Biphenyl-2-ol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria (OECD 471, Ames test): negative in S. typhimurium strains TA98, TA100, TA1535 and TA1537 and E. coli strain WP2uvrA with and without metabolic activation

Gene mutation in mammalian cells (OECD 476, HPRT test): negative in CHO-WB1 cells with and without metabolic activation

Cytogenicity in mammalian cells (OECD 487, Micronucleus test): negative in Chinese hamster V79 cells with and without metabolic activation

Link to relevant study records

Reference

Reference 1

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

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

Target gene:

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

Species / strain / cell type:

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

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

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

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

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

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

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

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".

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(1984)

Deviations:

no

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

Specific details on test material used for the study:

- Storage condition of test material: refrigerator

Target gene:

HPRT locus

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

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.

Species / strain:

mammalian cell line, other: CHO-WB1

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.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

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

Conclusions:

Interpretation of results (migrated information):
negative

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 Jun - 07 Oct 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

adopted 29 Jul 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

Not applicable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Chinese hamster lung fibroblasts (V79), ATCC, CCL-93
- Suitability of cells: cell type selected is listed as one of the recommended cell types in OECD guideline 487
- Absence of Mycoplasma contamination: confirmed

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: MEM (minimum essential medium) supplemented with 10% FBS (fetal bovine serum), penicillin/streptomycin solution, L-glutamine, amphotericin and HEPES; 5% carbon dioxide atmosphere (95% air); 37 °C
The treatment media for the short-exposure experiment did not contain FBS, whereas the media for the long-exposure experiment contained FBS and cytochalasin B.

Cytokinesis block (if used):

1.5 µg/mL cytochalasin B (CytoB)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: Cofactor supplemented post mitochondrial fraction (S9 mix)
- 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). An appropriate quantity of the S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. Cofactors were added to the S9 mix in 100 mM sodium-phopshate buffer (pH 7.4).
- Concentration or volume of S9 mix and S9 in the final culture medium: 5%
- 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:
- without and with metabolic activation: 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45 and 0.50 mM
Experiment II:
- without metabolic activation: 0.025, 0.050, 0.100, 0.125, 0.150, 0.175, 0.200, 0.225, 0.250 and 0.300 mM

The concentrations were chosen based on a pre-experiment conducted under identical conditions as described for main experiment, testing the following concentrations with and without S9 mix: 0.039, 0.078, 0.156, 0.313, 0.625, 1.25, 2.5, 5.0, 7.5 and 10 mM
The concentration of 10 mM was considered to be the highest test concentration used in this test system following the recommendation of the corresponding OECD testing guideline 487.

The following concentrations were selected for the microscopic analyses of micronuclei frequencies. The selection of the maximum concentration was based on cytotoxicity for all experimental conditions.
Experiment I with short-term exposure (4 h):
without metabolic activation: 0.15, 0.30 and 0.35 mM
with metabolic activation: 0.20, 0.35 and 0.40 mM
Experiment II with long-term exposure (24 h):
without metabolic activation: 0.050, 0.100 and 0.175 mM

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the cells and the S9 activity.
- Justification for percentage of solvent in the final culture medium: final concentration: 1% v/v DMSO; the concentration is acceptable according to the osmolality and pH value examinations performed.

Untreated negative controls:

yes

Remarks:

cell culture medium (MEM)

Negative solvent / vehicle controls:

yes

Remarks:

MEM with 1% DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

all PC dissolved in MEM

Positive control substance:

colchicine

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicates for each concentration except in the pre-experiment
- Number of independent experiments: 2, plus pre-experiment

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: ca. 50 000 cells per culture flask
- Test substance added in treatment medium (Exp. I) or complete medium (Exp. II)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 48 h attachment period
- Exposure duration/duration of treatment: Experiment I: The cells were incubated with the test item for 4 h in presence or absence of metabolic activation (short-term exposure). At the end of the incubation, the treatment medium was removed and the cells were washed twice with PBS. Subsequently, the cells were incubated in complete culture medium + 1.5 µg/mL cytochalasin B for 20 h at 37 °C.
Experiment II: After an attachment period of approx. 48 h the test item was added in complete culture medium. 1 h later 1.5 µg/mL cytochalasin B were added and the cells were incubated for 23 h at 37 °C (long-term exposure).

FOR MICRONUCLEUS ANALYSIS:
- Cytokinesis block method: 1.5 µg/mL cytochalasin B for the 24 h long-term exposure treatment
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): At the end of the cultivation, the complete culture medium was removed. Subsequently, cells were trypsinated and resuspended in about 9 mL complete culture medium. The cultures were transferred into tubes and incubated with hypotonic solution (0.4 % KCl) for some minutes at room temperature. After the treatment with the hypotonic solution the cells were fixed with methanol + glacial acetic acid (3+1). The cells were resuspended gently and the suspension was dropped onto clean glass slides. Consecutively, the cells were dried on a heating plate. Finally, the cells were stained with acridine orange solution.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): at least 2000 binucleated cells per concentration (1000 binucleated cells per slide)
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): Scoring was performed according to the criteria of Fenech (2000), i.e. clearly surrounded by a nuclear membrane, having an area of less than one-third of that of the main nucleus, being located within the cytoplasm of the cell and not linked to the main nucleus via nucleoplasmic bridges. Mononucleated and multinucleated cells and cells with more than six micronuclei were not considered.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cytokinesis block proliferation index (CBPI)
- Any supplementary information relevant to cytotoxicity: CBPI was determined in 500 cells per culture and cytotoxicity is expressed as % cytostasis, which indicates the inhibition of cell growth of treated cultures in comparison to control cultures. For more information please refer to "Any other information on materials and methods incl. tables".

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The frequency of micronucleated cells was reported as % micronucleated cells.

Evaluation criteria:

A test item is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative/solvent control
- the increase is concentration-related in at least one experimental condition when evaluated with an appropriate trend test
- any of the results are outside the distribution of the historical negative/solvent control data (e.g. Poisson-based 95% control limits).
When all of these criteria are met, the test item is considered able to induce chromosome breaks and/or gain or loss in this test system.
A test item is considered to be clearly negative if in all experimental conditions examined none of the criteria mentioned above are met.

Statistics:

The non-parametric Chi square Test was performed to verify the results in both experiments, confirming statistical significance as p < 0.05. The Chi square Test for trend was performed to test whether there is a concentration-related increase in the micronucleated cells frequency in the experimental conditions.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytostasis: Exp.I(-S9): ≤ 0.15 mM: ≤ 30%; 30 mM: 43%; 0.35 mM: 60% Exp.I(+S9): ≤ 0.20 mM: ≤ 30%; 35 mM: 38%; 0.40 mM: 67% Exp.II(-S9): ≤ 0.050 mM: ≤ 30%; 0.100 mM: 37%; 0.175 mM: 59%

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: a pH of 7.25 was maintained in both experiments
- Data on osmolality: Exp I: 482 mOsm/kg for the solvent control and 465 mOsm/kg for the test item; Exp II: 481 mOsm/kg for the solvent control and 480 mOsm/kg for the test item
- Precipitation and time of the determination: not observed for any concentration in any of the main experiments. Precipitation of the test item was noted at 2.5 mM and higher without and at 1.25 mM and higher with metabolic activation in the pre-experiment only.

RANGE-FINDING/SCREENING STUDIES:
The highest dose group evaluated in the pre-experiment was 0.313 mM without and 0.625 mM with metabolic activation. At higher concentrations no intact cells were found on the slides. The results of the pre-experiment are accessible in the attachment "result tables and figures".

