Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three Ames tests, two Chromosome Aberration tests, one mammalian cell mutation test in V79 cells and one study of Sister Chromatid Exchange are available in vitro.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in accordance with OECD TG 473 and GLP
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Principles of method if other than guideline:
Test method was in accordance with the OECD 473 Test Guideline and the Guidelines for Screening Mutagenicity Testing of Chemicals (Chemical Substances Control Law of Japan)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
- Analytical purity:99.96
Target gene:
Chromosomal aberrations in Chinese hamster lung cells (CHL/u)
Species / strain / cell type:
other: Chinese hamster lung cells (CHL/u)
Metabolic activation:
with and without
Metabolic activation system:
Rat liver induced with phenobarbital and 5,6-benzoflavone S-9
Test concentrations with justification for top dose:
0.03; 0.06 and 0.12 mg/mL with and without S-9
Vehicle / solvent:
dimethyl sulphoxide (DMSO)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S-9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S-9
Details on test system and experimental conditions:
No further information
Evaluation criteria:
According to criteria set out in OECD Test Guidelines No 473 and Guidelines for Screening Mutagenicity Testing of Chemicals (Chemical Substances Control Law of Japan)
Statistics:
Chromosome analysis conducted using one-sided Fisher's exact probability test. Significant results tabulated for p<0.01
Key result
Species / strain:
other: chinese hamster lungs cells (CHL/U)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
clastogenicity and polyploidy with or without metabolic activation at 0.03 or 0.06 mg/mL and above
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
A significant increase in frequency of cells with chromosomal aberrations was noted at all three treatments, 0.03, 0.06, 0.12 mg/mL, without metabolic activation and in the two higher dose levels with metabolic activation (Chromosome analysis conducted using one-sided Fisher's exact probability test. Significant results tabulated for p<0.01). A significant increase in polyploidy was also observed at 0.06 or 0.12 mg/mL, with and without metabolic activation.
Conclusions:
Interpretation of results: positive

A statistically significant (p<0.01: Fisher's Exact Test) increase in frequency of cells with chromosomal aberrations was noted at all three treatments, 0.03, 0.06, 0.12 mg/mL, without metabolic activation and in the two higher dose levels with metabolic activation.
A statistically significant increase in polyploidy (p<0.01: Fisher's Exact Test) was also observed at 0.06 or 0.12 mg/mL, with and without metabolic activation.

A positive response for induction of chromosomal aberrations was observed in chinese hamster lung cells treatd with 4,4'-biphenyldiol, typically at dose levels of 0.06 mg/mL and above, the response was typified by clastogenic and polyploidy effects.
Executive summary:

The cytogenicity of biphenyl-4,4'-diol (biphenol) was investigated in Chinese Hamster cells (CHL/IU) cells in vitro. A significant (p<0.01) increase in frequency of cells with chromosomal aberrations was noted at all three treatments, 0.03, 0.06, 0.12 mg/mL, without metabolic activation and in the two higher dose levels with metabolic activation. A significant increase in polyploidy was also observed at 0.06 or 0.12 mg/mL, with and without metabolic activation.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
Good quality published study assessing mammalian cell mutation in V79 cells.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
Target gene:
Gene mutations resulting in 6 thioguanine resistance
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
Male SD rat liver S9 fraction
Test concentrations with justification for top dose:
Concentrations tested were stated to be up to the cytotoxic limit: the concentration range tested was 20-1000 uM in the absence and presence of metabolic activation.
Vehicle / solvent:
No data
Untreated negative controls:
no
Negative solvent / vehicle controls:
other: reference to solvent control data from other studies
True negative controls:
other: negative responses obtained with other substances evaluated
Positive controls:
other: positive responses obtained with other substances evaluated
Positive control substance:
not specified
Details on test system and experimental conditions:
A total of 1.5 x 10e6 cells and 30 mL medium were put into 15-cm Petri dishes. After 18 hours, the test substance was added for 24 hours. The frequency of mutants resistant to 6-thioguanine (7 1g/mL) was determined after an expression period of 6 days. At least 5 concentrations were used, up to the limits of toxicity (1000 uM).
Evaluation criteria:
No details presented in the publication but methods followed were consistent with standard test guidelines. Genotoxicity evaluated based on frequency of mutants resistant to 6-thioguanine.
Statistics:
No information
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
tested up to 1000 uM 4,4'-dihydroxyphenyl, which was the cytotoxic limit
Vehicle controls validity:
not specified
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No mutagenic activity is reported: the substance was therefore concluded to have a very low potency (calculated as the induced mutation frequency)

It is concluded that the substance showed a comparatively low level of cytotoxicity and did not induce gene mutations under the conditions of this study.

Conclusions:
Interpretation of results: negative

No evidence for the induction of gene mutations in V79 cells was seen with 4,4'-dihydroxyphenyl under the conditions of this assay.
Executive summary:

4,4'-dihydroxyphenyl was investigated for its potential to induce gene mutations in V79 cells (HPRT locus). Cells were exposed to concentrations of up to 1000 uM (the cytotoxic limit) in the presence and absence of metabolic activation (rat liver S9 fraction). There was no evidence for the induction of forward mutations under the conditions of this assay.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
23-SEP-2013 to 19-FEB-2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This GLP-compliant study was conducted according to standardised guidelines (i.e., OECD 473, EU B.10 and EPA OPPTS 870.5375).
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM (minimal essential medium with Earle's salts) containing a L-glutamine source and supplemented with 10% (v/v) foetal calf serum (FCS), 1% (v/v) penicillin/streptomycin (10 000 IU / 10 000 μg/mL), 1% (v/v) amphotericin B (250 μg/mL); during exposure to the test substance in the presence of S9 mix MEM medium was used without FCS supplementation.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from induced rats (exogenous metabolic activation)
Test concentrations with justification for top dose:
- 1st experiment
* 4-hour exposure, 18-hour sampling time, without S9 mix: 0, 3.91, 7.81, 15.63, 31.25, 62.5, 125, and 250 μg/mL
* 4-hour exposure, 18-hour sampling time, with S9 mix: 0, 15.63, 31.25, 62.5, 125, 250, and 500 μg/mL

The slides of the 1st experiment either in the absence or presence of metabolic activation were not scorable due to technical reasons. Therefore, a repeat experiment, designated 2nd experiment, was performed.

