5-Brom-2-[(3,4,5-trifluorphenoxy)-difluormethyl]-1,3-difluorbenzene

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 Oct 2017 - 31 Jan 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

5-[(4-bromo-2,6-difluorophenyl)difluoromethoxy]-1,2,3-trifluorobenzene

EC Number:

610-623-5

Cas Number:

511540-64-0

Molecular formula:

C13 H4 Br F7 O

IUPAC Name:

5-[(4-bromo-2,6-difluorophenyl)difluoromethoxy]-1,2,3-trifluorobenzene

Test material form:

solid

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

Blood samples were drawn from healthy non-smoking donors not receiving medication. For this study, blood was collected from a male donor (23 years old) for Experiment I and from a male donor (34 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 chromosomal aberrations in their peripheral blood lymphocytes.
Human lymphocytes were stimulated for proliferation by the addition of the mitogen phytohemagglutinin to the culture medium for a period of 48 hours. The cell harvest time point was approximately 1.5 x AGT (average generation time). Any specific cell cycle time delay induced by the test item was not accounted for directly.

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (Phenobarbital/β-naphthoflavone induced rat liver S9, mixed with S9 cofactor solution)

Test concentrations with justification for top dose:

Experiment I: Exposure period 4 h
with S9-mix: 4.4, 7.7, 13.5, 23.7, 41.5, 72.6, 127, 222, 667, 2000 µg/mL
without S9-mix: 4.4, 7.7, 13.5, 23.7, 41.5, 72.6, 127, 222, 667, 2000 µg/mL
Experiment II: Exposure period 22 h
without S9-mix: 6.4, 12.7, 16.6, 21.5, 28.0, 36.4, 47.3, 61.5, 80.0, 120 µg/mL

Vehicle / solvent:

DMSO (0.5 % v/v)

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

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.

Pre-experiment
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Cytotoxicity is characterized by the percentages of mitotic suppression in comparison to the controls by counting 1000 cells per culture in duplicate. The experimental conditions in this pre-test phase were identical to those required and described below for the main experiment.
The pre-test was performed with 10 concentrations of the test item separated by no more than a factor of √10 and solvent and positive controls. All cell cultures were set up in duplicate. Exposure time was 4 hrs (with and without S9 mix). The preparation interval was 22 hrs after start of the exposure.

Cytogenetic Experiment
Pulse exposure
About 48 hrs after seeding, 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test item concentration. The culture medium was replaced with serum-free medium containing the test item. For the treatment with metabolic activation, 50 μL S9 mix per mL culture medium was added. After 4 hrs the cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded and the cells were resuspended in and washed with "saline G" (pH 7.2, containing 8000 mg/L NaCl, 400 mg/L KCl, 1100 mg/L glucose •H2O, 192 mg/L Na2HPO4 • 2 H2O and 150 mg/L KH2PO4). The washing procedure was repeated once as described. After washing, the cells were resuspended in complete culture medium (with 10 % FBS) and cultured until preparation of the cells.
Continuous exposure (without S9 mix)
About 48 hrs after seeding, 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test item concentration. The culture medium was replaced with complete medium (with 10 % FBS) containing the test item. The culture medium was not changed until preparation of the cells.
Preparation of metaphases
Cultures were treated with the metaphase-arresting substance colcemid (final concentration: 0.2 μg/mL) approximately three hours before the requested harvest time. The cultures were harvested by centrifugation 22 hrs after beginning of treatment. The supernatant was discarded and the cells were resuspended in hypotonic solution (0.0375 M KCl). Then the cell suspension was allowed to stand at 37 °C for 20 minutes. After removal of the hypotonic solution by centrifugation (approx. 900 x g), the cells were fixed with a mixture of methanol and glacial acetic acid (3+1 parts, respectively). A small amount of cell suspension was then dropped onto clean, wet microscope slides and allowed to dry. The slides were stained with Giemsa, and, after drying covered with a cover slip. All slides were labelled with a computer-generated random code to prevent scorer bias.

2000 μg/mL were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 4.4 to 2000 μg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test for toxicity, precipitation of the test item was observed at the end of treatment at 41.5 μg/mL and above. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.
Clear toxic effects were observed after 4 hours treatment in the absence of S9 mix at 72.6 μg/mL, only. 120 μg/mL were chosen as top treatment concentration for Experiment II.

Evaluation of cytotoxicity and cytogenetic damage
Evaluation of the slides was performed according to the standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik" using microscopes with 100 x oil immersion objectives.
Cytotoxicity is characterized by the percentages of mitotic suppression in comparison with the controls by counting 1000 cells per culture in duplicate.
At least 150 well-spread metaphases were evaluated per culture for structural aberrations. Only metaphases containing a number of centromeres equal to a number of 46 ± 2 were included in the analysis. Breaks, fragments, deletions, exchanges and chromosomal disintegrations are recorded as structural chromosomal aberrations. Gaps were recorded as well, but they are not included in the calculation of the aberration rates since gaps are achromatic lesions of unknown biological relevance for which a clear relationship to treatment cannot be established.

