Sodium 2-biphenylate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Reference 2

Reference Type:

secondary source

Title:

O-Phenylphenol and its Sodium and Potassium Salts: A Toxicological Assessment

Author:

Bomhard, E. M. et al.

Year:

2002

Bibliographic source:

Crit. Rev. Toxicol. 32(6):551-626

Reference 3

Reference Type:

secondary source

Title:

Analysis of Genotoxicity and the Carcinogenic Mode of Action for Ortho-Phenylphenol

Author:

Brusick, D.

Year:

2005

Bibliographic source:

Environ Mol Mutagen 45:460-481

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(1984)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: New and Revised Health Effects Test Guidelines, October 1984 (U.S.) Environmental Protection Agency, Washington DC (PB 84-233295), HG - Gene Muta - Somatic Cells, October 1984

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Biphenyl-2-ol

EC Number:

201-993-5

EC Name:

Biphenyl-2-ol

Cas Number:

90-43-7

Molecular formula:

C12H10O

IUPAC Name:

2-phenylphenol

Test material form:

solid: crystalline

Details on test material:

- Name of test material (as cited in study report): Preventol o extra, o-Phenylphenol, 1,1'-Biphenyl-2-ol
- Physical state: white crystalline solid
- Analytical purity: 99.9%
- Purity test date: 4 Jul 1989, approved 18 May 1991
- Lot/batch No.: 430 501-00 Pa. 18

Specific details on test material used for the study:

- Storage condition of test material: refrigerator

Method

Target gene:

HPRT locus

Species / strain

Species / strain / cell type:

mammalian cell line, other: CHO-WB1

Details on mammalian cell type (if applicable):

- Type and identity of media: Ham's F12 medium supplemented with L-glutamine (1 mM), penicillin (50 U/mL), streptomycin (50 µg/mL) and FCS (final concentration 10%)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Co-factor supplemented microsomal S9 homogenate from Aroclor 1254 induced male Wistar rats

Test concentrations with justification for top dose:

-S9: 6.25, 12.5, 25.0, 50.0, 75.0 and 100.0 µg/mL
+S9: 12.5, 25.0, 50.0, 75.0, 100.0 and 115.0 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Remarks:

-S9: ethylmethanesulfonate 0.9 mg/mL; +S9: 7,12-dimethylbenzanthracene 20 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 5 h
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 14 days

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: 3 replicates

DETERMINATION OF CYTOTOXICITY
- Method: relative cloning efficiency

Evaluation criteria:

- An assay will be considered positive if a dose-dependent and reproducible increase in mutant frequency is observed. It is desirable to obtain this dose-relationship for at least 3 doses. The mutagenic response should be at least twice that of the negative controls. If a reproducible increase greater than two times the minimum criterion is observed for a single dose near the highest testable concentration, the test article is also considered mutagenic.
- An assay will be considered equivocal if there is a no dose-dependency but one or more doses induce a mutant frequency which is considered significant and/or is at least twice that of the negative controls.
- An assay will be considered negative if none of the doses tested (for a range of applied concentrations which extends to toxicity causing about 30% survival or less) induces a reproducible mutant frequency which is considered significant.

Statistics:

The number of mutations per 1 Mio cells will be governed by the Poisson distribution provided that the following assumptions are met, as in the case of mutation experiments such as the HGPRT-assay:
- The mean number of spontaneous mutations must be small relative to the maximum possible number of events per sampling units.
- An occurrence of the event must be independent from prior occurrences within the sampling unit.
A Poisson heterogeneity test is used to determine whether or not there are statistically significant increases in mutant frequency.
If the test is first taken as a global test with subsequent comparisons among less than s mean with s being the number of groups including tthe negative controls (provided the global test yielded a significant result), the type I error rate may be adjusted to account for the multiplicity tests.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: at 75 µg/mL and above (+/-S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at concentrations >250 µg/mL (preliminary dilution trial)

RANGE-FINDING/SCREENING STUDIES: Based on the results of a preliminary cytotoxicity test, concentrations of 6.25-100 µg/mL (-S9) and 12.5-115 µg/mL (+S9) were selected for the mutation tests.