- 2nd experiment
* 4-hour exposure, 18-hour sampling time, without S9 mix: 0, 7.81, 15.63, 31.25, 62.5, 125, and 250 μg/mL
* 4-hour exposure, 18-hour sampling time, with S9 mix: 0, 31.25, 62.5, 125, 250, 500, and 1000 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO (1% (v/v))
- Justification for choice of solvent/vehicle: due to the limited solubility of the test substance in water, dimethyl sulfoxide (DMSO) was selected as vehicle, which was demonstrated to be suitable in the V79 in vitro cytogenetic assay and for which historical control data were available.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Remarks:
EMS = ethylmethanesulphonate (500 µg/mL); CPP = cyclophosphamide (0.5 µg/mL)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 14 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 2-3 hours

SPINDLE INHIBITOR: colcemide (10 µg/mL)
STAIN: 7.5% (v/v) Giemsa/Titrisol solution pH 7.2

NUMBER OF REPLICATIONS:
All cultures were prepared in duplicate.

NUMBER OF CELLS EVALUATED:
A sample of 100 metaphases for each vehicle control culture was analysed for chromosomal aberrations. Only 50 metaphases per culture in all dose groups and positive control groups were scored due to clearly increased aberration rates.
As a rule, the first 100 consecutive well-spread metaphases of each culture were analysed for structural chromosome aberrations on coded slides for all test groups. Only metaphases containing 20–22 chromosomes were counted. In the case of clearly increased aberration rates, the number of metaphases to be analysed for this test group can be reduced to at least 50 metaphases per culture.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (based on 1000 cells/culture) and cell counts

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Other: aneuploidy, cell morphology, pH value, osmolarity, solubility
Evaluation criteria:
ACCEPTANCE CRITERIA
The V79 in vitro cytogenetic assay was considered valid if the following criteria were met:
- The quality of the slides must allow the identification and evaluation of a sufficient number of analysable metaphases.
- The numbers of cells with structural/numerical aberrations in the negative control had to be within the range of the historical negative control data.
- The positive control substances both with and without S9 mix had to induce a distinct increase of structural chromosome aberrations.

ASSESSMENT CRITERIA
The test substance was considered as “positive” if the following criteria were met:
- A statistically significant, dose-related and reproducible increase in the number of cells with structural chromosome aberrations (excl. gaps).
- The number of aberrant cells (excl. gaps) exceeded both the concurrent negative/vehicle control value and the historical negative control data range.

A test substance generally was considered as “negative” if the following criteria were met:
- The number of cells with structural aberrations (excl. gaps) in the dose groups was not statistically significant increased above the concurrent negative/vehicle control value and was within the historical negative control data range.
Statistics:
The proportion of metaphases with structural aberrations was calculated for each group. A comparison of each dose group with the negative control group was carried out using Fisher's exact test for the hypothesis of equal proportions. This test was Bonferroni-Holm corrected versus the dose groups separately for each time and was performed one-sided.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
Results of the second experiment
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity indicated by clearly reduced cell numbers or mitotic rates was observed at least at the highest applied test substance concentration in all experimental parts of this study.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The test substance caused a clear, statistically significant and biologically relevant increase in the number of structurally aberrant metaphases incl. and excl. gaps either without S9 mix or after adding a metabolizing system.
In the 2nd experiment all concentrations (31.25 - 125 μg/mL without S9 mix or 62.5 – 250 μg/mL with S9 mix) scored for cytogenetic damage showed aberration rates (20.0 – 49.0% aberrant metaphases, exclusive gaps) clearly exceeding either the concurrent vehicle control values or the historical negative control data range (0.0% - 5.5% aberrant metaphases, exclusive gaps). Additionally, high proportions of exchanges and multiple aberrant metaphases were found in all treated test groups.
With regard to these distinct findings no confirmatory experiment was performed.
No increase in the frequency of cells containing numerical chromosome aberrations was demonstrated in the absence and the presence of metabolic activation.

The test doses of the first experiment were nor scorable due to technical reasons.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolarity: osmolarity and pH values were not relevant influenced by test substance treatment.
- Precipitation: test substance precipitation in culture medium at the end of exposure period was observed from 125 μg/mL onward in the 1st experiment and from 250 μg/mL onward in the 2nd experiment.

RANGE-FINDING/SCREENING STUDIES:
In a pretest for toxicity based on the molecular weight of the test substance, 1900 μg/mL (approx. 10 mM) of test item was used as top concentration. The cells were prepared at a sampling time of 18 hours (about 1.5-fold cell cycle time) after 4 and 18 hours exposure time without S9 mix and after 4 hours exposure time with S9 mix.
As indication of test substance toxicity cell count and cell attachment (morphology) were determined for dose selection. Various additional parameters were checked or determined for all or at least some selected doses (i.e., pH, solubility).
In the pretest the pH values were not relevant influenced by the addition of the test substance preparation to the culture medium at the concentrations tested. In addition, no test substance precipitation in the vehicle DMSO was observed up to the highest required concentration of 1900 μg/mL. In culture medium test substance precipitation 4 hours after start of treatment occurred from 237.5 μg/mL onward in the absence of S9 mix and from 118.75 μg/mL onward in the presence of S9 mix. After 18 hours continuous treatment in the absence of S9 mix precipitation of the test substance in culture medium occurred at 118.75 μg/mL and above at the end of exposure period.
After 4 hours treatment in the absence of S9 mix cytotoxicity indicated by reduced cell numbers of about or below 50% of control was observed at 237.5 μg/mL and above. In addition, in the presence of S9 mix clearly reduced cell numbers were observed after treatment with 475 μg/mL and above. Besides, in the pretest with 18 hours continuous treatment in the absence of S9 mix, the cell numbers were clearly reduced after treatment with 475 μg/mL.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the 2nd experiment after 4 hours treatment at 18 hours preparation interval a clear suppression of the mitotic activity was observed at 250 μg/mL in the presence of S9 mix. In the experimental part without S9 mix the concentrations scored showed no clear cytotoxicity.
However, the highest applied concentrations in both parts of the 2nd experiment showing clearly reduced mitotic indices were not scorable for cytogenetic damage due to low metaphase numbers and/or poor metaphase quality.
In addition, a relevant growth inhibition indicated by cell numbers of below 50% of control was observed in both experiments in the absence and presence of S9 mix at the highest tested concentrations each. In detail, in the 2nd experiment cell numbers below 50% of control were obtained at 62.5 and 250 μg/mL in the absence of metabolic activation and at 1000 μg/mL in the presence of metabolic activation.
Cell attachment at the end of exposure was not influenced at any dose tested for structural chromosomal aberrations under all conditions.

Table 1: Summary table - experimental parts without and with S9 mix

Exp.

Schedule

Exposure/ preparation

period

Test groups

S9 mix

P

Genotoxicity

Cytotoxicity*

Aberrant cells [%]

Polyploid cells

[%]

Cell number

[%]

Mitotic index

[%]

incl.

gaps#

excl.

gaps#

with

exchanges

2

4/18 hrs

Vehicle control(1)

-

n.d.

