Biphenyl-2-ol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 Jun - 26 Jul 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21 July 1997, corrected 26 June 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany

Type of assay:

bacterial reverse 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:

[1,1'-biphenyl]-2-ol

Method

Target gene:

his operon for S. typhimurium strains
trp operon for the E. coli strain

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9: Eurofins Munich, Germany
- Method of preparation of S9 mix: Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and ß-naphthoflavone (100 mg/kg bw) for three consecutive days by the oral route. The protein concentration in the S9 preparation was 34.4 mg/mL.The S9 mix was prepared according to Ames et al. (1973). 100 mM of ice-cold sodium-ortho-phosphate-buffer (pH 7.4) was added to the following pre-weighed sterilised reagents to give final concentrations in the S9 mix of 8 mM MgCL2, 33 mM KCI, 5 mM glucose-6-phosphate, and 4 mM NADP.
This solution was mixed with the liver 9000 x g supernatant fluid in the following proportion: co-factor solution 9.5 parts, and liver preparation 0.5 parts. The S9 mix substitution buffer was used in the study as a replacement for S9 mix, without metabolic activation (-S9). Phosphate-buffer (0.2 M) contains per litre of purified water: 0.2 M NaH2P04 x H20 (120 mL), and 0.2 M Na2HP04 (880 mL). The two solutions were mixed and the pH was adjusted to 7.4. Sterilisation was performed for 20 min at 121 °C in an autoclave. This 0.2 M phosphate-buffer was mixed with 0.15 M KCI solution (sterile) in the following proportion: 0.2 M phosphate-buffer: 9.5 parts, and 0.15 M KCI solution: 0.5 parts.
- Quality controls of S9: 2-aminoanthracene and benzo[a]pyrene were used as quality controls and, additionally, a sterility test was performed.

Test concentrations with justification for top dose:

Experiment I: 3.16, 10.0, 31.6, 100, 316, 1000, and 2500 µg/plate
Experiment II: 1.0, 3.16, 10.0, 31.6, 100, 316, 1000, and 2500 µg/plate
2500 µg/plate was selected as the maximum concentration based on the results of a preliminary range-finding study with the following concentrations: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate. No precipitation was noted up to the highest concentration in the pre-experiment. However, cytotoxicity as indicated by the absence of a background lawn was observed for the 2500 µg/plate dose, justifying it as the choice for top dose in the two main experiments.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.
- Justification for percentage of solvent in the final culture medium: not specified

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

solvent control: DMSO

True negative controls:

yes

Remarks:

A. dest.

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other:

Remarks:

a) -S9: TA 98 (10 µg/plate), TA 1537 (40 µg/plate) b) +S9: TA 98 (2.5 µg/plate), TA 100 (2.5 µg/plate), TA 1535 (2.5 µg/plate), TA 1537 (2.5 µg/plate), WP2 uvrA (10 µg/plate)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicates
- Number of independent experiments: 2 independent experiments

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: approximately 10^9 cells/mL
- Experiment I: 100 µL test substance was added in agar according to the plate incorporation method.
- Experiment II: 100 µL test substance was added in agar according to the pre-incubation method.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 1 h
- Exposure duration/duration of treatment: 48 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition: Cytotoxicity was detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately < 0.5 in relation to the solvent control.

Evaluation criteria:

The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows: if in tester strains TA98, TA100 and E. coli WP2 uvrA (pKM101) the number of reversions is at least twice as high, or if in tester strains TA1535 and TA1537 the number of reversions is at least three times higher as compared to the reversion rate of the solvent control.
According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary. A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Statistics:

not performed

Results and discussion

Test results

Test results 1

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: +/-S9: 1000 µg/plate and higher Experiment II: +/-S9: 316 µg/plate and higher

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Reduction in number of revertants down to mutation factor of <0.5 in Experiment II for 31.6 µg/plate (+S9): not biologically relevant due to lack of a dose-response relationship and lack of concomitant clearing of background lawn

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I: +/-S9: 1000 µg/plate and higher Experiment II: +/-S9: 316 µg/plate and higher

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I: +/-S9: 316 µg/plate Experiment II: +/-S9: 316 µg/plate and higher

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I: -S9: 100 µg/plate and higher +S9: 1000 µg/plate and higher Experiment II: +/-S9: 316 µg/plate and higher

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I: -S9: 316 µg/plate and higher +S9: 1000 µg/plate and higher Experiment II: +/-S9: 316 µg/plate and higher

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: not observed

RANGE-FINDING/SCREENING STUDIES:
A preliminary range-finding study with the following concentrations was conducted: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate. No precipitation was noted up to the highest concentration in the pre-experiment (with and without metabolic activation). However, cytotoxicity as indicated by the absence of a background lawn was observed for the 2500 µg/plate dose.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: All criteria of validity were met. The negative control plates with and without metabolic activation were within the historical control data range with the exception of tester strain TA1535, without metabolic activation in Experiment I and with metabolic activation in Experiment II. Slightly lower spontaneous reversion counts of 3 (control range without metabolic activation 5 - 34, with metabolic activation 4 - 37) were observed in one single plate in each experiment. Since the data were considered acceptable for addition to the laboratory historical database, the observed slight decrease was regarded as not biologically relevant and did not influence the validity of the results.
For further details on the results, please refer to the tables in the attached file "Result Tables".

HISTORICAL CONTROL DATA
For details on the historical control data, please refer to the tables in the attached file "Historical Control data".

Applicant's summary and conclusion

Conclusions:

During the described mutagenicity test and under the experimental conditions reported, the test substance did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, the test substance is considered to be non-mutagenic in this bacterial reverse mutation assay.