Sodium 2-biphenylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD 471): negative with and without metabolic activation (RA from 2-phenylphenol, CAS 90-43-7)

HPRT test (OECD 476): negative with and without metabolic activation (RA from 2-phenylphenol, CAS 90-43-7)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

refer to analogue justification provided in IUCLID section 13

Reason / purpose for cross-reference:

read-across source

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

Conclusions:

Negative results were determined in a HPRT test with the source substance (CAS 90-43-7) with and without metabolic activation in CHO-WB1 cells. Applying the RA approach, similar results were expected for the target substance (CAS 132-27-4).

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

refer to analogue justification provided in IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I and II: at 500 µg/plate and ≥ 250 µg/plate and with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I and II: at 500 µg/plate and ≥ 250 µg/plate and with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I and II: at 500 µg/plate and ≥ 250 µg/plate and with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I and II: at 500 µg/plate and ≥ 250 µg/plate and with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I and II: at 500 µg/plate and ≥ 250 µg/plate and with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

The source substance (CAS 90-43-7) did not exhibit mutagenic properties in bacterial strains with and without metabolic activation. Applying the RA-approach, similar results are expected for the target substance (CAS 132-27-4).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

DNA damage in vivo (Alkaline Comet Assay, similar to OECD 489): negative in kidney and liver cells of CD-1 mice (RA from 2-phenylphenol, CAS 90-43-7)
Micronucleus Test in vivo (similar to OECD 474): negative in bone marrow and in urinary bladder epithelium of Fischer 344 rats (RA from 2-phenylphenol, CAS 90-43-7)

Bone marrow chromosome aberration test in vivo (OECD 475): negative in bone marrow of ICL-ICR mice

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

(number of animals not given)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

not specified

GLP compliance:

no

Type of assay:

other: mammalian bone marrow chromosome aberration test

Species:

mouse

Strain:

ICL-ICR

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 7 weeks
- Diet: Basal diet (CE-2. CLEA Japan Inc.), ad libitum
- Water: Tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 25 ± 1
- Humidity (%): 55 ± 5
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 15/9

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Distilled water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was dissolved in distilled water and orally administered with stomach tube at a volume of 10 mL/kg bw.

Duration of treatment / exposure:

6, 24 and 48 h, respectively

Frequency of treatment:

Single treatment

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

600 mg/kg bw/day (nominal)

Dose / conc.:

1 200 mg/kg bw/day (nominal)

Control animals:

yes

Tissues and cell types examined:

Bone marrow cells

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION: Chromosome stainings were routinally made.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not applicable

Table 1: Frequency of Chromosome Aberrations in Mice

				Gap		Break
Duration of Treatment (h)	Dose Level (mg/kg bw)	Number of analysed cells	Total aberrations	C1 a)	C2 b)	C1	C2
6	1200	200	1	1	0	0	0
	600	200	0	0	0	0	0
	300	200	1	1	0	0	0
	0	200	0	1	0	0	0
24	1200	200	2	2	0	0	0
	600	200	0	0	0	0	0
	300	200	1	0	0	0	0
48	1200	200	2	2	0	0	0
	600	200	0	0	0	0	0
	300	200	0	0	0	0	0
	0	200	0	0	0	0	0

a) Chromatid Type

b) Chromosome Type

Reference 2

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

refer to analogue justification provided in IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

At 2000 mg/kg bw clinical signs of systemic toxicity was noted. 2/12 animals of that high dose died. No cytotoxicity was noted in hepatocytes and kidney cells of any of the animals of the treatment and control groups (positive and vehicle controls).

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

Under the conditions of this test, the appropriate analogue substance did not exhibit DNA-damaging properties in CD-1 mice. Applying the RA-approach similar results are expected for the target substance.

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

refer to analogue justification provided in IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

in bone marrow cells

Toxicity:

no effects

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not examined

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

in urinary bladder epithelial cells

Toxicity:

no effects

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not examined

Conclusions:

Based on the results of the conducted study with the appropriate analogue substance OPP, increased micronuclei formation in urinary bladder epithelial cells of male F344 rats was observed only at dietary doses of 8000 and 12,500 ppm, which were shown to produce cytotoxic effects in the target tissue. At the same time, bone marrow cells of animals treated with 8000 ppm OPP did not show increased micronuclei formation. No positive control group was included into the test to clarify whether the test item reaches the bone marrow. However, Bomhard, E.M. et al. (2002) report, that there are toxicokinetic data, allowing the conclusion that OPP, as well as ist sodium salt SOPP and their metabolites, reach the bone marrow in sufficient quantities. Thus, the test item OPP was concluded to be not clastogenic under the conditions of this test. Applying the RA-approach, similar results are expected for the target substance.