STUDY RESULTS
- Cytotoxicity:
In experiment I without metabolic activation no increase of the cytostasis above 30% was noted up to 0.15 mM. At 0.30 mM a cytostasis of 43% and at 0.35 mM a cytostasis of 60% was noted.
In experiment I with metabolic activation no increase of the cytostasis above 30% was noted up to 0.20 mM. At 0.35 mM a cytostasis of 38% and at 0.40 mM a cytostasis of 67% was noted.
In experiment II without metabolic activation no increase of the cytostasis above 30% was noted up to 0.050 mM. At 0.100 mM a cytostasis of 37% and at 0.175 mM a cytostasis of 59% was observed. If cytotoxicity is observed, the highest concentration evaluated should not exceed the limit of 55% ± 5% cytotoxicity according to the OECD Guideline 487. Higher levels of cytotoxicity may induce chromosome damage as a secondary effect of cytotoxicity. The other concentrations evaluated should exhibit intermediate and little or no toxicity. However, OECD 487 does not define the limit for discriminating between cytotoxic and non-cytotoxic effects. According to laboratory experience this limit is a value of the relative cell growth of 70% compared to the negative/solvent control which corresponds to 30% of cytostasis.
- Clastogenicity / Aneugenicity:
Exp I: -S9: micronucleated cell frequency of the negative control (0.80%) was within the historical control limits of the negative control (0.30% - 1.44%) and the micronucleated cell frequency of the solvent control (0.65%) was within the historical control limits of the solvent control (0.34% – 1.49%). The mean values of micronucleated cells found after treatment with the test item were 0.75% (0.15 mM), 0.85% (0.30 mM) and 0.95% (0.35 mM). The numbers of micronucleated cells were within the historical control limits of the solvent control and did not show a biologically relevant increase compared to the concurrent solvent control.
+S9: micronucleated cell frequency of the negative control (0.75%) was within the historical control limits of the negative control (0.37% – 1.65%) and the micronucleated cell frequency of the solvent control (1.15%) was within the historical control limits of the solvent control (0.33% – 1.70%). The mean values of micronucleated cells found after treatment with the test item were 1.10% (0.20 mM), 1.05% (0.35 mM) and 1.35% (0.40 mM). The numbers of micronucleated cells were within the historical control limits of the solvent control and did not show a biologically relevant increase compared to the concurrent solvent control.
Exp II: -S9: micronucleated cell frequency of the negative control (0.70%) was within the historical control limits of the negative control (0.30% – 1.44%) and the micronucleated cell frequency of the solvent control (0.60%) was within the historical control limits of the solvent control (0.34% – 1.49%). The mean values of micronucleated cells found after treatment with the test item were 0.65% (0.050 mM), 0.55% (0.100 mM) and 0.73% (0.175 mM). The numbers of micronucleated cells were within the historical control limits of the solvent control and did not show a biologically relevant increase compared to the concurrent solvent control.
No statistically significant enhancement (p < 0.05) of cells with micronuclei was noted in the dose groups of the test item evaluated in experiment I and II with and without metabolic activation.
The Chi Square Test for trend was performed to test whether there is a concentration-related increase in the micronucleated cells frequency in the experimental conditions. No statistically significant increase in the frequency of micronucleated cells under the experimental conditions of the study was observed in experiment I and II.
For details on the results, please refer to the attachment "result tables and figures".

HISTORICAL CONTROL DATA
- Positive historical control data: provided
- Negative (solvent/vehicle) historical control data: provided
The historical control data is accessible in the attachment "historical control data".

Conclusions:

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce structural and/or numerical chromosomal damage in Chinese hamster V79 cells.
Therefore, the test substance is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in this in vitro Mammalian Cell Micronucleus Test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Cytogenicity in the bone marrow in vivo (OECD 474, Micronucleus assay): negative in the bone marrow of male Fisher 344 rats

Cytogenicity in the bone marrow in vivo (OECD 475, Chromosome aberration assay): negative in the bone marrow of male Wistar rats

Cytogenicity in the target organ urinary bladder in vivo (no guideline followed): positive for micronuclei induction in male Fisher 344 rats

DNA damage in somatic cells in vivo (OECD 489, Comet assay): negative in the liver and kidney of male CD-1 mice

DNA adduct formation in vivo (no guideline followed): negative for DNA adduct formation in epithelial urinary bladder cells after subchronic exposure in male CDF[F-344]/BR rats

DNA damage in germ cells in vivo (OECD 478, Dominant lethal assay): negative in germ cells of male SLC-C3H mice

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non-GLP, in principle similar to guideline, basic data given

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

(additional examination on bladder epithelial cells, data on animal number only given for test in urothelium, only one dose group for test in bone marrow , no individual animal data given, no justification for test item administration via diet)

GLP compliance:

not specified

Type of assay:

micronucleus assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): 2-Phenylphenol, OPP
- Analytical purity: >99%
- Supplier: Aldrich Chemical Co., Milwaukee, WI, USA

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratory, Raleigh, NC, USA
- Age at study initiation: 8 weeks
- Housing: in polycarbonate cages
- Diet: Lab diet 5001 (PMI Nutrition International, Richmond, IN, USA), ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 50
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

- Vehicle used: none

Duration of treatment / exposure:

15 days

Frequency of treatment:

daily

Post exposure period:

none

Remarks:

Doses / Concentrations:
8000 ppm
Basis:
nominal in diet
for micronucleus test in bone marrow cells

Remarks:

Doses / Concentrations:
2000, 4000, 8000, and 12,500 ppm
Basis:
nominal in diet
for micronucleus test in urinary bladder epithelial cells

Remarks:

Doses / Concentrations:
148±14, 320±28, 644±74, and 1114±150 mg/kg bw/day
Basis:
actual ingested
for micronucleus test in urinary bladder epithelial cells

No. of animals per sex per dose:

- 4 males per dose for the micronucleus test in urinary bladder epithelial cells
- no data given for the micronucleus test in bone marrow

Control animals:

yes

Tissues and cell types examined:

1. Tissue: bone marrow; cell type: bone marrow cells
2. Tissue: urinary bladder; cell type: bladder epithelial cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
- data from earlier Japanese studies and more recent data from Dow and Bayer Chemical Companies

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
Animals were fed for 15 days and sacrificed on Day 15. 24 h before sacrifice, animals were injected 50 mg/kg bw BrdU in DMSO/saline (2:1).

DETAILS OF SLIDE PREPARATION:
Bladder cells:
Bladder cells were removed by scraping the luminal epithelial cells. The cells were then subjected to hypotonic treatment (0.075 M KCl) for 15 min at room temperature prior to fixing with Carnoy's fixative (methanol:acetic acid 3:1). The fixed cells were dropped onto slides, allowed to air-dry, and stored at -20 °C unter nitrogen atmosphere.
The cells were stained with DAPI in phenylenediamine antifade mounting medium.

Bone marrow cells:
At sacrifice, the femoral bone marrow cells were harvested using standard procedures (Hayashi, M. et al., 1983). The slides were fixed in 100% methanol at -20 °C for 20 min, air dried and stored at -20 °C in a nitrogen atmosphere until use.
Bone marrow preparations were stained with acridine orange (acridine orange solution, 0.1% aqueous stock diluted 1:30 with Sorenson's phosphate buffer, pH 6.8) for 2 min and rinsed twice for 3 min each in phosphate buffer (pH 8.0).

METHOD OF ANALYSIS:
Analysis was conducted by means of fluorescence microscopy.
For each bladder 2000 nuclei were scored for micronuclei. A number of binucleated cells were seen. Due to the difficulty in distinguishing binucleated cells from two adjactent cells, each nucleus was scored individually.
Each slide with bone marrow cells was scored for the number of micronucleated polychromatic erythrocytes (MNPCE) per 2000 polychromatic erythrocytes (PCE). The ratio of PCE to normochromatic erythrocytes (NCE) was determined for each rat on the basis of the number of mature cells (NCE) encountered while accumulating 200 PCE.

OTHER:
Animals examined for OPP-induced micronuclei formation in urinary bladder epithelial cells received BrdU (50 mg/kg bw) 24 h prior to sacrifice. Using BrdU incorporation and the labelling index*, replicating cells were identified, indicating cytotoxicity in the target tissue.
* ratio of BUdR-positive nuclei to the total number of cells counted

Hayashi, M. et al. (1983). Mutat Res 120:241-247.

Statistics:

The frequencies of micronuclei, PCE/NCE ratios and MNPCE per treatment group were compared by analysis of variance with the Fischers protected least significant difference (PLSD) being used as a post-hoc test. Critical values were determined using a 0.05 probability of Type I error.

