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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:
experimental study
Adequacy of study:
key study
Study period:
31 March 2014 - 04 June 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
[4-(4-phenoxybenzoyl)phenyl](4-phenoxyphenyl)methanone
EC Number:
620-097-9
Cas Number:
54299-17-1
Molecular formula:
C32H22O4
IUPAC Name:
[4-(4-phenoxybenzoyl)phenyl](4-phenoxyphenyl)methanone
Test material form:
solid: particulate/powder

Method

Target gene:
Thymidine Kinase
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium containing L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium
pyruvate (200 µg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Test concentrations with justification for top dose:
0.012, 0.025, 0.05, 0.1, 0.2 and 0.4 mM for the first experiment (3-hour treatment),
0.02, 0.04, 0.08, 0.15, 0.3 and 0.6 mM for the second experiment (24 hour treatment).
Vehicle / solvent:
- Vehicle used: ethanol
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: methylmethanesulfonate (-S9 mix); cyclophosphamide (+S9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 and 24 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 11-12 days

SELECTION AGENT (mutation assays): trifluorothymidine

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth.
Evaluation criteria:
Positive result defined as:
- At least at one dose-level the mutation frequency minus the mutation frequency of the vehicle control (IMF) equals or exceeds the global evaluation factor (GEF) of 126 E-6
- A dose-related trend is demonstrated by a statistically significant trend test
- Unless clearly positive, the reproducibility should be confirmed

Negative results defined as:
- No evidence of mutagenicity at concentrations inducing moderate cytotoxicity (10% < Adj. RTG <20%), or
- If there is no culture with 10% < Adj. RTG <20%:
¿ at least one negative data point with 20% < Adj. RTG <25% + negative data from 20% < Adj. RTG <100%, or
¿ at least one negative data point with 1% < Adj. RTG <10% + negative data from 25% < Adj. RTG <100%

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
whitout S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: At the concentration of 0.8 mM, the pH of the culture medium was approximately 7.1 (as for the vehicle control)
- Effects of osmolality: the osmolality was equal to 402 mOsm/kg H2O (396 mOsm/kg H2O for the vehicle control).
- Precipitation: = 0.16 mM

RANGE-FINDING/SCREENING STUDIES:
Using a test item concentration of 75 mg/mL and a treatment volume of 0.5% (v/v) in the culture medium (i.e. 100 µL/20 mL culture medium for the 3-hour treatments and 250 µL/50 mL culture medium for the 24 hour treatment), the final concentration of 0.8 mM (corresponding to 376.4 µg/mL) was achievable. Thus the concentrations selected for the treatments of the preliminary test were 0.002, 0.016, 0.08, 0.16, 0.4 and 0.8 mM.

At the concentration of 0.8 mM, the pH of the culture medium was approximately 7.1 (as for the vehicle control) and the osmolality was equal to 402 mOsm/kg H2O (396 mOsm/kg H2O for the vehicle control).

At the end of the treatment periods (3- or 24-hour treatments), a slight to strong precipitate was observed in the culture medium at concentrations = 0.16 mM.

Following the 3-hour treatment without S9 mix, a moderate toxicity was induced at concentrations of 0.002 mM and at concentrations = 0.08 mM, as shown by a 40 to 48% decrease in the Adjusted Relative Total Growth (Adj. RTG).
Following the 24-hour treatment without S9 mix, a moderate to marked toxicity was induced at concentrations = 0.16 mM, as shown by a 49 to 80% decrease in the Adj. RTG.
Following the 3-hour treatment with S9 mix, a slight toxicity was induced at the highest concentration of 0.8 mM, as shown by a 29% decrease in the Adj. RTG.

MAIN EXPERIMENTS
The Cloning Efficiencies (CE2), the mutation frequencies and the suspension growths of the vehicle controls were as specified in the acceptance criteria. For the positive control cultures, the increase in the mutation frequencies met also the acceptance criteria. The study was therefore considered to be valid.
Since the test item was found cytotoxic and poorly soluble in the preliminary test, the selection of the highest concentrations to be used in the main experiments was based on the level of precipitate and cytotoxicity, according to the criteria specified in the international guidelines.