4.5

3.0

1.0

0.0

100.0

100.0

7.81 µg/mL

-

-

n.d.

n.d.

n.d.

n.d.

80.9

n.d.

15.63 µg/mL

-

-

n.d.

n.d.

n.d.

n.d.

64.0

n.d.

31.25 µg/mL

-

-

32.0(s)

32.0(s)

29.0(s)

0.0

66.2

66.3

62.50 µg/mL

-

-

29.0(s)

29.0(s)

24.0(s)

0.0

49.0

69.2

125.00 µg/mL

-

-

32.0(s)

29.0(s)

25.0(s)

0.0

58.5

66.3

250.00 µg/mL

-

+

n.s.

n.s.

n.s.

n.s.

38.9

n.s.

Positive control(2x)

-

n.d.

28.0(s)

26.0(s)

12.0(s)

0.0

n.t.

69.2

2

4/18 hrs

Vehicle control(1)

-

n.d.

6.0

2.0

0.0

0.0

100.0

100.0

31.25 µg/mL

-

-

n.d.

n.d.

n.d.

n.d.

103.1

n.d.

62.50 µg/mL(x)

-

-

22.0(s)

20.0(s)

17.0(s)

0.0

88.5

110.1

125.00 µg/mL(x)

-

-

28.0(s)

27.0(s)

25.0(s)

0.0

57.2

102.9

250.00 µg/mL(x)

-

-

49.0(s)

49.0(s)

36.0(s)

0.0

68.0

23.7

500.00 µg/mL

-

-

n.s.

n.s.

n.s.

n.s.

64.2

n.s.

1000.00 µg/mL

-

+

n.s.

n.s.

n.s.

n.s.

45.9

n.s.

Positive control(3x)

-

n.d.

29.0(s)

29.0(s)

19.0(s)

0.0

n.t.

61.9

P = Precipitation occurred at the end of exposure period

= Relative values compared with the respective vehicle control

# = Inclusive cells carrying exchanges

(x) = Evaluation of a sample of 100 metaphase only due to strong clastogenicity

n.d. = not determined

n.s. = Not scorable due to poor metaphase quality and/or strong cytotoxicity

n.t. = Not tested

(s) = Aberration frequency statistically significant higher than corresponding control values

(1) = DMSO 1% (v/v)

(2) = EMS 500 μg/mL

(3) = CPP 0.5 μg/mL

Conclusions:
Interpretation of results: positive with and without metabolic activation

Under the experimental conditions applied, the test substance was a chromosome-damaging (clastogenic) substance under in vitro conditions using
V79 cells in the absence and the presence of metabolic activation.
Executive summary:

The objective of this study was to evaluate the potential of the test item to induce structural chromosome aberrations (clastogenic activity) and/or changes in the number of chromosomes (aneugenic activity) in Chinese hamster lung fibroblast V79 cells in vitro, according to the OECD guideline 473 and in compliance with GLP.

 

The test item, suspended in dimethyl sulfoxide (DMSO), was tested at concentrations up to 1000 µg/mL in two independent experiments, both with and without a liver metabolizing system (S9 mix). Lung fibroblast cultures were exposed to the test item or the vehicle control for 4 hours (± S9 mix). Following exposure to the test item or vehicle control, the cultures were incubated in fresh medium at +37°C until harvest. Harvest times were 18 hours after the beginning of treatment. About two or three hours before harvest, each culture was treated with colcemide solution (10 µg/mL) to block cells at the metaphase-stage of mitosis. The cells were then stained with 7.5% (v/v) Giemsa/Titrisol solution pH 7.2. The cytotoxicity was indicated by the reduction of mitotic index (MI).

 

The frequencies of cells with structural chromosome aberrations of the vehicle and positive controls (ethylmethylsulphonate and cyclophosphamide) were as specified in acceptance criteria. The study was therefore considered to be valid.

 

The slides of the first experiment either in the absence or presence of metabolic activation were not scorable due to technical reasons. Therefore, a repeat experiment, designated 2nd experiment, was performed.

 

In the second experiment, the test substance caused a clear, statistically significant and biologically relevant increase in the number of structurally aberrant metaphases incl. and excl. gaps either without S9 mix or after adding a metabolizing system. No increase in the frequency of cells containing numerical chromosome aberrations was demonstrated. Cytotoxicity indicated by clearly reduced cell numbers or mitotic rates was observed at least at the highest applied test substance concentration in all experimental parts of this study.

 

Under the experimental conditions described, the test substance was considered to have a chromosome-damaging (clastogenic) effect under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Published Ames test; guideline-comparable study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
Conventional plate incorporation or pre-incubation methods.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
The target was histidine dependence in S.typhimurium.
Species / strain / cell type:
other: TA97, TA98, TA100, TA102, TA104 and TA1535
Metabolic activation:
with and without
Metabolic activation system:
NADPH-fortified postmitochondrial fraction (S9) from liver homogenate of Aroclor 1254-induced rats
Test concentrations with justification for top dose:
The dose range tested was 20-1000 ug/plate, with and without S-9
Vehicle / solvent:
No data
Untreated negative controls:
yes
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Details on test system and experimental conditions:
Standard plate incorporation and pre-incubation stages used in the standard methods devoloped by Ames et al.
Evaluation criteria:
significant increase in revertant colony numbers
Statistics:
No data
Key result
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
other: TA104
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
4,4'-dihydroxybiphenyl tested over the range 20-1000 µg/plate was negative for mutagenicity in all six tester strains with or without metabolic activation.

4,4'-dihydroxybiphenyl tested over the range 20-1000 µg/plate was negative for mutagenicity in all six tester strains with or without metabolic activation.

Conclusions:
Interpretation of results: negative

No evidence of mutagenic potential was found for 4,4-dihydroxybiphenyl when tested in six strains of S. typhimurium. No increase in revertant colonies when tested with or without metabolic activation.
Executive summary:

4,4-dihydroxybiphenyl was tested in six strains of S.typhimurium. The methods were based on those of Ames et al and included assays with or without metabolic activation. No evidence of mutagenic potential was found for 4,4-dihydroxybiphenyl when tested in six strains of S.typhimurium. No increase in revertant colonies when tested with or without metabolic activation.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Not stated
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Modern study conducted in accordance with OECD TG 471
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Analytical purity:99.96%
Target gene:
Bacterial reverse mutation assays use amino-acid requiring strains of Salmonella typhimurium to detect point mutations (substitution, addition or deletion of one or a few DNA base pairs). Bacterial reversion assays detect mutations which functionally reverse mutations present in the tester strains and restore the capability to synthesise an essential amino acid. The purpose of the reverse mutation assay is to establish the potential of the test item to induce gene mutations in bacteria (S. typhimurium) .