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

Sex:

male

Genotoxicity:

positive

Remarks:

in urinary bladder epithelial cells

Toxicity:

no effects

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not examined

Additional information on results:

MICRONUCLEI IN BLADDER EPITHELIAL CELLS
Based on the food consumption of the animals, the daily intake of OPP at dietary concentracions of 2000, 4000, 8000, and 12,500 ppm OPP was 148±14, 320±28, 644±74 and 1114±150 mg/kg bw/day.
- Systemic toxicity: Animals fed with 8000 ppm OPP and above lost weight, initially seen at day 2 of treatment. By Day 8 all animals gained weight at a similar rate regardless of treatment. At termination, mean body weights were comparable to the control.
- Genetic toxicity: The frequencies of micronuclei in bladder epithelial cells in controls was 0.19±0.02%, whereas the frequencies in the 8000 and 12,500 ppm OPP groups were significantly increased to 0.77±0.02% and 0.56±0.14%, respectively (n=4). The micronucleus frequencies in the rats fed with 8000 ppm OPP were modestly higher than those in the rats fed with with 12,500 ppm. Animals treated with 2000 and 4000 ppm OPP did not show an increase in micronuclei in the bladder epithelial cells over the control animals.
BrdU labelling revealed a 11-14 fold increase in cell proliferation in urinary bladder epithelial cells on animals treated with 8000 and 12,500 ppm OPP. No increase in in cell proliferation was noted in animals treated with 2000 and 4000 ppm OPP. The authors concluded that the increased cell proliferation noted at 8000 and 12,500 ppm is the result of OPP-induced superficial cytotoxicity of the urothelium with regenerative hyperplasia.

MICRONUCLEI IN BONE MARROW CELLS
- Induction of micronuclei (for Micronucleus assay): MNPCE frequencies in the bone marrow of control animals were 0.04±0.05% and did not differ significantly from those of animals fed 8000 ppm OPP (0.05±0.07%).
- Ratio of PCE/NCE: OPP treatment did not reduce the PCE:NCE ratio in bone marrow cells as compared to the control animals.

Conclusions:

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. Under the conditions of the test, the test item was considered positive for induction of micronuclei in the target organ urinary bladder and negative for clastogenicity in bone marrow in vivo.

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

Reference 2

Endpoint:

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

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study according to an generally accepted test procedure

Principles of method if other than guideline:

single cell gel/comet assay in rodents for detection of DNA damage, GLP study according to a test procedure scientifically acceptable

GLP compliance:

yes

Type of assay:

mammalian comet assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): Preventol O extra, OPP, o-Phenylphenol
- Physical state: solid
- Analytical purity: 99.8%
- Purity test date: 29 June 1998, approved until 12 June 2000
- Lot/batch No.: E 0008
- Stability under test conditions: The test item was analysed to be stable in the vehicle at concentrations ranging from 25-200 mg/mL for at least 24 h.
- Storage condition of test material: at room temperature, protected from light

Species:

mouse

Strain:

CD-1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga GmbH, Sulzfeld, Germany
- Age at study initiation: 5-8 weeks
- Weight at study initiation: 24-35 g
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Fasting period before study:
- Housing: individually in Makrolon type II cages with bedding of soft wood granules, type S 8/15 (Rettenmaier und Soehne, Fuellstoff-Fabriken, Ellwangen-Holzmuehle, Germany)
- Diet: Altromin 1324 Standard diet (Altromin GmbH, Lage, Germany), ad libitum
- Water: Tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.5-23
- Humidity (%): 40-62
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: olive oil
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw
- Lot/batch no. (if required): 2047001 (Henry Lamotte GmbH)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item and the positive control substance ere suspended in olive oil. For each animal the respective amount of test substance was sepataely suspende in a syringe.

Duration of treatment / exposure:

3, 8, and 24 h

Frequency of treatment:

single oral dose

Post exposure period:

none

Remarks:

Doses / Concentrations:
250 and 2000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

4 males per dose per treatment period

Control animals:

yes, concurrent vehicle

Positive control(s):

none; no data; ethylmethanesulphonate
- Route of administration: oral (gavage)
- Doses / concentrations: 400 mg/kg bw
- Duration of treatment / exposure: 3 h

Tissues and cell types examined:

Primary hepatocytes and kidney cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The dose selection was based on the publication of Sasaki et al. (1997) Mutat Res 395: 189-198

DETAILS OF SLIDE PREPARATION:
- Hepatocytes: After perfusion, incisions were made into each of the liver lobes. Subsequentially, liver cells were carefully collected with the help of a metal comp. The obtained cell suspension was filtered over fine gauze, filled up to 25 mL with cold WEI (Williams Medium E with 1% L-glutamine, 0.1% gentamycin sulfate, without foetal calf serum) and kept on ice for 5-10 min. Afterwards, the resulting supernatant was discarded and the pellet resuspended in 25 mL of cold WEI. Subsequent centrifugation occurred for 3 min at 50 x g and <15°C. Resuspending of the pellet in WEI and second centrifugation were performed as described above. The resulting pelet was resuspended in 5-15 mL WEI. An aliquot was used for the determination of cell viability and cell count by the method of trypan blue exclusion. The obtained viability value of the cell suspension after perfusion is a measure for the substance induced cytotoxicity during in vivo exposure.
- Kidney cells: Both kidneys were cut into half and the cortex region was isolated. The isolated tissue was cut into small pieces with a scalpel, suspended with a pipette and subsequently transferred to 50 mL of a digestion buffer. This buffer containes 25 mL collagenase solution and 25 mL trypsin solution (0.25% trypsin w/v in HBSS. Tissue pieces were incubated under stirring in a waterbath (37°C) for 20 min. After addition of 1 mL foetal calf serum (FCS) the cell suspension was filtered over gross gauze. Bovine serum albumine (10% w/v, 1 mL) was added and the cell suspension centrifuged at 68 x g and 4°C for 5 min. The pellet was resuspended in 25 mL cold HBSS and centrifuged again at 120 x g. The resulting pellet was resuspended in 10 mL cold HBSS: An aliquot was used for the determination of cell viability and cell count by the method of trypan blue exclusion. The obtained viability value of the cell suspension after perfusion is a measure for the substance induced cytotoxicity during in vivo exposure.

METHOD OF ANALYSIS:
The Comet Assay was performed according to the method of Singh et al. (1988) Exp. Cell Res. 175:184-191 with minor modifications. The different steps are comparable to the procedure described by Sasaki et al. (1997) Mutat Res 395: 189-198. Aliquots of the liver and kidney cell suspensions not greater than 100 µL were taken to reach an approximate cell number of 3-5E+04 liver cells and 8-10E+04 kidney cells, respectively. Cells were centrifuged at 180 x g for 6 min. The resulting pellet was mixed with 50 µL of 0.7% low melting agarose (LMA). The cell/LMA suspension was carefully pipetted onto slides already covered with two layers of 0.5% normal melting gaarose (NMA). Afterwards, a second LMA layer (0.7%, 50 µL) was placed on top. After lysis (1 h or overnight) buffer containing NA2EDTA, sacosinate, DMSO and freshly added Triton X-100; without proteinase K), alkaline treatment (20 min, NaOH and Na2EDTA, pH≥13) was performed followed by electrophoresis (60 min, 18 V, 300 mA) and neutralisation (Tris base, pH 7.5). Subsequently, slides were stained with ethidium bromide.
Evaluation of comets was performed using image analysis (Perceptive Instruments, Havehill, UK). Tail length, defined as distance between the middle of the head and the end of the tail, was used as assessment parameter. 50 cells/slide and 2 slides/animal were scored (100 cells total).

Evaluation criteria:

A response is considered positive, if a chemical induces a dose dependent mean increase of the tail length per dose group of more than 25% above the negative control group. Increases <25% compared to the negative control are considered negative. However, these criteria may be overruled by good scientific judgement.