COMPARISON WITH HISTORICAL CONTROL DATA:
Historical control data are available for the negative, vehicle and positive controls and are based on the results of 24 experiments performed from June 1988 to December 1989.
- Negative control: 8.0±7.3 (range 0.6-33.5) (-S9); 6.2±5.7 (range 0.6-26.3) (+S9)
- Vehicle control (DMSO): 7.4±7.3 (range 0.2-31.3) (-S9); 7.1±6.2 (range 0.6-25.3) (+S9)
- Positive cotrols: 289.4±156.0 (range 49.7-769.2) (-S9, ethylmethanesulfonate); 99.7±65.2 (range 20.2-441.4) (+S9, 7,12-dimethylbenzanthracene)
The results of the negative and vehicle controls, both with and without metabolic activation, are within the range of the historical control data. This is also true for the positive control in the presence of S9 mix. The positive control results without metabolic activation (Trial 1 and 3) exceed the historical control data; however, this is considered to have no impact on the reliability of the test.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The highest dose tested both with and without metabolic activation was too cytotoxic to be examined and was therefore not cloned. Only in Trial 1 with metabolic activation, this respective test concentration could be evaluated.

ADDITIONAL INFORMATION ON MUTAGENICITY:
Statistically significant increases were noted in few samples treated with the test item. However, these statistically significant increases were either observed in the presence of strong cytotoxicity or could not be confirmed by the duplicate treatment of the respective trial. In any case, there was no clear dose-relationship. Thus, the test item can be concluded to be not mutagenic to CHO-WB1 cells under the conditions of this test.

Any other information on results incl. tables

Table 1: Results of the Mammalian Cell Gene Mutation Assay - 5 h Exposure - Without Metabolic Activation

Concentration [µg/mL]	Survival to treatment [% of vehicle control]	Mutants Frequency x 1E-06	Survival to treatment [% of vehicle control]	Mutants Frequency x 1E-06	Survival to treatment [% of vehicle control]	Mutants Frequency x 1E-06
	Trial 1		Trial 2		Trial 3
Negative control #	96.8	11.3 6.3	107.3	2.5 3.8	80.3	4.3 1.5
Vehicle control ##	100.0	5.2 7.9	100.0	1.5 3.2	100.0	5.2 1.4
Positive control ###	15.9	855.2* 547.3*	11.2	511.8* 511.0*	6.5	819.1* 842.4*
6.25	85.0	6.5 8.0	79.7	6.5 0.0	78.8	6.7 4.1
12.5	84.5	7.1 9.8	76.6	3.7 2.3	97.1	4.0 7.7
25.0	83.6	3.5 7.2	37.8	13.2* 16.9*	67.5	3.3 9.4*
50.0	40.7	9.2 1.8	46.6	3.4 10.5*	87.6	4.3 2.1
75.0	0.6	23.4* 0.0	2.2	1.1 0.9	31.4	1.3 2.8
100.0	n.c.		n.c.		n.c.

# Negative control: culture medium

## Vehicle control: DMSO

### Positive control: ethylmethanesulfonate 0.9 mg/mL

n.c. = not cloned due to cytotoxicity

* Significant increase, p≤0.05

 

Table 2: Results of the Mammalian Cell Gene Mutation Assay - 5 h Exposure - With Metabolic Activation

Concentration [µg/mL]	Survival to treatment [% of vehicle control]	Mutants Frequency x 1E-06	Survival to treatment [% of vehicle control]	Mutants Frequency x 1E-06	Survival to treatment [% of vehicle control]	Mutants Frequency x 1E-06
	Trial 1		Trial 2		Trial 3
Negative control #	102.5	2.2 2.0	122.9	0.9 1.8	114.5	3.1 1.0
Vehicle control ##	100.0	2.1 2.3	100.0	1.8 1.7	100.0	1.7 2.1
Positive control ###	85.0	48.9* 25.1*	101.2	31.8* 22.2*	145.3	41.2* 49.8*
12.5	c		102.6	8.1* 3.0	150.4	2.9 4.5
25.0	107.0	8.8* 1.5	107.3	2.7 11.2*	159.8	0.7 1.8
50.0	111.3	2.5 4.2	125.7	7.8* 2.7	113.0	2.2 0.9
75.0	86.3	1.3 1.1	48.1	0.9 4.4	65.4	0.9 3.4
100.0	103.3	0.7 0.7	31.6	1.0 0.8*	18.1	1.9 0.0
115.0	53.5	7.1 2.2	n.c.		n.c.

# Negative control: culture medium

## Vehicle control: DMSO

### Positive control: 7,12-dimethylbenzanthracene 20 µg/mL

n.c. = not cloned due to cytotoxicity

c = contaminated

* Significant increase, p≤0.05

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative