2-hydroxy-1-(4-(4-(2-hydroxy-2-methylpropionyl)benzyl)phenyl)-2-methylpropan-1-one

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:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

444-860-9

EC Name:

-

Cas Number:

474510-57-1

Molecular formula:

C21 H24 O4

IUPAC Name:

2-hydroxy-1-(4-{[4-(2-hydroxy-2-methylpropanoyl)phenyl]methyl}phenyl)-2-methylpropan-1-one

Details on test material:

Physical state: solid, white

Method

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Hams F12 medium + supplements
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction from phenobarbital and beta-naphthoflavone induced rat liver

Test concentrations with justification for top dose:

first experiment +/- S9 mix: 7.8, 15.6, 31.3, 62.5, 125, 250 µg/ml
second experiment +/- S9 mix: 6.25, 12.5, 25, 50, 100, 150, 200 µg/ml

Vehicle / solvent:

Due to the limited solubility of the test substance in water, dimethyl sulfoxide (DMSO) was
selected as the vehicle, which had been demonstrated to be suitable in the CHO/HPRT
assay and for which historical data are available.
The final concentration of the vehicle DMSO in the culture medium was 1% (v/v).

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4h
- Expression time (cells in growth medium): 3 days (4 hour treatment) or 2 days (24 hour treatment) at 37°C
- Selection time (if incubation with a selection agent): 6-7 days in TG-medium
- Fixation: At the end of the selection period, the medium will be removed and the remaining colanies will be fixed with methanol, stained with Giemsa and counted

SELECTION AGENT (mutation assays): TG-medium

NUMBER OF REPLICATIONS: two independent experiments, every sample in triplicate

NUMBER OF CELLS EVALUATED: all colonies are counted

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

OTHER EXAMINATIONS:
- pH
- osmolarity
- solubility
- cell morphology

Evaluation criteria:

A finding is assessed as positive if the following criteria are met:
• Increase in the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and our historical negative control data range
• Evidence of the reproducibility of any increase in mutant frequencies.
• A statistically significant increase in mutant frequencies and the evidence of a doseresponse relationship.

Statistics:

An appropriate statistical trend test was performed to assess a dose-related increase of
mutant frequencies. The number of mutant colonies obtained for the test substance treated
groups was compared with that of the respective vehicle control groups. A trend is judged as
statistically significant whenever the p-value (probability value) is below 0.10 and the slope is
greater than 0. However, both, biological and statistical significance will be considered
together.

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 125 ug/ml onward

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Evaporation from medium: no
- Precipitation: no
- Other confounding effects: no

RANGE-FINDING/SCREENING STUDIES: In the pretest for toxicity based on the purity and the molecular weight of the test substance 3 600 μg/mL (approx. 10 mM) of the test item was used as top concentration both with andwithout S9 mix at 4 hour exposure time and without S9 mix at 24 hour exposure time.

COMPARISON WITH HISTORICAL CONTROL DATA: historical negative control data from May 1994 - December 2011

ADDITIONAL INFORMATION ON CYTOTOXICITY: Cytotoxic effects indicated by clearly reduced cloning efficiencies of below 20% of control
were observed in all experimental parts scored for gene mutations. In culture medium test substance precipitation occurred at 450 μg/mL and above at the beginning and at the end of treatment in the absence and presence of S9 mix. Cytotoxicity indicated by reduced relative cloning efficiency of about or below 20% relative survival was observed at 112.5 μg/mL and above under all treatment conditions.

After 4 hours of treatment the morphology and attachment of the cells treated with at least the highest applied concentration was adversely influenced in all experimental parts scored for gene mutations. This occurred in samples regardless of the presence of metabolic activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

The test material was found not to be mutagenic under the conditions of the test.

Executive summary:

In this guideline (OECD 476) study conducted with GLP certification, the test material (EC: 444-860-9) was considered to be non-genotoxic. The HPRT test was conducted in Chinese Hamster Ovary (CHO) cells with and without metabolic activation. Based on the solubility properties of the test substance and according to an initial rangefinding cytotoxicity test doses up to 250 µg/ml were tested in presence and absence of a metabolic activation system.