The S. typhimurium histidine (his) reversion system measures his- ® his+ reversions. The S. typhimurium strains are constructed to differentiate between base pair (TA 100, TA 1535, TA 102) and frameshift (TA 98, TA 1537) mutations.
These assays directly measure heritable DNA mutations of a type which is associated with adverse effects. Point mutations are the cause of many human
genetic diseases and there is substantial evidence that somatic cell point mutations in oncogens and tumour suppressor genes are involved in cancer in humans and experimental systems.
The tester strains have several features that make them more sensitive for the detection of mutations. The specificity of the strains can provide useful information on the types of mutations that are induced by mutagenic agents.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
other: E. coli strain is defined as E. coli WP2 uvrA
Metabolic activation:
with and without
Metabolic activation system:
S-9
Test concentrations with justification for top dose:
Experiment I without S-9: 0, 78.1, 156, 313, 625, 1250, 2500 and 5000 ug/plate
Experiment I with S-9: 0, 78.1, 156, 313, 625, 1250, 2500 and 5000 ug/plate
Experiment II without S-9: 0, 78.1, 156, 313, 625, 1250, 2500 and 5000 ug/plate
Experiment II with S-9: 0, 78.1, 156, 313, 625, 1250, 2500 and 5000 ug/plate
Vehicle / solvent:
dimethyl sulphoxide (DMSO)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
furylfuramide
Remarks:
for strains TA100, TA98 and WP2 uvrA without S-9 Migrated to IUCLID6: AF2: 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
for TA1535 without S-9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
for strain TA1537 without S-9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2AA: 2-aminoanthracene
Remarks:
for all five strains, with S-9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

S-9 obtained from rat liver induced with phenobarbital and 5,6-benoflavone

triplicates plates prepared for each test concentration (only one prepared for the cytotoxicity tests)

each assay was replicated.
Evaluation criteria:
According to criteria set out in OECD Test Guidelines No 471 and Guidelines for Screening Mutagenicity Testing of Chemicals (Chemical Substances Control Law of Japan)
Statistics:
No details
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Growth inhibition observed at 1250 ug/plate in TA100 and at 2500 ug/plate in TA1535, TA98 and TA1537 with and without S-9.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

4,4'-biphenyldiol did not induce gene mutations in any of the tester strains, Salmonella typhimurium TA100, TA98, TA1535 or TA1537 or E.coli WP2 uvrA, with or without metabolic activation. Growth inhibition was observed at 1250 ug/plate and above in TA100 and at 2500 ug/plate and above in strains TA1535, TA98 and TA1537 with and without metabolic activation

[µg/plate]

Experiment 1

TA100

TA1535

WP2uvrA

TA98

TA1537

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

0

142

160

9

10

21

34

25

40

7

14

78.1

155

160

NT

NT

NT

NT

NT

NT

NT

NT

156

141

186

9

9

24

28

23

44

8

12

313

134

162

6

6

20

226

26

41

8

9

625

125

129

6

8

23

19

24

44

5

9

1250

83

123

5

4

18

17

22

31

7

8

2500

0

13

0

2

10

8

0

4

3

2

5000

NT

NT

0

0

9

6

0

0

0

0

+control

602

821

627

338

178

796

380

441

226

268

 

Experiment 2

0

118

128

8

8

41

46

23

40

12

13

78.1

112

165

NT

NT

NT

NT

NT

NT

NT

NT

156

114

160

9

11

44

42

27

47

7

12

313

104

151

9

10

39

44

24

37

8

12

625

78

119

6

9

43

36

21

35

9

11

1250

45

97

4

6

34

33

24

32

6

11

2500

0

2

0

2

33

29

2

8

1

2

5000

NT

NT

0

0

29

29

0

0

0

0

+control

431

440

601

345

176

777

422

434

3313

253

NT: Not tested

Conclusions:
Interpretation of results: negative

No evidence of mutagenicity was seen under the conditions of this assay.
Executive summary:

In a standard bacterial reverse mutation assay 4,4'-biphenyldiol did not induce gene mutations in any of the tester strains, Salmonella typhimurium TA100, TA98, TA1535 or TA1537 or E.coli WP2 uvrA, with or without metabolic activation.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other:
Remarks:
Only four tester strains were used rather than five as required by test guidelines.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
The methods used were in accordance with the standard bacterial reverse mutation test described by Ames et al.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
Point mutations at GC base pairs
Species / strain / cell type:
other: TA100, TA98, TA97 and TA102
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Microsomes from PB- or 3-MC induced rat liver and a NADPH-generating system
Test concentrations with justification for top dose:
No details are given in the paper relating to test concentrations used in the Salmonella reverse mutation assays. The assays were conducted to compare results with an embryotoxicity assay in S.granularis (sea urchin). The concentrations used in that assay were in the 10-6 to 10-5 range since 10-7 was ineffective and 10-4 proved to be embryotoxic. It is unclear whether the same concentrations were used in the Salmonella assay.
Vehicle / solvent:
DMSO, the solvent level was 0.01%
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
not specified
Positive control substance:
not specified
Remarks:
The paper indicates the methods of Ames were followed in this assay using pre-incubation and plate incorporation and presumably the relevant positive and negative controls, but no details of either are presented.
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation and preincubation replicates but no details were presented in the publication

DURATION
- Preincubation period: no data
- Exposure duration: no data
- Expression time (cells in growth medium): no data
- Selection time (if incubation with a selection agent): no data
- Fixation time (start of exposure up to fixation or harvest of cells): no data

Salmonella mutagenicity assays were conducted according to Ames standard methods including both plate incorporation and liquid incubation. The tester strains were TA100, TA98, TA97 and TA102. Duplicate assays were completed, with and without metabolic activation. The bacteria were routinely checked for their spontaneous reversion rate and for the response to known mutagens.

The salmonella assay was conducted for comparative purposes to determine whether diphenyl and diphenyl ether and their hydroxy derivatives had mutagenesis and embryogenesis effects in prokaryotic and eukaryotic organisms.

No additional details provided for the Salmonella tests.
Evaluation criteria:
No details provided. The assay was conducted to standard Ames methods and it can be presumed the evaluation was in accordance with Ames standard methods also.
Statistics:
No details
Key result
Species / strain:
other: TA100, TA98, TA97 and TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
Diphenyl, diphenyl ether and their hydroxy derivatives including 4,4'-dihydroxydiphenyl, gave negative results in all of the reverse mutation assays in all tester strains irrespective of the plate incorporation or liquid incubation methods used, with or without metabolic activation.
Conclusions:
Interpretation of results: negative with or without metabolic activation

Negative results in all tester strains for 4,4'-dihydroxydiphenyl, with or without metabolic activation.
Executive summary:

In a standard Ames test, using only four tester starins, TA100, TA98, TA97 and TA102, negative results were obtained in all tester strains for 4,4'-dihydroxydiphenyl, with or without metabolic activation.

