Methanone, 1,1'-(1,4-phenylene)bis[1-(4-fluorophenyl)-

Genetic toxicity: in vitro

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Recent study complated under cGLP conditions within the Eu according to an approved methodology.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD487

Deviations:

yes

Remarks:

Treatment, recovery and harvest time modified to optimum

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Bis(4-fluorophenyl) ketone

EC Number:

206-466-3

EC Name:

Bis(4-fluorophenyl) ketone

Cas Number:

345-92-6

IUPAC Name:

bis(4-fluorophenyl)methanone

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Batch Number:4EDP0024
Purity: >99.5%
Molecular Weight 218.201g/mol

Method

Species / strain

Species / strain / cell type:

lymphocytes:

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Exogenous metabolic activation (S9)

Test concentrations with justification for top dose:

14.2 to 2190.0ug/mL

Vehicle / solvent:

DMSO

Controls

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulfoxide

Positive controls:

yes

Positive control substance:

mitomycin C

Details on test system and experimental conditions:

Human lymphocytes.
Blood samples were drawn from healthy non-smoking donors not receiving medication. For this study, blood was collected from a female donor (31 years old) for Experiment IA, from a male donor (23 years old) for Experiment IB and from a female donor (25 years old) for Experiment II. The lymphocytes of the respective donors have been shown to respond well to stimulation of proliferation with PHA and to positive control substances. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes.
Human lymphocytes were stimulated for proliferation by the addition of the mitogen PHA to the culture medium for a period of 48 hours. The cell harvest time point was approximately 2 – 2.5 x AGT (average generation time). Any specific cell cycle time delay induced by the test item was not accounted for directly.

Culture conditions
Blood cultures were established by preparing an 11 % mixture of whole blood in medium within 30 hrs after blood collection. The culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 μg/mL), the mitogen PHA (3 μg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL).
All incubations were done at 37 °C with 5.5 % CO2 in humidified air.

Evaluation criteria:

Evaluation of cytotoxicity and cytogenetic damage
Evaluation of the slides was performed using NIKON microscopes with 40 x objectives. The micronuclei were counted in cells showing a clearly visible cytoplasm area. The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976). The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus. At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. The frequency of micronucleated cells was reported as % micronucleated cells. To describe a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is expressed as % cytostasis. A CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis.
n3)x(MUNC2)x(BINC1)x(MONCCBPI++=
CBPI Cytokinesis-block proliferation index n Total number of cells MONC Mononucleate cells BINC Binucleate cells MUNC Multinucleate cells
Cytostasis % = 100 – 100 [(CBPIT – 1) / (CBPIC – 1)]
T Test item C Solvent control

Statistics:

Data Recording
The data generated were recorded in the laboratory protocol. The results are presented in tabular form, including experimental groups with the test item, solvent, and positive controls.

Results and discussion

Test results

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At the highest evaluated concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Three independent experiments were performed. In Experiment IA, the exposure period was 4 hours with and without S9 mix. In Experiment IB, the exposure period was 4 hours with S9 mix. In Experiment II, the exposure period was 20 hours without S9 mix. The cells were prepared 40 hours after start of treatment with the test item.
In each experimental group two parallel cultures were analysed. 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. To determine a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is described as % cytostasis.
The highest treatment concentration in this study, 2190.0 μg/mL (approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the OECD Guideline 487 for the in vitro mammalian cell micronucleus test.
Visible precipitation of the test item in the culture medium was observed microscopically in Experiment IA at 24.9 μg/mL and above in the absence of S9 mix and at 43.6 μg/mL and above in the presence of S9 mix, in Experiment IB at 30.0 μg/mL and above in the presence of S9 mix and in Experiment II at 34.2 μg/mL and above in the absence of S9 mix at the end of treatment.
No relevant influence on osmolarity or pH value was observed. The osmolarity is generally high compared to the physiological level of approximately 300 mOsm. This effect however, is based on a final concentration of 1% DMSO in medium. As the osmolarity is measured by freezing point reduction, 1% of DMSO has a substantial impact on the determination of osmolarity.
In the absence and presence of S9 mix, clear cytotoxicity was observed at the highest evaluated concentrations (Table 3 – Table 5).
In the absence and the presence of S9 mix, no relevant increase in the number of micronucleated cells was observed after treatment with the test item. The micronucleus rates of the cells after treatment with the test item (0.05 – 1.25 % micronucleated cells) were close to the range of the solvent control values (0.10 – 1.05 % micronucleated cells) and within the range of the laboratory historical control data (see Appendix 1). However, in Experiment IA in the absence of S9 mix one single statistically significant increase in micronucleated cells (1.25 %) was observed after treatment with 24.9 μg/mL. This value is in the range of the laboratory historical solvent control data (0.15 – 1.40 % micronucleated cells) and therefore considered biologically irrelevant. In Experiment IB in the presence of S9 mix one single statistically significant increase in micronucleated cells (0.65 %) was observed after treatment with 30.0 μg/mL. This value is also in the range of the laboratory historical solvent control data (0.15 – 1.70 % micronucleated cells) and therefore considered biologically irrelevant.
Either Demecolcin (125.0 ng/mL), MMC (2.0 μg/mL) or CPA (17.5 μg/mL) were used as positive controls and showed distinct increases in cells with micronuclei.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, bis(4-fluorophenyl)ketone is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to cytotoxic and precipitating concentrations.