Biphenyl-2-yl-(9,9-dimethyl-9H-fluoren-2-yl)-(9,9’-spirobifluoren-2-yl)-amine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item was not mutagenic in bacterial cells and did not show a clastognic potential in mammalian cells.

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:

September 13 - November 11, 2011

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:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Chemical name: biphenyl-2-yl-(9,9-dimethyl-9H-fluoren-2-yl)-(9,9’-spirobifluoren-2-yl)-amine
Batch No.: OS11008922
Appearance: pale yellow powder without visible impurities
Supplier: Merck KGaA, Darmstadt
Analytical Report: Merck KGaA, Darmstadt, WL-AC6, Dr. Ulrich Engel
Released until: August 15, 2016
Solvent: dimethyl sulfoxide (DMSO) purity ≥ 99.9 % grade: for analysis

Target gene:

Salmonella: histidine operon
E coli. tryptophane operon

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

- Type and identity of media:
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Species / strain / cell type:

S. typhimurium TA 102

Details on mammalian cell type (if applicable):

- Type and identity of media:
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from Aroclor 1254 pretreated rats

Test concentrations with justification for top dose:

1st series: 5.00, 15.8, 28.1, 50.0, 158, 500, 1580 µg/plate
2nd series: 15.8, 28.1, 50.0, 88.9, 158 µg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: Sodium azide, 2-Aminoanthracene, Cumene hydroperoxide, 9-Aminoacridine, Daunomycin, 4-Nitroquinoline-N-oxide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 3-5 hours
- Exposure duration: about 2 days


SELECTION AGENT (mutation assays): Histidine, Tryptophane

NUMBER OF REPLICATIONS:
Negative controls 6
Test material 3
Positive controls 3


NUMBER OF CELLS EVALUATED: about 1e9


DETERMINATION OF CYTOTOXICITY
- Method: other: background cytotox

Rationale for test conditions:

According to guideline

Evaluation criteria:

- valid assay (cf. laboratory historical data)
- no or weak increase in revertant colonies = negative
- clear, dose dependent (over at least two concentrations), and reproducible increase in revertant colonies= positve

Statistics:

not applied

Key result

Species / strain:

S. typhimurium TA 1535

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

Key result

Species / strain:

S. typhimurium TA 1537

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

Key result

Species / strain:

S. typhimurium TA 102

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

Key result

Species / strain:

S. typhimurium TA 100

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

Key result

Species / strain:

S. typhimurium TA 98

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Evaporation from medium: no
- Water solubility: ok
- Precipitation:yes ( ≥ 158 µg/plate)
- Other confounding effects: no

Conclusions:

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

Executive summary:

The investigations for the mutagenic potential of the test item were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 102, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series were performed. In the two series with S9 mix, 10 % S9 in the S9 mix were used in the first and 30 % in the second series, respectively. The test item was dissolved in dimethyl sulfoxide (DMSO) and tested at concentrations ranging from 5.00 to 1580 μg/plate. Precipitation of the test material on the agar plates occurred at concentrations greater or equal than 158 μg/plate. Toxicity to the bacteria was not observed. Daunomycin, sodium azide, 9-aminoacridine, 4-nitroquinolin-N-oxide and cumene hydroperoxide served as strain specific positive control test materials in the absence of S9 mix. 2-Aminoanthracene and benzo[a]pyrene were used for testing the bacteria and the activity of the S9 mix. Each treatment with the test materials used as positive controls led to a clear increase in revertant colonies, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.

With and without addition of S9 mix as the external metabolizing system 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 18, 2013 - Mar 18, 2014

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)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Appearance: white solid
Stability: chemically stable under standard ambient conditions (room temperature)
Released until: August 15, 2016

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).

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 mix

Test concentrations with justification for top dose:

28.1, 88.9, 281 µg/mL

Vehicle / solvent:

DMSO, 1%

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

other: Griseofulvin

Details on test system and experimental conditions:

No. of slides per concentration:
Solvent control: 4
Others: 2
No. of metaphases evaluated per slide: 100 or 200 (for structural aberrations) 1000 (for polyploidy)

Preparation times:
- S9 mix: 25 and 31 hours
+ S9 mix:: 25 hours
Exposure times:
- S9 mix: 5, 25, and 31 hours
+ S9 mix: 5 hours
Solvent for the test material: DMSO
Concentrations evaluated:
Test item first series (with and without S9 mix): 28.1; 88.9; and 281 µg/mL



Positive controls: - S9 mix: 250 and 500 µg Ethylmethansulfonat (EMS)/mL
31.6 and 88.9 µg Griseofulvin (GRIS)/mL

+S9 mix: 2.00 µg Cyclophosphamide (CPA)/mL

Rationale for test conditions:

Guideline settings are applied

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.

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 V79 cell cultures with the test item did not relevantly increase the proportion of cells with aberrant chromosomes. The test item was thus not clastogenic in this in vitro test system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available data, the test substance is not considered to be classified as mutagenic according to Regulation (EC) No 1272/2008.