Endpoint:
in vitro DNA damage and/or repair study
Remarks:
Type of genotoxicity: other: Sister Chromatid Exchange (SCE)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
Results from a published paper
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)
GLP compliance:
no
Type of assay:
sister chromatid exchange assay in mammalian cells
Target gene:
Induction of sister chromatid exchange
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
Human peripheral T-lymphocytes obtained from one adult male
Additional strain / cell type characteristics:
other: Primary culture
Metabolic activation:
not applicable
Test concentrations with justification for top dose:
0.1-500 uM range
Vehicle / solvent:
Dissolved in DMSO and diluted with RPMI 1640 to give a final concentration of 0.33% DMSO
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
RPMI 1640
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Benzene
Details on test system and experimental conditions:
Heparinised whole blood was obtained from a single healthy adult male donor. The whole blood was processed on Ficoll-Paque density gradients and mononuclear lymphocyte cultures were prepared. Two to four cultures per treatment were prepared by inoculating 10E6 MNL in 1.9 ml of complete medium (RPMI, heat inactivated foetal bovine serum, penicillin, streptomycin sulphate and L-gutamine). T-lymphocytes were stimulated by addition of concanavalin and the cultures were incubated at 37°C for 24 h. 5-Bromo-2'deoxyuridine was added after 24 hours and the test materials then added to the cultures at various concentrations (for 4,4'-biphenol the concentration range was 0.1 to 500 µM). Following 4 hour treatment with demecolcine the cultures were harvested at 72 hours. The slides were stained by a modified fluorescence plus Giemsa technique.
Evaluation criteria:
Fifty second division metaphases, 200 consecutive metaphases and 2000 nuclei were analysed from each treatment for SCE frequency, cell cycle kinetics and mitotic index
Statistics:
All cytogenetic data were analysed for normality and analysed using one-way analysis of variance. A one tailed Dunnett's multiple range test was used to compare SCE frequency.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
not applicable
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
4,4'-biphenol was found to be significantly different from the concurrent solvent control (p<0.05) for SCE, mitotic index and cell cycle kinetics. 4,4'-biphenol caused a minimal but statistically significant increase in SCE that showed no concentration dependency. There was also evidence of mitotic inhibition and significant cell cycle delay caused by the biphenol derivatives.

Chemical

Concentration

(mm)

SCEs/metaphase

Mitotic index (%)

Cell cycle kinetics (%)

First division

Second

division

Third division

Fourth

division

DMSO (vehicle control)

 

9.27±0.48

4.30 ±1.33

17.0 ±4.9

42.0 ± 5.7

33.0 ± 7.1

8.0 ± 4.3

4,4'-Biphenol

 

 

0.1

9.46 ± 0.65

3.55 ±1.20

28.0 ±7.1

36.0 ±4.2

35.5 ±12.0

0.5 ± 0.7

0.5

9.22 ± 0.59

3.45 ± 0.21

22.5 ±0.7

31.5 ±2.1

42.0 ± 7.1

4.0 ± 5.7

5.0

11.62 ±0.72ab

2.45 ± 0.12a

38.5 ±6.4a

47.0 ± 2.9

13.0 ±6.7

1.5 ±1.0

7.0

9.78 ± 0.08

1.40 ±0.00

44.5 ±12.0

40.0 ± 8.5

15.0 ±2.8

0.5 ± 0.7

10.0

10.46 ±0.59

2.00 ±0.00

57.5 ±3.5

40.5 ± 3.5

1.5 ±0.7

0.5 ± 0.7

50.0

Cytotoxic

[a]significantly different to solvent control value (P<0.05 ANOVA)

[b]significantly different to solvent control value (P<0.05 Dunnett's test)

Conclusions:
Interpretation of results: ambiguous

4,4'-biphenol caused a marginal but statisticallly significant increase in SCE frequency; however the magnitude of chnage was minimal, was without a clear association with concentration and was associated with cytotoxicity. The result of the study is therefore considered to be ambiguous; the endppoint assessed is also of unclear toxicological significance.
Executive summary:

Under the conditions of this assay, 4,4'-biphenol was shown to induce a marginal but statistically significant increase in SCE frequency in cultured human T-lymphocytes. (p<0.05 at a concentration of 5 µm and greater), inhibited mitotic activity and delayed cell cycle kinetics in this assay, although no concentration relationship was established. The result of the study is therefore considered to be ambiguous; the endppoint assessed is also of unclear toxicological significance.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Two in vivo studies of the induction of micronuclei in mouse bone marrow are available.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06-NOV-2014 to 14-SEP-2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This GLP-compliant study was conducted according to standardised guidelines (i.e., OECD 474, EU B.14 and EPA OTS 798.5395).
Reason / purpose:
exposure-related information
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH
- Age at study initiation: 5-8 weeks
- Weight at study initiation: 28.26 g (mean)
- Assigned to test groups randomly: yes, according to a randomization plan prepared with an appropriate computer program
- Fasting period before study: no data available
- Housing: single housing in polycarbonate cages, type II
- Diet: standardized pelleted feed (Maus/Ratte Haltung "GLP",Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water: muncipal water from bottles, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24°C
- Humidity: 30-70%
- Air changes: no data available
- Photoperiod: 12 hrs dark / 12 hrs light (light from 6 am to 6 pm; dark from 6 pm to 6 am)

IN-LIFE DATES: From 10-NOV-2014 to 11- NOV-2014 (all test substance concentrations, vehicle, positive control) and From 10-NOV-2014 to 12- NOV-2014 (highest test substance concentration, vehicle
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO and corn oil (final ratio 2:3)
- Justification for choice of solvent/vehicle: the choice was done based on preliminary solubility testing in which DMSO was the most suitable vehicle. It was the only one tested with which a homogeneous suspension was obtained at the highest required dose. Using the vehicles DMSO and corn oil subsequently a homogeneous suspension of the test substance was obtained. Therefore, a mixture of DMSO and corn oil (ratio 2:3) was selected as vehicle, which was demonstrated to be suitable in the mouse micronucleus test and for which historical control data were available.
- Concentration of test material in vehicle: 50, 100, and 200 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL
- Lot/batch no., purity: no data available
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
In the two lower dose groups the substance to be administered was dissolved in DMSO (125 mg/mL or 250 mg/mL) and then emulsified in corn oil (ratio 2:3, each). Besides, in the top dose a suspension in DMSO (500 mg/mL) was obtained which was subsequently suspended in corn oil (ratio 2:3).
To achieve homogeneity of the test substance in the vehicle, the test substance preparation of the high dose in DMSO was stirred with an ultraturrax and treated with ultrasonic waves. Then, it was shaken thoroughly to obtain a homogeneous suspension in corn oil. The preparations of the two lower dose groups were shaken thoroughly to obtain solutions in DMSO and emulsions in corn oil, each.
All test substance formulations were prepared immediately before administration.