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

Table 1.   Table for In Vivo Comet assay results of liver and kidney cells
Organ	Dose group		Tail length [µm, mean* ± SD]
			3 h		8 h			24 h
Liver	Neg. control		34.17 ± 3.24		34.58 ± 2.33			34.77 ± 1.80
	OPP
	250 mg/kg		40.02 ± 0.84		32.63 ± 3.56			35.30 ± 4.51
	2000 mg/kg		35.93 ± 3.70		31.98 ± 2.14			36.80 ± 3.33
	EMS400 mg/kg		48.60 ± 4.58		48.72 ± 3.57			45.30 ± 1.87
Kidney	Neg. control		23.23 ± 1.99		23.48 ± 0.87			20.56 ± 0.45
	OPP
	250 mg/kg		20.93 ± 1.11		21.76 ± 1.02			21.65 ± 2.57
	2000 mg/kg		19.71 ± 1.00		21.54 ± 1.35			23.27 ± 3.37
	EMS400 mg/kg		34.60 ± 2.25		33.90 ± 2.11			32.75 ± 4.05
* 100 cells evaluated
Table 2.   Table for Cytogenetic In-Vivo-Test: Cytotoxicity
				negative control		250	2000		positive control
Number of cells evaluated				100
Cytotoxicity: Liver cells
Sacrifice Time: 3h		Absolute Viability^a		79.0±8.04		77.6±5.04	76.2±5.06		74.9±3.16
		Relative Viability^b[%]		100		98.1	96.4		94.8
Sacrifice Time: 8h		Absolute Viability^a		81.0±5.48		77.0±2.27	73.5±1.30		78.1±4.68
		Relative Viability^b[%]		100		95.1	90.8		96.4
Sacrifice Time: 24h		Absolute Viability^a		82.9±3.61		77.7±4.96	74.2±1.72		72.3±9.77
		Relative Viability^b[%]		100		93.7	89.5		87.2
Cytotoxicity: Kidney cells
Sacrifice Time: 3h		Absolute Viability^a		92.3±1.56		91.6±2.758	86.2±6.37		89.9±2.53
		Relative Viability^b[%]		100		99.2	93.4		97.4
Sacrifice Time: 8h		Absolute Viability^a		92.7±2.86		91.3±2.11	90.1±2.69		93.9±2.31
		Relative Viability^b[%]		100		98.5	97.2		101.3
Sacrifice Time: 24h		Absolute Viability^a		92.5±1.63		92.5±1.73	90.0±1.87		93.3±2 43
		Relative Viability^b[%]		100		100	97.3		100.9
^a= mean viability of cell preparation per dose group after perfusion ^b= relative to negative control animals

Conclusions:

Interpretation of results (migrated information): negative

Reference 3

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Remarks:

Rodent Dominant lethal test

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 478 (Genetic Toxicology: Rodent Dominant Lethal Test)

Version / remarks:

adopted 29 Jul 2016

Deviations:

yes

Remarks:

- choice of vehicle/neg. ctrl. (5% gum arabic) - only 2 instead of 3 dose levels - dose levels differ by > factor 4 - pos. ctrl. EMS only administered once instead of 5 times - no information on the laboratory's proficiency or historical control data

Qualifier:

no guideline available

Principles of method if other than guideline:

- Principle of test: investigating chromosomal aberrations in germ cells in vivo using mice
- Short description of test conditions: The test substance was administered orally to male mice for 5 consecutive days. Afterwards, males were mated with untreated virgin mice, and the dominant lethality induction rate was determined over a period of 6 weeks.
- Parameters analysed / observed: dominant lethal mutations resulting in embryonic or fetal death

GLP compliance:

no

Type of assay:

rodent dominant lethal assay

Species:

mouse

Strain:

C3H

Remarks:

SLC - C3H

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 9 - 10 weeks
- Assigned to test groups randomly: not specified
- Housing: no information on housing details
- Diet: Oriental Yeast, MF; ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 ± 2
- Humidity (%): 55 ± 10
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): not specified

Route of administration:

oral: gavage

Vehicle:

- Vehicle/solvent used: 5% gum arabic solution

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
No details provided in the study report.

Duration of treatment / exposure:

5 days

Frequency of treatment:

once daily

Post exposure period:

6 weeks mating period

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

500 mg/kg bw/day

No. of animals per sex per dose:

15 males per dose group and control

Control animals:

yes, concurrent vehicle

Positive control(s):

ethylmethanesulphonate (EMS)
- Route of administration: intraperitoneal
- Doses / concentrations: 300 mg/kg bw
- Exposure frequency: once

Tissues and cell types examined:

The numbers of corpora lutea, implantations, live pups, and early and late dead embryos were counted.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The LD50 value of the test substance in SLC-C3H male mice ranged from 2000 to 3000 mg/kg bw. In this test, 500 mg/kg bw a day was taken to be the high dose.

TREATMENT AND SAMPLING TIMES
After completion of treatment, each male was mated with two syngeneic untreated virgin females each week for 6 weeks. The presence or absence of plug were observed every morning, and females with cavities were transferred to a cage for one animal on the 0th day of gestation. Females with no plug for 1 week were weighed and transferred to a single cage at the end of the week. Females were killed on the 12th to 13th day of gestation, and the number of corpora lutea, implantations, live pups, and early and late dead embryos were counted. For females without plugs, the date of pregnancy was estimated based on the amount of weight gain. Records were aggregated weekly for each group to determine the dominant lethality induction rate.

METHOD OF ANALYSIS:
Dominant lethality induction rate was calculated (for details see section: "any other information on materials and methods incl. tables", table 1).

OTHER:
Clinical observations, body weight measurements and autopsies were performed.

Evaluation criteria:

not reported

Statistics:

A statistical analysis not further specified was performed.
Means and standard deviations were calculated.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

500 mg/kg bw dose: acute effects (depression) and reduced body weight

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY
- Statistical evaluation: The pregnancy rate in the test substance-treated group was almost the same as that in the control group from 1 to 6 weeks, but decreased significantly (56.7%) in the positive control group at 2 weeks. In the 100 mg/kg bw test substance administration group, a decrease in the average number of offspring was observed 6 weeks after the administration. However, no increase in the number of embryos dying early was seen. There was no difference between the control group in the average number of corpora lutea, average number of implantations, average number of offspring, and frequency of early dead embryos in the 500 mg/kg bw administration group, and no significant increase in the dominant lethality was seen to be induced from week 1 to 6. On the other hand, in the administration group used as a positive control, the average number of offspring decreased and the number of early dead embryos increased significantly from week 1 to 3, and the strongest (68.9%) dominant lethal inducing effect was observed 2 weeks after administration. After 4 weeks, the number of early dead embryos decreased and returned to normal levels. Regarding early embryonic deaths, in the positive control group, a significant increase was observed in week 1 to 3, but there was no difference between the test substance--administered group and the control group, and no dose-response relationship was obtained. For details on the study results, see section "any other information on results incl. tables", tables 1 - 2.
- Clinical observations: Male animals in the high-dose group showed signs of depression immediately after administration. However, no effects on mating ability were noted. In the low-dose group, no acute symptoms were observed.
- Body weight measurements: Weight loss was observed in the high-dose group (92% of that of the control group).
- Autopsy: No abnormalities were noted.

Table 1: Results of the dominant lethal mutation test with the test substance in mice

Week	Test compound	Dose level mg/kg bw/day	No. of females mated	No. of females with implants	No. of corpora lutea per pregnancy^1	No. of implants per pregnancy^1	No. of live embryos per pregnancy^1	Dead implants (%)	Dominant lethal mutations (%)
1	Test substance	0	30	30	10.4 ± 1.3	9.4 ± 1.4	8.5 ± 1.7	9.9
		100	30	29	11.1 ± 1.6	9.1 ± 1.4	7.9 ± 1.7	13.2	7.1
		500	30	26	10.5 ± 1.3	9.3 ± 1.9	8.2 ± 2.0	12.3	3.5
	EMS	300	30	30	10.9 ± 1.3	9.3 ± 1.7	7.4 ± 1.8*	20.4***	12.9
2	Test substance	0	30	27	11.3 ± 1.5	9.8 ± 1.4	9.0 ± 1.5	8.0
		100	30	27	10.9 ± 1.6	9.4 ± 1.3	8.8 ± 1.4	6.7	2.2
		500	30	29	11.3 ± 1.2	10.1 ± 1.1	9.1 ± 1.4	9.9	-1.1
	EMS	300	30	17	9.7 ± 3.0	7.1 ± 2.0***	2.8 ± 2.0***	60.0***	68.9
3	Test substance	0	30	28	10.5 ± 1.1	9.4 ± 1.2	8.5 ± 1.3	9.9
		100	30	26	10.8 ± 1.6	9.7 ± 1.0	8.3 ± 1.5	13.5	1.4
		500	30	28	10.6 ± 1.4	9.1 ± 1.7	7.8 ± 1.8	14.9	8.4
	EMS	300	30	27	10.3 ± 1.3	8.7 ± 1.9	7.0 ± 2.1**	19.7**	17.7
4	Test substance	0	30	28	10.7 ± 1.2	9.4 ± 1.0	8.2 ± 1.1	12.5
		100	30	26	10.8 ± 1.2	9.4 ± 1.2	8.6 ± 1.7	9.0	-4.9
		500	30	23	10.4 ± 0.8	9.3 ± 1.1	8.3 ± 1.4	10.7	-1.2
	EMS	300	30	27	11.1 ± 1.2	9.9 ± 1.4	9.0 ± 1.5	9.4	-9.8
5	Test substance	0	30	29	10.2 ± 1.3	9.4 ± 1.3	8.1 ± 1.8	14.6
		100	30	28	10.3 ± 1.1	9.5 ± 1.1	8.6 ± 1.3	9.0	-6.2
		500	30	27	10.6 ± 1.0	9.4 ± 1.4	8.3 ± 1.2	11.8	-2.5
	EMS	300	30	23	10.6 ± 1.2	9.6 ± 1.1	8.3 ± 1.6	13.1	-2.5
6	Test substance	0	30	26	11.0 ± 1.4	10.6 ± 1.2	9.3 ± 1.5	7.7
		100	30	23	11.3 ± 1.0	9.6 ± 1.3	8.4 ± 1.4*	11.8	9.7
		500	30	26	11.0 ± 1.1	9.8 ± 1.2	8.5 ± 1.6	13.3	8.6
	EMS	300	30	28	10.4 ± 1.3	9.3 ±1.0*	7.8 ± 1.5***	15.8**	16.1

¹: Mean ± S.D

*:p<0.05; **:p<0.01; ***:p<0.001

 

 

Table 2: Percentage of mouse litters with early deaths after treatment of males with the test substance or positive control

Test compound	Dose level mg/kg bw/day	week
		1	2	3	4	5	6
	Litters with one or more early deaths (%)
Test substance	0	56.7	48.1	64.3	57.1	72.4	53.9
	100	65.5	48.1	69.2	46.2	53.6	69.6
	500	65.4	65.5	71.4	60.9	59.3	76.9
EMS	300	86.2 *	100 **	85.2	63.0	73.9	75.0
	Litters with two or more early deaths (%)
Test substance	0	26.7	22.2	17.9	39.3	34.5	15.4
	100	27.6	11.1	34.6	26.9	14.3	34.8
	500	26.9	27.6	39.3	21.7	25.9	34.6
EMS	300	58.6 *	94.1 ***	55.6 **	14.8	30.4	46.4 *

* p<0.05; ** p<0.01; ***p<0.001

Conclusions:

Under the conditions of this test, it is concluded that there is no dominant lethal mutagenesis effect of the test substance, thus providing in vivo evidence that the substance is not a germ cell mutagen.

Reference 4

Endpoint:

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

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study which meets basic scientific pinciples

Qualifier:

no guideline available

Principles of method if other than guideline:

Special investigation of urinary bladder effects in male rats after subchronic treatment with 2-phenylphenol.

GLP compliance:

no

Type of assay:

other: DNA adducts

Specific details on test material used for the study:

- Name of test material (as cited in study report): technical grade ortho-phenylphenol (OPP), 2-Biphenylol
- Composition of test material, percentage of components: 50% of test material was synthesized by Bayer AG (Leverkusen, Germany), 50% of test material was synthesized by the Dow Chemical Company (Midland, MI, USA)
- Stability under test conditions: confirmed in stability in rodent diet at room and freezer temperatures for 14 and 28 days, respectively
- Storage condition of test material: under freezer conditions (approximately -23 °C)

Species:

rat

Strain:

other: CDF[F-344]/BR

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SASCO, Inc., Madison, WI
- Age at study initiation: 5-7 weeks
- Housing: individually in suspended stainless steel wire-mesh cages
- Diet: Purina Mills Rodent Lab Chow 5001-4 in "etts" form (Purina Mills, St. Louis, MO), ad libitum
- Water: tap water (municipal water supply od Kansas City, MO), ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26
- Humidity (%): 40-70
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

acetone/corn oil mixture

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Acetone/corn oil mixture was used to dissolve the test substance

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Storage temperature of food: under freezer conditions

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily

Post exposure period:

none

Remarks:

Doses / Concentrations:
800, 4000, 8000, 12,500 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

22 males (12 for postlabelling analyses and 10 for cell proliferation determination)

Control animals:

yes, concurrent vehicle

Tissues and cell types examined:

During weeks 12-13 and 13-14 of the study, urine was collected for metabolite and general urinalysis determinations, respectively. In addition, urinary bladders were collected during week 14 to perform a 32P-postlabelling analysis on the urothelium while histopathological evaluations included determination of a BrdU-labelling index as well as light and scanning electron microscopy (SEM only on 0 and 8000 ppm group).

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

reduced body weights in 8000 and 12,500 ppm groups

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

not examined

Additional information on results:

- Body weight gain was reduced in both the 8000- and the 12,500-ppm-dose groups; all other dose groups were unchanged. Food intake remained unaffected at all doses tested.
- Simple hyperplasia of the urothelium was observed histopathologically at ≥ 8000 ppm. Significant bladder changes were also noted by SEM (Table 1) .
- The glucuronide and sulphate conjugates of OPP and the hydroxylated metabolite, 2,5-phenylhydroquinone (PHQ), were found to be the major urinary metabolites. For all dose groups, the major conjugate present was the sulphate conjugate of OPP. Minute levels of free OPP and PHQ were observed in all dose groups, with free PHQ comprising 0.6-1.5% of the total metabolites measured.
- An increase in the BrdU-labelling index of the bladder epithelium was observed at ≥ 8000 ppm (Table 1).
- Following 13 weeks of dietary exposure to OPP, 32P-postlabelled rat urothelial DNA showed no evidence for formation of OPP-DNA adducts.

Table 1: Effects of OPP on the bladder urothelium after 13 weeks

Group	Bladder histology		Labelling index ± SE*	SEM classification^a
	Normal	Simple hyperplasia		1	2	3	4	5
0 ppm	10	0	0.10 ± 0.02	6^b	4
1000 ppm	10	0	0.12 ± 0.02
4000 ppm	10	0	0.12 ± 0.02
8000 ppm	8	2	0.33 ± 0.08*		2		2	6*
12,500 ppm	3*	7*	0.57 ± 0.12*

* Significantly different from control; p ≤ 0.05.

a: Scoring system: 1: completely normal bladder with flat surface composed of uniform polygonal cells without superficial necrotic or exfoliated cells; 2: similar, but with occasional foci of one to a few necrotic or exfoliated cells; 3: cobblestone appearance and/or more extensive and larger foci of necrosis/exfoliation; 4: extensive necrosis and appearance of rounded cells in addition to polygonal cells; 5: obvious piling up of round cells (hyperplasia), the cells usually having uniform and/or pleomorphic microvilli rather than microridges.

b: Each value represents the number of urinary bladders given a particular grading within each dose group.

Conclusions:

Interpretation of results (migrated information): negative
OPP caused an increase of mitotic activity and hyperplasia of the urothelium at dose levels ≥8000 ppm that also caused evident toxicity. No DNA adducts were formed by OPP or its metabolites. The NOAEL is 4000 ppm (~285 mg/kg bw/day).