Bomhard, E. M. et al. (2002). Crit. Rev. Toxicol. 32(6):551-626.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Justification for read-across

An in vivo bone marrow chromosome aberration test and an unscheduled DNA synthesis test is available with sodium 2-biphenylate (CAS 132-27-4). In addition for the evaluation of the in vitro mutagenic potential, read-across from an appropriate structural analogue substance (2-phenylphenol, CAS 90-43-7) is conducted in accordance with Regulation (EC) No 1907/2006, Annex XI, 1.5 in order to fulfil the standard information requirements defined in Regulation (EC) No 1907/2006, Annex VII-VIII, 8.4. Common functional groups and structural similarities combined with similar toxicokinetic properties of the source and target substance are the basis of read-across. A detailed justification for the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).

In vitro mutagenicity/genotoxicity:

CAS 90-43-7

Genetic toxicity in bacteria (Ames)

Bacterial mutagenicity of the read-across substance 2-phenylphenol (CAS 90-43-7) was tested in a bacterial reverse mutation assay performed according to OECD guideline 471 (JBC, 1989). The assay was performed with a standard battery of Salmonella typhimurium tester strains including TA 1535, TA 1537, TA 98 and TA 100, and Escherichia coli WP2uvrA, with and without metabolic activation. The highest concentration of 500 µg/plate was chosen and tested in all bacterial strains based on the results of a range finding study. The test substance did not exhibit mutagenic properties in the absence or presence of metabolic activation. Toxicity indicated by reduced number of revertants was observed in all tester strains at 500 µg/plate without metabolic activation and ≥ 250 µg/plate with metabolic activation. The positive control substances induced a distinct increase in the number of revertants in all strains with and without metabolic activation thereby showing the validity of the assay. The solvent control was also shown to be valid.

Based on the results of the conducted study, 2-phenylphenol (CAS 90-43-7) is not considered to exhibit mutagenic properties in bacterial cells.

Genetic toxicity (mutagenicity) in mammalian cells

An in vitro mammalian cell gene mutation assay according to OECD guideline 476 and under GLP conditions was performed with the read-across substance 2-phenylphenol (CAS 90-43-7) in CHO-WB1 cells (Brendler, 1992). The cells were treated with the test substance for 5 h with and without metabolic activation (cofactor-supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male rats, treated with Aroclor 1254). 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. The test substance showed cytotoxic properties (measured as cloning efficiency) at 115 and 75 µg/mL with and without metabolic activation, respectively. 7,12-dimethylbenzanthracene and ethylmethanesulphonate were used as positive controls with and without S9 mix, respectively and caused a distinct increase of the mutation colonies. The results of the negative and vehicle controls, both with and without metabolic activation, are within the range of the historical control data. 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.

In vivo mutagenicity/genotoxicity

CAS 132-27-4

In vivo bone marrow chromosome aberration

An in vivo bone marrow chromosome aberration test was conducted similar to OECD 475 with the test substance 2-biphenylate (CAS 132-27-4) (Yoshida et al., 1979). In this study, male JCL-ICR mice were administered via gavage dose levels of 300, 600 and 1200 mg/kg bw. After 6, 24 and 48 h, the animals were sacrificed by decapitation and bone marrow cells were collected. Chromosome stainings were routinally made (not further specified). The presence of structural and numerical aberrations was examined with metaphases from first-division cell. No induction of chromosome aberration was observed at all dose levels and for all sampling times. Therefore, the test substance was concluded to be not clastogenic under the conditions of this test.

In vivo Unscheduled DNA Synthesis Test

In an available unscheduled DNA Synthesis Test sodium 2-biphenylate (CAS 132-27-4) was used as reference substance and the information given in this study report was taken into account for the evaluation of the genotoxic potential (Klein, 1986). No separate study summary is provided in the dossier. Sodium 2-biphenylate (CAS 132-27-4) was administered via gavage at a dose level of 0.1 mg/kg bw. The results give rise to an increase in UDS induction, while at the same time DNA repair remains unaffected. According to the study director, the demonstrated increase in the percentage of S-phase cells is more suggestive of an effect on tissue than of DNA damage.

CAS 90-43-7

In vivo mammalian alkaline comet assay

In the GLP compliant Comet assay OPP was tested in vivo in male CD1 mice (Brendler-Schwaab, S., 2000; Bomhard, E. M. et al., 2002 and Brusick, D., 2005). Ethylmethanesulfonate (EMS) served as a positive control. Control mice were given vehicle (olive oil). Mice received a single oral dose of 250 and 2000 mg/kg bw, respectively. For EMS, 400 mg/kg bw were used. Four mice were used per dose and time point. Hepatocytes and kidney cells were prepared 3, 8, and 24 h after administration. Cells were pelleted by centrifugation and embedded into agarose slides. Electrophoresis was performed after alkaline treatment. DNA in the gel was visualized by ethidium bromide staining. Evaluation of “comets” was performed using digital image analysis. Fifty cells per slide and two slides per animal were scored. The test is considered positive if an increase in tail length of ≥ 25% is observed. Mice treated with 2000 mg/kg bw OPP showed the following compound-related symptoms until sacrifice: apathy, semi-anaesthetized state, roughened fur, pallor, staggering gait, sternal recumbency, spasm, shivering, languor, wide-legged gait and slitted eyes. 2 of 12 treated males died during the test period, due to acute toxicity of 2000 mg/kg bw OPP. No cytotoxicity was noted in hepatocytes and kidney cells of any of the animals of the treatment and control groups (positive and vehicle controls). The positive control EMS caused a significant increase of the Comet tail length. No increases in Comet tail length were observed in hepatocytes and kidney cells in both OPP dose groups.

Mammalian erythrocyte micronucleus test

An in vivo micronucleus test is available for OPP (Balakrishnan, S. and Eastmond, D.A., 2006). Fisher 344 rats were daily treated with the test item at dietary doses of 2000, 4000, 8000, or 12,500 ppm, respectively, for 15 days. Animals were evaluated for micronuclei formation in urinary bladder epithelial cells. Cytotoxicity in the target tissue indicated as increased cell proliferation was examined by means of BrdU incorporation. For comparison, further animals were treated with 8000 ppm OPP and bone marrow cells were evaluated for OPP-induced micronuclei. Increased micronuclei formation in urinary bladder epithelial cells was observed only in male F344 rats dosed with 8000 and 12,000 ppm OPP, which were shown to produce cytotoxic effects in the target tissue. At the same time, bone marrow cells of animals treated with 8000 ppm OPP did not show increased micronuclei formation. No positive control group was included into the test to clarify whether the test item reaches the bone marrow. However, Bomhard, E.M. et al. (2002) report, that there are toxicokinetic data, allowing the conclusion that OPP, as well as its sodium salt Na OPP and their metabolites, reach the bone marrow in sufficient quantities. Thus, the test item was concluded to be not clastogenic under the conditions of this test.

Overall evaluation:

In their publications, Bomhard, E. M. et al. (2002) and Brusick, D. (2005) reviewed and extensively discussed the in vitro and in vivo genotoxicity data available for OPP, the sodium salt Na OPP and any of their enzymatic or non-enzymatic breakdown products. More than 130 studies are available to determine if OPP, Na OPP or its metabolites directly react with DNA to induce mutation, changes in chromosome structure or number, DNA repair, or nonspecific DNA damage including strand breakage or covalent binding. Genotoxicity data for OPP and Na OPP are not only numerous but heterogeneous, requiring weight-of-evidence methods to arrive at a conclusion regarding their genotoxic properties and potential. Evidence derived from the available studies leads to the conclusion that study results showing OPP/Na OPP directly interacting with DNA are equivocal. Clastogenicity was the most consistent type of genetic toxicity produced by OPP/Na OPP (and their breakdown products) and was consistently associated with other intracellular pre-neoplastic toxicity produced at super-threshold concentrations. The weight of evidence from the combined database supports the hypothesis that OPP/Na OPP-induced DNA damage is a threshold-dependent response associated with target tissue toxicity, most likely induced by their breakdown products phenylhydroquinone and phenylbenzoquinone. It is possible that this threshold-dependent clastogenicity could contribute to the carcinogenic mode of action for OPP or Na OPP (Brusick, D., 2005).

Reference

Bomhard, E. M. et al. (2002) O-Phenylphenol and its Sodium and Potassium Salts: A Toxicological Assessment. Crit. Rev. Toxicol. 32(6):551-626.

Brusick, D. (2005) Analysis of Genotoxicity and the Carcinogenic Mode of Action for Ortho-Phenylphenol. Environ Mol Mutagen. 45(5):460 -481.

Justification for classification or non-classification

The available data on genetic toxicity with the read-across substance 2-phenylphenol (CAS 90-43-7) and the registered substance sodium 2-biphenylate (CAS 132-27-4) do not meet the criteria for classification according to Regulation (EC) No 1272/2008.