For the determination of the test substance concentrations in the vehicle, 6 samples of each dose (including three retain samples) were taken from the test substance preparations after treatment of the last animal (approximately 1 hour) and then were kept deep-frozen. The determination of the concentrations in the vehicle, three samples per dose, was carried out by means of high performance liquid chromatography (HPLC). The homogeneity of the samples and the stability of the test substance in the vehicle were confirmed indirectly based on these data.
The concentration control analyses of all concentrations revealed that the values were in the expected range of the target concentrations, i.e., were in a range of 90%-110% of the nominal concentration, or above. Based on the recovery rates it had to be considered that the test substance was stable at room temperature in the vehicle DMSO/corn oil at least over a period of 40 minutes.


EXPOSURE OF TARGET CELLS
> To fulfill the recommendations of the current OECD Guideline No. 474 an additional bioavailability study (BASF - Project No.: 99M0276/13M370) was performed to demonstrate the occurrence of the test substance in the plasma samples and to prove the exposure of the bone marrow of the animals. Mice of both sexes were given orally 2000 mg/kg body weight of the test substance. Blood was taken from vena facialis 2 hours after administration (about 100 μL per animal). Four hours after administration, the animals were sacrificed by decapitation. gain, blood samples were taken (about 500 μL per animal). Blood was collected with EDTA, centrifuged for 2 minutes at 20000 x g at 4°C. The supernatant, plasma was kept frozen at about -80°C prior to analysis.
Definite test substance concentrations in the samples were determined.
> Results: No clinical signs of toxicity were observed until sacrifice. The plasma level was in a concentration range of 293.67 to 1113.34 ng/mL depending on the animal and time point. The bioavailability of the test substance in blood after oral administration was clearly demonstrated
For more details, see study described in the endpoint "Toxicokinetics", from BASF, study number 99M0276/13M370; author: Dr. M. Schulz.
Duration of treatment / exposure:
Single oral exposure
Frequency of treatment:
Single oral administration
Post exposure period:
Animals were sacrificed 24 and 48 hours after oral administration.
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 per group
Control animals:
other: DMSO/corn oil (ration 2:3)
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): routinely used in the experimental laboratory
- Route of administration: oral
- Doses / concentrations: 20 mg/kg or 2 mg/mL in deionized water
Tissues and cell types examined:
Bone marrow of the two femora
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The doses were selected in accordance with the requirements set forth in the test guidelines and based on the results of a preliminary range finding test. The pretest was performed following the method described for the main experiment. The test substance was administered by gavage (orally) to NMRI mice of both sexes (3/dose and sex). Then, the animals were examined for clinically evident signs of toxicity several times. About 48 hours after administration the surviving animals were sacrificed by isoflurane anaesthesia followed by cervical dislocation.
Based on the data of the pretest 2000 mg/kg bw was selected as the highest dose in the present cytogenetic study. 1000 mg/kg and 500 mg/kg body weight were administered as further doses.

TREATMENT AND SAMPLING TIMES:
Animals were sacrificed 24 and 48 hours after administration in the highest dose group and in the vehicle controls. In the medium and low dose groups and in the positive control group, the 24-hour sacrifice interval was only investigated.
All animals were anaesthetized with isoflurane. Subsequently, they were sacrificed either by decapitation in the course of the blood sampling or by cervical dislocation (only positive control group). Then, the two femora were prepared by dissection and all soft tissues were removed. After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube using a cannula filled with foetal calf serum (FCS with EDTA) which was pre-heated up to 37°C (about 2 mL/femur).

DETAILS OF SLIDE PREPARATION:
The slides were stained with eosin and methylene blue for about 5 minutes. After briefly rinsing in deionized water, the preparations were soaked in deionized water for about 2-3 minutes. Subsequently, the slides were stained with Giemsa solution for about 15 minutes. After rinsing twice in deionized water and clarifying in xylene, the preparations were mounted in Corbit-Balsam.

METHOD OF ANALYSIS:
In general, 2000 polychromatic erythrocytes (PCEs) were evaluated for the occurrence of micronuclei from each animal of every test group, so in total 10000 PCEs were scored per test group. The normocytes erythrocytes (i.e., normocytes; NCEs) with and without micronuclei occurring per 2000 polychromatic erythrocytes were also recorded. The following parameters were recorded:
- Number of PCEs
- Number of PCEs containing micronuclei
- Number of NCEs
- Number of NCEs containing micronuclei
- Ratio of PCEs to NCEs
- Number of small micronuclei (d < D/4) and of large micronuclei (d ≥ D/4) [d = diameter of micronucleus, D = cell diameter]

Slides were coded before microscopic analysis.
Evaluation criteria:
ACCEPTANCE CRITERIA:
The mouse micronucleus test was considered valid if the following criteria were met:
- The quality of the slides must allow the evaluation of a sufficient number of analysable cells; i.e., ≥ 2000 PCEs per animal and a clear differentiation between PCEs and NCEs.
- The ratio of PCEs/NCEs in the concurrent vehicle control animals had to be within the normal range for the animal strain selected.
- The number of cells containing micronuclei in vehicle control animals had to be within the range of the historical vehicle control data for PCEs.
- The positive control substance had to induce a distinct increase in the number of PCEs containing small and/or large micronuclei

ASSESSMENT CRITERIA:
A finding was considered positive if the following criteria were met:
- A statistically significant and dose-related increase in the number of PCEs containing micronuclei.
- The number of PCEs containing micronuclei had to exceed both the concurrent vehicle control value and the range of the historical vehicle control data.

A test substance was considered negative if the following criteria were met:
- The number of cells containing micronuclei in the dose groups was not statistically significant increased above the concurrent vehicle control value and was within the range of the historical vehicle control data.
Statistics:
The asymptotic U test according to MANN-WHITNEY (modified rank test according to WILCOXON) was carried out to clarify the question whether there were statistically significant differences between the untreated control group and the treated dose groups with regard to the micronucleus rate in polychromatic erythrocytes. The relative frequencies of cells containing micronuclei of each animal were used as a criterion for the rank determination for the U test. Statistical significances were identified as follows:
* p ≤ 0.05
** p ≤ 0.01
However, both biological relevance and statistical significance were considered together.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw
- Solubility: no data available
- Clinical signs of toxicity in test animals: the recommended highest oral dose of 2000 mg/kg bw was survived by all animals showing weak signs of toxicity. However, there were no distinct differences in clinical observations between male and female animals.
- Evidence of cytotoxicity in tissue analysed: not applicable (i.e., acute oral toxicity study)
- Rationale for exposure: according to OECD guideline 474
- Harvest times: About 48 hours after administration the surviving animals were sacrificed by isoflurane anaesthesia followed by cervical dislocation.