Reference 5

Endpoint:

genetic toxicity in vivo, other

Remarks:

in vivo mammalian somatic cell study: cytogenicity / urinary bladder micronucleus test

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: Micronucleus test in urinary bladder
The study was conducted to determine cellular and genetic alterations in the rat bladder following treatment with OPP and to investigate the influence of the sodium salt on the tumorigenicity of OPP. Sodium OPP (SOPP) has been consistently shown to induce tumours in the urinary tract of male F344 rats, whereas OPP has produced more variable results. Tumours in SOPP-fed rats occurred at lower comparable doses, with shorter latency periods and were more malignant than those that occurred in the positive studies of OPP-treated rats. Sodium salts of several acids have produced effects on urothelial proliferation and tumorigenesis in the rat bladder, suggesting that an elevation in urinary pH and sodium concentration may act as promoter in the development of urinary bladder cancers in the rat.
- Short description of test conditions: Male Fisher F344 rats were administered 2% OPP, 2% OPP + 2% sodium chloride or 2% sodium chloride in their diet for 14 days. 24 h prior to sacrifice, the animals were administered 5-bromo-2'deoxyuridine (BrdU) by intraperitoneal injection. Upon sacrifice, the bladder epithelial cells were evaluated for induction of cell proliferation and micronucleus formation. To further determine changes in chromosome number, fluorescence in situ hybridisation (FISH) was used with a DNA probe for rat chromosome 4.
- Parameters analysed / observed: Body weight, micronuclei formation and proliferation of epithelial cells in the urinary bladder

GLP compliance:

no

Type of assay:

other: mammalian urinary bladder micronucleus test

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Sprague-Dawley (Indianapolis, USA)
- Age at study initiation: 16 weeks
- Assigned to test groups randomly: yes
- Housing: in polycarbonate cages
- Diet: Formulab rat chow (PMI Feeds, St. Louis, Missouri, USA), ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 50
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

The test material was incorporated into the diet.

Details on exposure:

DIET PREPARATION
Diets containing 2% OPP, 2% OPP + 2% sodium chloride and 2% sodium chloride were prepared by mixing the compound with water and Formulab rat chow.

Duration of treatment / exposure:

14 days

Frequency of treatment:

continuously via the diet

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

with and without addition of 2% sodium chloride
Note: The animals were fed with 2% OPP in the diet. Based on Derelanko (2008), 2% in the diet correspond to 20000 ppm. With the conversion factor of 10 for young rats, 2% in the diet therefore correspond to 20000 ppm / 10 = 2000 mg/kg bw/day. (Derelanko, 2008: The Toxicologists Pocket Handbook, 2nd edition, Table 28).

No. of animals per sex per dose:

4 males

Control animals:

yes

yes, plain diet

Positive control(s):

none

Tissues and cell types examined:

urinary bladder cells

Details of tissue and slide preparation:

TREATMENT AND SAMPLING TIMES:
Rats were treated with OPP, OPP + 2% sodium chloride or 2% sodium chloride for 14 days. 24 h before sacrifice, the rats were administered 100 mg/kg bw BrdU in DMSO / saline (1:2) by intraperitoneal injection. The animals were sacrificed on study Day 14.

DETAILS OF SLIDE PREPARATION:
For each animal, the urinary bladder was removed, rinsed in ice-cold saline and inverted over PE160 tubing. The bladder was then tied securely with 00 silk thread and inflated with 0.9% sodium chloride using an 18 gauge needle and a 1 mL syringe. The epithelial cells were scraped off the exposed luminal surface on the inflated bladders, using a coverslip, into a clean Petri dish containing ice-cold saline. The cells were transferred to a 15 mL screw cap centrifuge tube and stored on ice. The dish was rinsed once with ~5 mL saline, which was than added to the tube containg the cells. After centrifugation and re-suspension in saline, single cell preparations were made by vigorously pipetting the cell suspension with a Pasteur pipette and by vortexing. Cell suspensions were transferred to glass slides using a Cytospin 2 cytocentrifuge. The slides were air-dried, fixed in 100% methanol for 30 min and stored under nitrogen in the presence of anhydrous calcium sulfate at -20°C until use.

BrdU LABELING AND MICRONUCLEUS ASSAY:
BrdU labeling was conducted by denaturating the cells in 0.07 N sodium hydroxide followed by neutralisation with phosphate buffered saline (PBS). The slides were then incubated with an anti-BrdU antibody diluted in 0.5% Tween-20 in PBS for 30 min. The antibody was detected by incubation with Texas Red-conjugated goat anti-mouse IgG for 30 min. Thereafter the slides were washed in PBS and DNA was counterstained with 4,6-diamnidino-2-phenylindole (DAPI).

FLUORESCENCE IN SITU HYBRIDISATION:
To facilitate the penetration of the FISH probe into aged slides, cells were incubated in 0.1% saponin for 30 min at room temperature, transferred to a pepsin solution (1 µg/mL in 0.01 N HCl) for 30 min at room temperature and incubated with 100 µL of 1 µg/mL proteinase K solution for 10 min at 37 °C. Thereafter, the slides were treated with 2% paraformaldehyde for 1 min at 4 °C. Target DNA was denaturated in 70% formamide, 2 x SCC (72 °C), the hybridisation mixture added and the target and probe incubated over night at 37 °C. The denatured hybridisation cocktail consisted of 1 µL digoxigenin-labelled probe, 1 µL sheared herring sperm DNA (1 mg/mL), 1 µL ddH2O and 7 µL MM2.1 hybridisation mix. Post hybridisation the slides were washed 3 times for 5 min at 65 °C in 0.1 x SCC, rinsed 3 times in PN buffer (0.1 M phosphate buffer containing 0.5% NP-40) at room temperature. The digoxigenin-labelled probe was detected using a FITC-conjugated sheep anti-digoxigenin antibody. DNA was counterstained with DAPI.

METHOD OF ANALYSIS:
The slides were scored for micronuclei by using a Nikon fluorescence microscope at 1250 x magnification. For each bladder, a total of 2000 cells were scored for the presence of micronuclei. Only micronuclei clearly deleniated from the main nucleus were scored as micronucleated cells.
For BrdU labeling, the DNA in the nucleus was scored as completely labeled if the entire nucleus was deep red or uniformly labeled. 2000 nuclei of each bladder were counted to determine the percentage of nuclei incorporating BrdU into their DNA. The replication index was calculated as the number of nuclei incorporating BrdU divided by the total number of nuclei counted.
The frequency of hybridisation regions per nucleus was determined from coded slides by scoring a minimum of 1000 cells / rat. A nucleus containing 3 or more chromosome 4 hybridisation regions was considered as a hyperdiploid cell.

Statistics:

The frequency of micronuclei, total BrdU labeling and hyperdiploidy in rat bladder cells were compared using the non-parametric Kruskal-Wallis ANOVA with the Mann-Whitney U-test used as post hoc test. Critical values were determined using a 0.05% probability of Type I error.

Sex:

male

Genotoxicity:

positive

Remarks:

2% OPP (corresponding to approx. 2000 mg/kg bw/day) without addition of salt in the diet

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not examined

Sex:

male

Genotoxicity:

positive

Remarks:

2% OPP (corresponding to approx. 2000 mg/kg bw/day) + 2% NaCl in the diet

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not examined

Sex:

male

Genotoxicity:

positive

Remarks:

2% NaCl in the diet

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not examined

Additional information on results:

RESULTS OF DEFINITIVE STUDY
Micronucleus test:
- Induction of micronuclei: The frequency of micronuclei in the control animals averaged 0.27%, whereas the animals administered sodium chloride in the absence of OPP already showed a statistically significant increase in micronucleated cells (0.72%) when compared to control animals.
OPP-treated rats showed a 4-fold, statistically significant increase in the frequency of micronuclei over those of control animals (1.05%) and rats administered OPP + sodium chloride showed an even higher, statistically significantly increased frequency of micronuclei over those of control animals (1.6%). For details, please refer to the pdf document on result Figure 1 provided under "Attachments".


