4-[Difluor(3,4,5-trifluorphenoxy)methyl]-3,5-difluor-4'-propyl-1,1'-biphenyl

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

OECD 471: negative

OECD 473: negative

OECD 476: negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002-07-30 to 2003-01-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

8 June 2000

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

HIS operon (S. thyphimurium)
TRP operon (E. coli)

Species / strain / cell type:

S. typhimurium TA 1535

Details on mammalian cell type (if applicable):

his G 46, uvrB, rfa

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain / cell type:

S. typhimurium TA 1537

Details on mammalian cell type (if applicable):

his C 3076, uvrB, rfa

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

his D 3052, uvrB, rfa + R-factor

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

his G 46, uvrB, rfa + R-factor

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain / cell type:

S. typhimurium TA 102

Details on mammalian cell type (if applicable):

his G 428, rfa + R-factor

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain / cell type:

E. coli WP2

Details on mammalian cell type (if applicable):

uvrA pkM101

Additional strain / cell type characteristics:

other: mutations in the tryptophan operon

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from Aroclor 1254-pretreated rats with standard co-factors

Test concentrations with justification for top dose:

The test material concentrations used were selected according to the EC and OECD guidelines for this test system and the requirements of the Labor Ministry of Japan:
1. Series: 5.00, 15.8, 50.0, 158, 500, 1580 and 5000 μg per plate (S9 10 %)
2. Series: 15.8, 50.0, 158, 500 and 1580 μg per plate (S9 30 %)

Vehicle / solvent:

acetone

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

cumene hydroperoxide

other: daunomycin

Remarks:

without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-aminoanthracene

Remarks:

with S9

Details on test system and experimental conditions:

The assessment of test material-induced effects is dependent on the number of spontaneous revertants of each bacterial strain (solvent controls) and the increase in the number of revertants at the test material concentration which shows the highest number of colonies. The following criteria, based upon the historical controls of the laboratory and statistical considerations, are established:

-----------------------------------------------------------------------------------------
Mean Number of Colonies Maximal Mean Number of Colonies over the Actual
(Solvent Control) Solvent Control
(Test Material)
-----------------------------------------------------------------------------------------

<=10 <=9 >=30
<=30 <=19 >=40
<=80 <=29 >=80
<=200 <=49 >=120
<=500 <=79 >=200
Assessment: No increase Clear increase

-----------------------------------------------------------------------------------------

All further results, ranging between "no" and "clear", are assessed as "weak in-creases".

Interpretations:
A test material is defined as non-mutagenic in this assay if:

- "no" or "weak increases" occur in the first and second series of the main experiment. ("Weak increases" randomly occur due to experimental variation.)

A test material is defined as mutagenic in this assay if:

- a dose-related (over at least two test material concentrations) increase in the number of revertants is induced, the maximal effect is a "clear increase", and the effects are reproduced at similar concentration levels in the same test system;

- "clear increases" occur at least at one test material concentration, higher concentrations show strong precipitation or cytotoxicity, and the effects are reproduced at the same concentration level in the same test system.

In all further cases, a third test series with the bacterial strain in question should be performed.
If the criteria for a positive test result are not fulfilled in at least two out of the three series, the test material is defined as being non-mutagenic in this test system.

Evaluation criteria:

details see results

Statistics:

n.a.

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Nov 22, 2016 - Sep 18, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro CA

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells:Hoffmann-La Roche, Pharma, Basel, at May 27, 1997
- Suitability of cells:
- Cell cycle length, doubling time or proliferation index: 16 to 17.5 hours
V79 cells have been successfully used in mutagenicity testing for many years. This cell line has a high proliferation rate and cloning efficiency. The cells have a relatively stable karyotype with a modal chromosome number of 22 ± 1, and an aberration rate of about 0-5 % of the metaphases. Since the cell line is not able to metabolize indirect mutagens to reactive forms, the test is performed both in the presence and absence of an external metabolizing system (liver S9 mix of rats pre-treated with Aroclor 1254).

Test concentrations with justification for top dose:

88.9, 158, 281 µg/mL

Vehicle / solvent:

Acetone (final concentration: 0.1%)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

No. of slides per concentration:
Solvent control: 4
Others: 2
No. of metaphases evaluated per slide: 150 (for structural aberrations) 1000 (for polyploidy)

Preparation times:
- S9 mix: 77 hours
+ S9 mix:: 77 hours
Exposure times:
- S9 mix: 5 and 29 hours
+ S9 mix: 5 hours
Solvent for the test material: Acetone
Concentrations evaluated:
Test Item: -/+S9 mix, 5 h: 112, 223, 445 and 889 μg/ml
- S9 mix, 29 h: 500, 1000 and 2000 μg/ml

Positive controls: - S9 mix: 0.15 µg Mitomycin C / mL
+S9 mix: 1.50 µg Cyclophosphamide (CPA)/mL

Evaluation criteria:

Step 1: Evaluation of Slide Quality
Step 2: Evaluation of Chomosomal Aberrations

A total of 100 or 200 well spread metaphases per culture (slide) were examined for cytogenetic damage at a magnification of 1000x, using an oil immersion phase contrast objective. Additionally, a total of 1000 metaphases were examined per culture (slide) for the occurrence of polyploidy.

Structural chromosome alterations are scored as follows:
Gap:
Achromatic region in chromatid(s) not greater than the width of a chromatid. Scored as gap (chromatid) or isogap (chromosomal).
Break:
Achromatic region in chromatid(s) greater than the width of a chromatid or a discontinuity with displacement. Scored as break (chromatid) or isobreak (chromosomal).
Exchange:
Aberrations arising from an exchange between one or two chromatids. These may be chromosome or chromatid interchanges. In studies of this type, where full karyotyping and chromosome banding are not performed, only asymmetrical or chromatid exchanges will normally be recognized.
Multiple aberrations:
Cells with more than five aberrations, gaps excluded.
Specific aberrations:
Atypic chromosomes, dicentric chromosomes and pulverized metaphases.
The position of each aberrant metaphase is recorded by Vernier reading.
Scoring for polyploid cells and endoreduplications and determination of mitotic index:
The frequency determination of polyploid cells and endoreduplications (chromosomes with 4, 8, 16 … chromatids) is based on scoring 1000 mitoses per slide, and estimation of the mitotic index on scoring 1000 cells per slide.

Statistics:

Fisher’s Exact Test

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

In conclusion, treatment of cultured human peripheral blood lymphocytes with the test material did not increase the proportion of cells with aberrant chromosomes under the experimental conditions. Thus, the test material is considered unable to induce chromosomal aberrations in this in vitro test system under the conditions described.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02.11.2016 - 24.03.2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD (2016). Guideline for the testing of chemicals, No 476: In vitro Mammalian
Cell Gene Mutation Tests using the Hprt and xprt genes

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: HPRT

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: D016030488
- Expiration date of the lot/batch: 30 April 2018
- Purity test date: 30 April 2017


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 15-25°C, protected from light
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: The solubility limit in culture medium was in the range of 32.63 to 65.26 μg/mL. The test material was soluble in
acetone at concentrations up to at least 208.82 mg/mL.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no reactivity

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no
- Preliminary purification step (if any): no
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid: Stock solutions were prepared by formulating the test material under subdued lighting in acetone, with the aid of vortex mixing, to give the maximum required concentration.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The master stock of L5178Y tk+/- (3.7.2C) mouse lymphoma cells originated from
Dr Donald Clive, Burroughs Wellcome Co. Cells supplied to Covance were stored as
frozen stocks in liquid nitrogen. Full details of the supplier are documented in central
records. Each batch of frozen cells was purged of mutants and confirmed to be
mycoplasma free. For each experiment, at least one vial was thawed rapidly, the cells
diluted in RPMI 10 and incubated at 37±1°C. When the cells were growing well,
subcultures were established in an appropriate number of flasks.

Metabolic activation:

with and without

Metabolic activation system:

S-9 from male Sprague Dawley rats induced with Aroclor 1254

Test concentrations with justification for top dose:

3.125 to 50 μg/mL., top dose was selcted based on precipitation limit in the test medium

Vehicle / solvent:

Acetone

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk
- Cell density at seeding (if applicable): 2 x 10 exp 5 cells/mL

DURATION
- Plating for Survival: 7 days
- Expression Period: 7 days
- Plating for Viability: 8 days
- Plating for 6TG Resistance: 9 days

SELECTION AGENT (mutation assays): 6TG

Rationale for test conditions:

According to guideline

Evaluation criteria:

For valid data, the test article was considered to be mutagenic in this assay if:
1. The MF at one or more concentrations was significantly greater than that of the negative control (p≤0.05)
2. There was a significant concentration-relationship as indicated by the linear trend analysis (p≤0.05)
3. If both of the above criteria were fulfilled, the results should exceed the upper limit of the last 20 studies in the historical negative control database
(mean MF +/- 2 standard deviations).
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis.

Statistics:

Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines (Robinson et al., 1990). The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of
variance.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative 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
- Water solubility: limited
- Precipitation: observed at highest tested concentration
- Definition of acceptable cells for analysis:
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES:

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells:

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture:
- Indication whether binucleate or mononucleate where appropriate:

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
- Negative (solvent/vehicle) historical control data:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: [complete, e.g. CBPI or RI in the case of the cytokinesis-block method; RICC, RPD or PI when cytokinesis block is not used]
- Other observations when applicable: [complete, e.g. confluency, apoptosis, necrosis, metaphase counting, frequency of binucleated cells]

Conclusions:

It is concluded that the test material did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to the limit of solubility, for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9) under the experimental conditions employed.

Executive summary:

The informtion for this endpoint study record was obtained from an experimental study. The OECD GLP criteria were met and the methods applied are fully compliant with OECD TG 476. From the results of this assay it is concluded that the test material did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to the limit of solubility, for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9) under the experimental conditions employed.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available