM (with and without metabolic activation, Experiment I)


- 0.0010, 0.0025, 0.0050, 0.010, 0.020, 0.040, 0.060, 0.080, 0.100, 0.125 mM (without metabolic activation, Experiment II)


- 0.0030, 0.0075, 0.015, 0.030, 0.045, 0.060, 0.075, 0.090, 0.011, 0.125 mM (with metabolic activation, Experiment II)


- 0.07, 0.08, 0.09 mM (without metabolic activation, Experiment III).


FAT 93504/B was tested up to cytotoxic concentrations.


Biologically relevant growth inhibition was observed in experiment I, II and III without metabolic activation. In experiment I without metabolic activation, the relative growth was 48.6 % for the highest concentration (0.125 mM) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 0.125 mM with a relative growth of 84.4 %. In experiment II without metabolic activation, the relative growth was 25.5 % for the highest concentration (0.125 mM) evaluated. The highest concentration evaluated with metabolic activation was 0.125 mM with a relative growth of 88.9 %. In experiment III without metabolic activation, the relative growth was 29.9 % for the highest concentration (0.09 mM) evaluated.


In experiment I without metabolic activation the highest mutation rate (compared to the solvent control values) of 1.91 was found at a concentration of 0.01 mM with a relative growth of 67.7 %.


In experiment I with metabolic activation the highest mutation rate (compared to the solvent control values) of 1.43 was found at a concentration of 0.080 mM with a relative growth of 83.9 %.



In experiment II without metabolic activation the highest mutation rate (compared to the solvent control values) of 3.34 was found at a concentration of 0.080 mM with a relative growth of 32.7 %.



In experiment II with metabolic activation the highest mutation rate (compared to the solvent control values) of 2.28 was found at a concentration of 0.015 mM with a relative growth of 113.3 %.


In experiment III without metabolic activation the highest mutation rate (compared to the solvent control values) of 1.22 was found at a concentration of 0.08 mM with a relative growth of 35.5 %.


The positive controls did induce the appropriate response. There was no evidence of a concentration related positive responseof induced mutant colonies over background. Hence, FAT 93504/B is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.