BrdU incorporation as measure for cell proliferation:
Animals administered OPP showed a 40-fold increase in BrdU incorporation when compared to control animals (total BrdU incorporation 13.1%). Animals administered OPP + sodium chloride showed a total BrdU incorporation of 4.1%, gaining statistical significance when compared to control animals. Also treatment with sodium chloride alone showed an increase in BrdU incorporation when compared to control animals (1.29%). For details, please refer to the pdf document on result Figure 2 provided under "Attachments".

Hyperdiploidy / Polyploidy:
There was no increased frequency of hyperdiploidy or polyploidy upon treatment with OPP at 2% in the diet (corresponding to approx. 2000 mg/kg bw/day), OPP + sodium chloride or with sodium chloride alone. Among 1000 cells scored, the frequency of hyperdiploidy / polyploidy in the control animals was 0.32%, whereas the frequency in animals administered 2% sodium chloride was 0.46%, which did not differ significantly when compared to control animals. Treatment with OPP and OPP + sodium chloride resulted in hyperdiploidy / polyploidy frequences of 0.37 and 0.5%. For details, please refer to the pdf document on result Table 1 provided under "Attachments".

Body weight development:

The animals administered OPP and OPP + sodium chloride lost weight during the initial 2 -3 days of treatment. After Day 4, all animals gained weight at a similar rate. There was no effect on body weight noted for the control animals fed plain diet or 2% sodium chloride in the diet.

Conclusions:

In the present study, male rats were administered OPP, OPP + 2% sodium chloride or 2% sodium chloride in the diet. Treatment with OPP and OPP + sodium chloride but also 2% sodium chloride alone was associated with a statistically significant increase in micronucleated cells in the rat bladder epithelial cells. Treatment with OPP or OPP + sodium chloride was further accompanied by an increase in cell proliferation as indicated by BrdU incorporation. FISH with a chromosome 4 specific probe, however, revealed no increased frequencies of hyperdiploidy or polyploidy.
Based on the experimental findings, high doses of OPP can cause micronuclei in the presence and absence of sodium salt and show a proliferating effect in the bladder. The results further indicate that also high sodium chloride concentrations can cause chromosomal damage in rat bladder cells.

Reference 6

Endpoint:

genetic toxicity in vivo, other

Remarks:

in vivo mammalian somatic cell study: cytogenicity / urinary bladder micronucleus test

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: Micronucleus test in urinary bladder
In order to investigate whether pH-dependent PHQ autoxidation plays an important role in the formation of damage in the bladder of OPP-treated cells, male F344 rats were fed a diet containing OPP and urine-modifying dietary salts for 15 days. Upon sacrifice, the bladder epithelial cells were evaluated for induction of cell proliferation and micronucleus formation. Further, to determine the origin of the micronuclei, the presence of centromeric anti-kinetochore proteins with the micronuclei was determined using a CREST antibody.
- Short description of test conditions: Fisher F344 rats were treated with the test item at 4000 and 8000 ppm for 15 days. The test item was incorporated into the diet supplemented with either 1% ammonium chloride or 3% sodium bicarbonate to produce either acidic or alkaline urinary pH.
- Parameters analysed / observed: Body weight, food and water consumption, urinary pH, urinary protein concentration, micronuclei formation in the urinary bladder

GLP compliance:

no

Type of assay:

other: mammalian urinary bladder micronucleus test

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories (Raleigh, North Carolina, USA)
- Age at study initiation: 8 weeks
- Assigned to test groups randomly: yes
- Housing: in groups of 4 in polycarbonate cages
- Diet: Lab diet 5001 (PMI Nutrition International Richmond, Indianapolis, USA), ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 50
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: feed

Vehicle:

The test material was incorporated into the diet.

Details on exposure:

DIET PREPARATION
The diets were supplemented with either 1% ammonium chloride, 3% sodium bicarbonate or no salt. In total, 9 types of diet were prepared:
1. no salt control
2. 1% ammonium chloride
3. 3% sodium bicarbonate
4. 4000 ppm test item
5. 4000 ppm test item + 1% ammonium chloride
6. 4000 ppm test item + 3% sodium bicarbonate
7. 8000 ppm test item
8. 8000 ppm test item + 1% ammonium chloride
9. 8000 ppm test item + 3% sodium bicarbonate

Duration of treatment / exposure:

15 days

Frequency of treatment:

continuosly via the diet

Dose / conc.:

4 000 ppm (nominal)

Remarks:

(equivalent to approx. 270 - 280 mg/kg bw/day)

Dose / conc.:

8 000 ppm (nominal)

Remarks:

(equivalent to approx. 550 - 600 mg/kg bw/day)

No. of animals per sex per dose:

4

Control animals:

yes, plain diet

other: in addition: diet with 1% ammonium chloride AND diet with 3% sodium bicarbonate

Positive control(s):

none

Tissues and cell types examined:

urinary bladder cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The dose levels of 1% ammonium chloride and of 3% sodium bicarbonate have been previously shown to cause acidic and alkaline urine in rats, respectively (Hasegawa et al., Jpn J Cancer Res (1991) 82:657-664)

TREATMENT AND SAMPLING TIMES:
Rats were treated with OPP in addition to the dietary salts for a period of 15 days. On study Day 15, the rats were sacrificed by CO2 asphyxiation. 24 h prior to sacrifice, the animals were administered 50 mg/kg bw BrdU in DMSO / saline (1:2) by intraperitoneal injection. Urinary bladder cells were harvested as described by Balakrishnan et al., Mutagenesis (2002) 17:89-93 (refer to endpoint study record 7.6.2, Balakrishnan, 2002, MNT in the bladder).

INDUCTION OF CELL PROLIFERATION BY BrdU LABELING AND MICRONUCLEUS ASSAY:
Replicating cells were studied using BrdU incorporation and determining the labeling index. BrdU labeling was conducted by denaturing the cells in 0.07 N NaOH followed by neutralisation with phosphate buffered saline (PBS). The slides were then incubated with an anti-BrdU antibody, diluted in 0.5% Tween-20 in PBS, in a humidified chamber for 30 min. The antibody was then detected by incubation with Cy3-conjugated goat antimouse IgG (10 µg/mL; Molecular Probes, Eugene, OR) in a humidified chamber for 30 min. After washing the slides in PBS, the DNA was counterstained with 4,6'-diamidino-2-phenylindole (DAPI, 1 µg/mL) in phenylenediamine antifade mounting medium.

CREST ANTIBODY LABELING OF BLADDER EPITHELIAL CELLS:
The bladder micronucleus assay with the CREST (calcinosis, Raynauld’s phenomenon, esophageal dysfunction, sclerodactyly and telangiectasia syndrome) antibody labeling method was used for the bladder cells with some modifications. In brief, the bladder epithelial cells were rinsed in PBS containing 0.01% Tween-20 for 5 min. The CREST antibody (in a 1:1 dilution with PBS; Antibodies Inc., Davis, CA) was applied to the slides for 180 min. The slides were rinsed twice in 0.1% Tween-20 for 2 min. The antibody was then labeled using fluoresceinated-goat anti-human IgG (1:120 dilution in PBS) for 75 min followed by a rinse in 0.1% Tween-20. The signal was amplified by adding another layer of fluoresceinated rabbit anti-goat IgG (1:120 dilution in PBS) and the nucleus was counterstained with DAPI (1 µg/ml in antifade).

Statistics:

Changes in dietary and urine parameters as well as in the frequencies of micronuclei, CREST-positive and CREST-negative micronuclei, and BrdU labeling were compared by analysis of variance with the Fishers Protected Least Significant Difference (PLSD) or a t-test being used as a post hoc test. Critical values were determined using a 0.05 probability of Type I error.