RESULTS OF DEFINITIVE STUDY (see in Table 1 below)
- Induction of micronuclei: the single oral administration of the vehicle DMSO/corn oil led to 1.3‰ PCEs containing micronuclei after the 24-hour sacrifice interval or to 1.8‰ after the 48-hour sacrifice interval, respectively.
After the single administration of the highest dose of 2000 mg/kg bw, 1.5‰ PCEs containing micronuclei were found after 24 and 48 hours, each. In the two lower dose groups, rates of micronuclei of 1.7‰ (1000 mg/kg group) and 1.2‰ (500 mg/kg group) were detected at a sacrifice interval of 24 hours in each case.
The positive control substance, cyclophosphamide, led to a statistically significant increase (17.8‰) in the number of PCEs containing exclusively small micronuclei, as expected.
The number of NCEs containing micronuclei did not differ to any appreciable extent in the vehicle control group or in the various dose groups at any of the
sacrifice intervals.
- Ratio of PCE/NCE: at 24-hour sacrifice interval a slight inhibition of erythropoiesis determined from the ratio of PCE/NCE was detected at all doses (500, 1000 and 2000 mg/kg bw) after test substance administration (87%, 89% and 87% of control, respectively).
- Statistical evaluation: there were thus no statistical significances or biologically relevant differences in the frequency of erythrocytes containing micronuclei either between the vehicle control groups and the three dose groups (500 mg/kg, 1000 mg/kg and 2000 mg/kg) or between the two sacrifice intervals (24 and 48 hours). The number of NCEs or PCEs containing small micronuclei (d < D/4) or large micronuclei (d ≥ D/4) did not deviate from the vehicle control values at any of the sacrifice intervals and was within the historical vehicle control data range
- Other: the administration of the test substance led to distinct clinical signs of toxicity in the top dose: piloerection, hunched posture and/or reduced general condition at several observations.

Table 1: Summary table – Induction of micronuclei in bone marrow cells

Test group

Sacrifice

interval

[hrs]

Animal

No.

Micronuclei in PCE

PCEs per

2000 erythrocytesc

Totala

[‰]

Large MNb

[‰]

Vehicle control

DMSO/corn oil

24

5

1.3

0.0

1437

Test substance

500 mg/kg bw

24

5

1.2

0.0

1254

Test substance

1000 mg/kg bw

24

5

1.7

0.1

1280

Test substance

2000 mg/kg bw

24

5

1.5

0.1

1256

Positive control

cyclophosphamide

20 mg/kg bw

24

5

17.8**

0.0

1430

Vehicle control

corn oil

48

5

1.8

0.0

1357

Test substance

2000 mg/kg bw

48

5

1.5

0.2

1301

PCE = polychromatic erythrocytes

NCE = normochromatic erythrocytes

bw = body weight

a = sum of small and large micronuclei

b = large micronuclei (indication for spindle poison effect)

c = calculated number of PCEs per 2000 erythrocytes (PCE + NCE) when scoring a sample of 10000 PCE per test group

* = p ≤ 0.05

** = p ≤ 0.01

Conclusions:
Under the experimental conditions applied, the test substance had no chromosome-damaging (clastogenic) effect nor did it lead to any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells of NMRI mice in vivo.
Executive summary:

The test substance was assessed for its potential to induce chromosomal damage (clastogenicity) or spindle poison effects (aneugenic activity) in NMRI mice using the micronucleus test method, according to OECD guideline 474 and in compliance with GLP.

 

The test substance, dissolved or suspended in DMSO and subsequently emulsified or suspended in corn oil, was administered once orally to male animals at dose levels of 500 mg/kg, 1000 mg/kg and 2000 mg/kg body weight (bw) in a volume of 10 mL/kg body weight in each case. The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration in the highest dose group of 2000 mg/kg body weight and in the vehicle controls. In the test groups of 1000 mg/kg and 500 mg/kg body weight and in the positive control group, the 24-hour sacrifice interval was investigated only. After staining of the preparations, 2000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 2000 polychromatic erythrocytes were also recorded.

 

As vehicle control, male mice were administered merely the vehicle, DMSO/corn oil (ratio 2:3), by the same route and in the same volume as the animals of the dose groups, which gave frequencies of micronucleated polychromatic erythrocytes within the historical vehicle control data range. The positive control substance cyclophosphamide led to the expected increase in the rate of polychromatic erythrocytes containing only small micronuclei.

 

A slight inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes (slightly below 90% of control, each) was detected after test substance administration at all doses at 24-hour sacrifice interval. According to the results of the present study, the single oral administration of the test item did not lead to any biologically relevant increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was close to the range of the concurrent vehicle controls in all dose groups and at all sacrifice intervals and within the range of the historical vehicle control data.

In an additional study, the exposure of the bone marrow was confirmed by the analysis of the test substance in plasma after oral dosing of animals (see study described in the section "Toxicokinetics", from BASF, study number 99M0276/13M370, author:   Dr. M. Schulz).