Experiments without S9 mix
At the end of the 3- and 24-hour treatments, a slight to strong precipitate was observed in the culture medium, at concentrations = 0.15 mM.
Following the 3-hour treatment, a slight toxicity was induced at concentrations = 0.2 mM, as shown by a 25 to 27% decrease in Adj. RTG.
Following the 24-hour treatment, a slight toxicity was induced at the concentration of 0.6 mM, as shown by a 34% decrease in Adj. RTG.
Following the 3- and 24-hour treatments, no noteworthy increase in the mutation frequency was noted at any tested concentrations.

Experiments with S9 mix
At the end of the 3-hour treatments, a slight to strong precipitate was observed in the culture medium, at concentrations = 0.15 mM.
In the first and second experiments, no noteworthy toxicity was induced, as shown by the absence of any noteworthy decrease in Adj. RTG .
Following the 3-hour treatments, no noteworthy increase in the mutation frequency was noted at any tested concentrations.

Applicant's summary and conclusion

Conclusions:
EKKE did not show any mutagenic activity in the mouse lymphoma assay, in the presence or in the absence of a rat metabolizing system.
Executive summary:

The potential of EKKE to induce mutations at the TK (Thymidine Kinase) locus was evaluated in L5178Y TK+/-mouse lymphoma cells. The study was performed according to international guidelines (OECD guideline No. 476 and GLP. After a preliminary toxicity test, the test item EKKE was tested in two independent experiments, with and without a metabolic activation system (S9 mix) prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.

Cultures of 20 mL at 5 x 105cells/mL (3-hour treatments) or cultures of 50 mL at 2 x 105cells/mL (24-hour treatment) were exposed to the test or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 2%). During the treatment period, the cells were maintained as suspension culture in RPMI 1640 culture medium supplemented by heat inactivated horse serum at 5% (3-hour treatment) or 10% (24-hour treatment) in a, 5% CO2humidified incubator. For the 24-hour treatment, flasks were gently shaken at least once. Cytotoxicity wasmeasured by assessment of Adjusted Relative Total Growth (Adj. RTG), Adjusted Relative Suspension Growth (Adj. RSG) andCloning Efficiency following the expression time (CE2). The number of mutant clones (differentiating small and large colonies) was evaluated after expression of the mutant phenotype.

Since the test item was found cytotoxic and poorly soluble in the preliminary test, the selection of the highest concentrations to be used in the main experiments was based on the level of precipitate and cytotoxicity, according to the criteria specified in the international guidelines. The Cloning Efficiencies (CE2), the mutation frequencies and the suspension growths of the vehicle controls were as specified in the acceptance criteria. For the positive control cultures, the increase in the mutation frequencies met also the acceptance criteria. The study was therefore considered as valid. At the end of the culture periods (short and long treatment times), a slight to strong precipitate was observed in the culture medium at concentrations=0.15 mM.

Using a treatment volume of 0.5% (v/v) in the culture medium, the selected concentrations were as follows:

.            0.012, 0.025, 0.05, 0.1, 0.2 and 0.4 mM for the first experiment (3-hour treatment),

.            0.02, 0.04, 0.08, 0.15, 0.3 and 0.6 mM for the second experiment (24-hour treatment).

Experiments without S9 mix

Following the 3-hour treatment, a slight toxicity was induced at concentrations=0.2 mM, as shown by a 25 to 27% decrease in Adj. RTG. Following the 24-hour treatment, a slight toxicity was induced at the concentration of 0.6 mM, as shown by a 34% decrease in Adj. RTG. Following the 3- and 24-hour treatments, no noteworthy increase in the mutation frequency was noted at any tested concentrations.

Experiments with S9 mix

In the first and second experiments, no noteworthy toxicity was induced, as shown by the absence of any noteworthy decrease in Adj. RTG. Following the 3-hour treatments, nonoteworthy increase in the mutation frequency was noted at any tested concentrations.

EKKE did not show any mutagenic activity in the mouse lymphoma assay, either in the presence or absence of a rat metabolizing system.