2-[(1-amino-9,10-dihydro-4-hydroxy-9,10-dioxo-2-anthryl)oxy]ethyl phenyl carbonate

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:

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 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:

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.

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; 300 µg/mL

Controls 2

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.