Rationale for test conditions:

Acceptability Criteria
The chromosomal aberration assay is considered acceptable if it meets the following criteria (OECD guideline 473):
a) The number of aberrations found in the solvent controls falls within the 95% control limits of the distribution of the laboratory’s historical negative control database. If they fall outside those limits, they are acceptable as long as these data are not extreme outliers and there is evidence that the test system is ‘under control’ and technical or human failure can be excluded.
b) The rate of chromosomal aberrations in the positive controls was statistically significantly increased compared with the concurrent negative control and compatible with those generated in the historical positive control data base.
c) Cell proliferation criteria in the solvent control were fulfilled by ensuring sufficient number of cells has reached mitosis and cytotoxicity levels were acceptable. For primary lymphocyte cultures the mitotic index (MI) is an appropriate measure of cytotoxicity.
d) The test item is tested with and without metabolic activation for 4 h, and sampled to a time equivalent to about 1.5 normal cell cycle lengths after the beginning of treatment, as well as a long treatment experiment without metabolic activation was performed. For the long treatment the cells were exposed to the test item continuously until sampling for a time equivalent to 1.5 normal cell cycle lengths. All three experimental conditions will be tested unless one part turns out positive.
e) At least three test concentrations that meet the acceptability criteria were evaluated and at least 300 well-spread metaphases were counted.

Evaluation criteria:

Interpretation of Results
A test substance is classified as non-clastogenic if:
a. none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b. there is no concentration-related increase when evaluated with an appropriate trend test,
c. all results are inside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits)

A test substance is classified as clastogenic if all of the following criteria are met:
a. at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b. the increase is dose-related when evaluated with an appropriate trend test,
c. any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits)
A test substance is classified as clastogenic if all of the following criteria are met:
a. at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b. the increase is dose-related when evaluated with an appropriate trend test,
c. any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits)

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: no influence
- Data on osmolality: no influence
- Precipitation and time of the determination: Experiment I (+/- S9 mix) at 41.5 µg/mL and above; Experiment II (-S9 mix) at 120 µg/mL and above; precipitation occured at the end of the experiment
- Definition of acceptable cells for analysis: At least 150 metaphases per culture were evaluated for structural chromosomal aberrations. 1000 cells were counted per culture for determination of the mitotic index

RANGE-FINDING/SCREENING STUDIES (if applicable):
2000 μg/mL were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 4.4 to 2000 μg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test for toxicity, precipitation of the test item was observed at the end of treatment at 41.5 μg/mL and above. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.
Clear toxic effects were observed after 4 hours treatment in the absence of S9 mix at 72.6 μg/mL, only. 120 μg/mL were chosen as top treatment concentration for Experiment II.

STUDY RESULTS
- Concurrent vehicle negative and positive control data
Vehicle control: DMSO
Positive control: EMS (- S9 mix), CPA (+ S9 mix)

For all test methods and criteria for data analysis and interpretation:
- Statistical analysis: Statistical analysis is confirmed by Fisher's exact test (modified) (p < 0.05)

Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements:
o For lymphocytres in primary cultures: mitotic index (MI)
- Genotoxicity results (for both cell lines and lymphocytes)
o Definition for chromosome aberrations, including gaps : Breaks, fragments, deletions, exchanges and chromosomal disintegrations are recorded as structural chromosomal aberrations. Gaps were recorded as well, but they are not included in the calculation of the aberration rates since gaps are achromatic lesions of unknown biological relevance for which a clear relationship to treatment cannot be established.
o Number of cells scored for each culture and concentration, number of cells with chromosomal aberrations and type given separately for each treated and control culture, including and excludling gaps : At least 150 well-spread metaphases were evaluated per culture for structural aberrations. Please refer to the attached backgroudn material for the number of cells with chromosomal aberrations
o Changes in ploidy (polyploidy cells and cells with endoreduplicated chromosomes): No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: Please refer to the attached background material.
- Negative (solvent/vehicle) historical control data: Please refer to the attached background material.

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro.
Therefore, the test item is considered to be non-clastogenic in this chromosome aberration test, when tested up to moderate cytotoxic or precipitating concentrations.

Executive summary:

The test item, dissolved in DMSO, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro in two independent experiments.

In each experimental group two parallel cultures were analyzed.Per culture at least 150 metaphases were evaluated for structural chromosomal aberrations.

The highest applied concentration in this study (2000 μg/mL of the test item) was chosen with respect to the current OECD Guideline 473.

Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation in accordance with OECD Guideline 473

In Experiment I in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed precipitation. In Experiment II in absence of S9 mix after continuous treatment, moderate cytotoxicity was observed at the highest evaluated concentration. The next higher tested concentrations, which were separated by a smaller factor than requested by the guideline, showed clear cytotoxicity exceeding the requested range. 

Either with or without metabolic activation neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item.

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with structural chromosome aberrations.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro.

Therefore, the test item is considered to be non-clastogenic in this chromosome aberration test, when tested up to moderate cytotoxic or precipitating concentrations.