Thus, under the experimental conditions of this study, the test substance did not induce cytogenetic damage in bone marrow cells of NMRI mice in vivo.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Published, guideline-comparable study. However, Some important deficiencies in methodology and reporting have been detected: primarily, neither body weights nor details on the housing and feeding conditions were reported; no positive controls were included, and no details on bone marrow cell sample preparation were provided. Furthermore, only one dose of 10 mg/kg bw was tested, which did not represent the maximum tolerated dose (MTD), as required in OECD Guideline No. 474
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Principles of method if other than guideline:
Mouse bone marrow micronucleus assay
GLP compliance:
no
Type of assay:
micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
Three month old mice were supplied by the Angelini breeding unit in Rome. Pregnant mice were tested on the 13th day of gestation
Route of administration:
oral: gavage
Vehicle:
20% v/v ethanol
Details on exposure:
A single dose of p,p'-biphenol was administered orally by gavage at a dose level of 40 mg/kg bw. Four animals were killed at varying timepoints after exposure, foetal liver and adult bone marrow samples were collected and processed according to standard micronucleus test methods. Harvesting times for male mice were: 0, 6, 12, 15, 18, 24, 30, 36, 40, 48 hr post-treatment; for virgin females samples were collected at 0, 15, 18, 24, 30, 36, 40 hr post-treatment; for pregnant females 0, 15, 18, 24, 30, 36, 40 hr post-treatment and foetal samples were collected at 0, 9, 12, 15, 18, 21, 24, 27, 30 and 36 hr post-treatment.
Duration of treatment / exposure:
Single oral exposure. Mice were terminated at various timepoints, between 0 and 48 hours, following exposure.
Frequency of treatment:
Single oral administration
Post exposure period:
Four mice were terminated at various timepoint following exposure and foetal liver or adult bone marrow samples obtained
Dose / conc.:
40 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Groups of 40 males and up to 28 pregnant females
Control animals:
no
Positive control(s):
No data
Tissues and cell types examined:
Bone marrow analysis for pregnant adult females and foetal liver
Details of tissue and slide preparation:
Bone marrow smears prepared and analysed - 3000 polychromatic erythrocytes scored per animal and numbers of normochromatic cells were also recorded. 3000 liver cells from a foetal litter pool were similarly scored.
Evaluation criteria:
Not specified in the report
Statistics:
Significance levels of induced micronuclei assessed by Kastenbaum and Bowman tables. The PCE:NCE ratio was analysed by t-test after arc-sin transformation.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
p,p-biphenol was not active on micronucleus induction in maternal bone marrow but was found to be cellularly toxic at the tested dose level. High cell toxicity expressed as the PCE:NCE ratio, was observed in foetal cells.
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
not applicable
Additional information on results:
Micronucleus induction in dams by p,p'-biphenol was 1-3 fold that in controls. Toxicity assessed as a reduction in the PCE/NCE ratio was 30-60% for dams treated with p,p'-biphenol (for benzene there was no effect on the ratio). No foetal transplacental induction of micronuclei by p,p'-biphenol was apparent in liver cells. Toxicity assessed as a reduction in the PCE/NCE ratio was more than 60% for foetuses treated with p,p'-biphenol.
Conclusions:
Interpretation of results: negative
p,p'-biphenol was investigated to determine the potential for inducing micronuclei in bone marrow cells in male, virgin female and pregnant female mice. The same genotoxic effect was also investigated in foetal liver cells. The investigation gave a negative response for p,p'-biphenol, with no biologically significant induction of micronuclei in adult bone marrow or foetal liver cells.
Executive summary:

p,p'-biphenol, was investigated to determine the potential to induce bone marrow micronuclei in adult mice (treated dams) and also to look for a transplacental micronucleus effects in foetal liver cells. The test substance was administered by oral gavage to the dams (treated male mice and virgin mice were maintained as controls) and at varying points following treatment (from 0 to 40 hours post-treatment) groups of 4 mice were terminated and bone marrow samples collected from the adults and liver samples from the foetuses. 3000 polychromatic erythroctes were scored for each animal at each time point and also for each pooled foetal liver sample at each time point. NCEs were also counted and the PCE:NCE ratio determined as a measure of cellular toxicity. The investigation gave a negative response for p,p'-biphenol, with no biologically significant induction of micronuclei in adult bone marrow or foetal liver cells.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Studies in vitro

No evidence of mutagenicity is reported in an Ames test (Japanese NIHS, 2004) using S. typhimurium strains TA98, TA100, TA1535 and TA1537 and E. coli WP2 uvrA in the presence and absence of metabolic activation. Pagano et al (1988) also report a negative response in an Ames test using S. typhimurium strains TA97, TA98, TA100, and TA102 in the presence and absence of metabolic activation. Glatt et al (1989) similarly report a negative response in an Ames test using S. typhimurium strains TA97, TA98, TA100, TA102, TA104 and TA1535 in the presence and absence of metabolic activation.

A study of cytogenicity performed in Chinese Hamster Lung cells (Japanese NIHS, 2004) notes statistically significant increases in the frequency of structural and numeric changes in the presence and absence of metabolic activation. A positive response is also reported by Schulz et al. (2014) in test performed according to OECD Guideline 473 (In vitro Mammalian Chromosome Aberration Test) in Chinese hamster lung fibroblasts (V79) and in compliance of Godd Laboratory Practices (GLP).

Glatt et al (1989) report a negative response in a mammalian cell mutation assay performed in V79 cells (HPRT locus).

Finally, Erexson et al (1985) report an equivocal response in a Sister Chromatid Exchange (SCE) assay performed in cultured human peripheral lymphocytes; a marginal but statistically significant increase in SCE frequency was observed at concentrations also reducing mitotic activity and without a clear concentration-response relationship. The toxicological significance of this endpoint is unclear.

Studies in vivo

Ciranni et al (1988) report a negative response in a mouse bone marrow micronucleus assay. In this study animals were dose at 40 mg/kg bw in a single oral dose. p,p'-biphenol was investigated to determine the potential for inducing micronuclei in bone marrow cells in male, virgin female and pregnant female mice. Some important deficiencies in methodology and reporting have been detected: primarily, neither body weights nor details on the housing and feeding conditions were reported; no positive controls were included, and no details on bone marrow cell sample preparation were provided. Furthermore, only one dose of 40 mg/kg bw was tested, which did not represent the maximum tolerated dose (MTD), as required in the current OECD Guideline No. 474.

A negative response is also reported by Schulz et al. (2015) in a mouse bone marrow micronucleous assay conducted in compliance with GLP and according to OECD 474, EU B.14 and EPA OTS 798.5395 guidelines. In this study three dose groups were tested: 500, 1000, and 2000 mg/kg body weight (nominal concentration). This study confirms the results published by Ciranni et al.

Results therefore indicate that the clastogenic potential of biphenyl-4,4'-diol seen in vitro is not expressed in vivo.


Justification for selection of genetic toxicity endpoint
The battery of in vitro studies gives negative indications of gene mutation but some indication for the induction of chromosomal aberrations. No evidence of chromosomal aberration was seen in in vivo micronucleus studies. It is therefore concluded that biphenyl-4,4'-diol is not mutagenic.

Short description of key information:
Three Ames tests, two Chromosome Aberration tests, one mammalian cell mutation test in V79 cells and one study of Sister Chromatid Exchange are available in vitro. Two in vivo studies of the induction of micronuclei in mouse bone marrow are also available. Studies in vitro indicate some clastogenic activity but this is not seen in vivo.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The results of in vitro studies with biphenyl-4,4'-diol indicate some clastogenic activity; however this activity is not apparent in two higher tier studies in vivo. The available data therefore indicate that the substance is not mutagenic: no classification for genetic toxicity is therefore proposed under the CLP Regulation.