Key result

Sex:

male

Genotoxicity:

positive

Remarks:

8000 ppm test item without salt enrichment

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not examined

Key result

Sex:

male

Genotoxicity:

positive

Remarks:

≥ 4000 ppm test item + 3% sodium bicarbonate

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not examined

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

4000 or 8000 ppm test item + 1% ammonium chloride

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not examined

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

4000 ppm test item without salt enrichment

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not examined

Additional information on results:

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels: chromosomal breakage and loss
- Induction of micronuclei: The frequency of micronuclei in the control animals treated with or without salts was very low in the range of 0.15 - 0.19%. The frequencies of micronuclei induced in the 8000 ppm OPP and 8000 ppm OPP + sodium bicarbonate-treated animals were statistically significantly increased to 0.51 ± 0.13% and 0.59 ± 0.18%, respectively. The mean frequencies of micronuclei in the rats treated with 4000 ppm OPP + sodium bicarbonate (0.36 ± 0.11) were statistically significantly higher than those in the rats treated with the sodium bicarbonate-enriched control diet. The micronucleus frequencies in rats treated with test item in the presence of 1% ammonium chloride doses were similar to the frequencies seen in the control animals and significantly lower than those observed after treatment with test item at 8000 ppm or at test item in the presence of 3% sodium bicarbonate. Results including statistical evaluation are presented in Figure 6 and in Table 3 attached as pdf document under "Attached documents".
- CREST staining to determine the origin of micronuclei in the bladder cells: The statistically significant increase in micronuclei at 8000 ppm and at 4000 and 8000 ppm in the presence of 3% sodium bicarbonate was due to both, chromosomal breakage and chromosomal loss. Approximately, 50-60% of the micronuclei detected in the bladder epithelial cells from animals in the three increased treatment groups were CREST-negative indicating that they originated from chromosome breakage. The other doses had inadequate numbers of micronuclei upon which to confidently estimate changes in proportions. An approximately 10-fold increase in CREST-positive micronuclei was also seen. For details, please refer to Table 3 which can be found attached to this chapter as pdf documents ("Attached documents").

Systemic toxicity:

Body weight: All animals gained weight as expected. There was no difference in weight gain between animals of different treatment groups.

Food consmption: There were modest differences in food measurements and it could be inferred that the animals on the diet with OPP and salts consumed more diet that the rats fed only the salt-enriched diet. For details, please refer to Table 1 which is attached as pdf document under "Attachments". The differences were explained by the rat's aversion to combinations of OPP and salts so that they nibbled and crumbled rather than actually consuming the food pellets.

Water consumption: There was a statistically significant decrease in water consumption in rats fed sodium bicarbonate-enriched diets. For details, please refer to Table 1 which is attached as pdf document under "Attachments".

Urinalysis:

In rats fed sodium bicarbonate-enriched diets the mean urinary pH was significantly increased, independent of the treatment with the test item.

For animals administered diet with no salts (plain diet or test item-enriched diet), the urinary pH was in the range of 7.2 - 7.3. Animals administered ammonium chloride-enriched diets showed an urinary pH of 5.7 - 5.8. Administration of sodium bicarbonate in the diet increased the mean urinary pH to 7.8 - 8.0.

Treatment with the test item was associated with a decrease in urinary proteins for normal and for salt-enriched diet. The decreases were seen with all treatments with the test item, except for 8000 ppm + 3% sodium bicarbonate, where the total urinary protein values were comparable to those of control levels. For details on urinalysis, please refer to Table 2 which is attached as pdf document under "Attachments".

The metabolite PHQ was identified in the urine, showing a dose-related increase in total PHQ levels but no dose-related pattern for free PHQ levels. More than 95% of PHQ was present as its acid-hydolysable (sulfate and glucuronide) conjugated forms. The treatment with salts had no impact on the metabolism of OPP to PHQ.

Cell proliferation (BrdU incorporation):

One rat each of the 4000 OPP + 1% ammonium chloride group and the 4000 OPP + 3% sodium bicarbonate group did not show any BrdU labeling in the bladder and were therefore not included in the analysis. Plain and salt-enriched diet treatment without test item averaged in 0.5 - 1.0% BrdU-labeled cells.

The incorporation of BrdU in the rats treated with 8000 ppm OPP and 8000 ppm OPP + sodium bicarbonate (averaging 7.5 ± 4.2 and 4.9 ± 1.8% respectively) was statistically significantly greater than that of the controls. In contrast, the BrdU-labeling in the rats fed the 8000 ppm OPP +1% ammonium chloride diet was similar to the controls and significantly lower than the 8000 and 8000 + sodium bicarbonate treated rats (refer to Figure 5 attached as pdf under "Attachments"). The labeling in the rats treated with 4000 ppm OPP with or without sodium bicarbonate, although increased, was quite variable (means 2.2 ±2.1 and 1.6 ±0.35%, respectively) and thus was not significantly elevated over the controls.

Conclusions:

In the present study, male rats were fed with the test item at 4000 and 8000 ppm in a diet supplemented with either no salt, 1% ammonium chloride or 3% sodium bicarbonate. Statistically significant increases in bladder cell proliferation as detected by BrdU incorporation and micronucleus formation were observed in the bladder cells of OPP-treated animals with either neutral or alkaline urinary pH after sodium bicarbonate treatment. No such effects were noted in animals with acidified urine administered OPP and 1% ammonium chloride treatment in the diet. Under the conditions of the test, OPP is considered a generic toxicant in urinary bladder cells at moderate and high dietary doses under neutral and alkaline conditions.

Reference 7

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosomal Aberration Test)

Version / remarks:

adopted 29 Jul 2016

Deviations:

yes

Remarks:

4 animals/dose instead of at least 5; animals 4 weeks old instead of 6-10; vehicle (5% gum arabic); 5-day treatment regimen; no info on clin. obs., bw measurements, blood sampling or cytotoxicity; no info on proficiency investigations or hist. ctrl. data

Qualifier:

no guideline available

Principles of method if other than guideline:

In a cytogenetic study, 4-week-old Wistar rats were treated orally with OPP in daily doses of 50, 100, 200, 400 and 800 mg/kg bw for 5 days or in single doses of 250, 500, 1000, 2000 and 4000 mg/kg bw/day. They were killed 24 h after the final treatment. The bone marrow cells were examined for chromosomal abnormalities.

GLP compliance:

no

Type of assay:

mammalian bone marrow chromosome aberration test

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Central Institute for Experimental Animals, Shizuoka Prefecture, Japan
- Age at study initiation: 4 weeks
- Weight at study initiation: not reported
- Assigned to test groups randomly: not specified
- Diet: solid feed (Oriental MF); ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 ± 1
- Humidity (%): 55 ± 5
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 10 / 14

Route of administration:

oral: gavage

Vehicle:

- Vehicle/solvent used: 5% gum arabic

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: not further specified

Duration of treatment / exposure:

5 consecutive days OR as single doses

Frequency of treatment:

once every 24 h for the 5-day treatment regimen

Post exposure period:

24 h after receiving the final administration

Dose / conc.:

250 mg/kg bw (total dose)

Remarks:

single dose

Dose / conc.:

500 mg/kg bw (total dose)

Remarks:

single dose

Dose / conc.:

1 000 mg/kg bw (total dose)

Remarks:

single dose

Dose / conc.:

2 000 mg/kg bw (total dose)

Remarks:

single dose

Dose / conc.:

4 000 mg/kg bw (total dose)

Remarks:

single dose

Dose / conc.:

50 mg/kg bw/day

Remarks:

5-day treatment

Dose / conc.:

100 mg/kg bw/day

Remarks:

5-day treatment

Dose / conc.:

200 mg/kg bw/day

Remarks:

5-day treatment

Dose / conc.:

400 mg/kg bw/day

Remarks:

5-day treatment

Dose / conc.:

800 mg/kg bw/day

Remarks:

5-day treatment

No. of animals per sex per dose:

4

Control animals:

yes

Positive control(s):

Mitomycin C
- Route of administration: intraperitoneal
- Doses/concentrations: 5 mg/kg bw

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In respect of the choice of appropriate doses, a reference LD50 value of 2700 mg/kg bw to 3000 mg/kg bw derived by single oral administration in mature rats and the results of a preliminary dose-setting study served as a basis for the doses selected in the main study.

DETAILS OF SLIDE PREPARATION: 2 mg/kg bw of Colchicine was intraperitoneally administered 3 h before the animals were sacrificed. The rats were anesthetized and killed using ether 24 h after they received the (final) test substance administration. Subsequently, bone marrow cord follicles were taken from the femur. The cells collected were fixed by hypotonic treatment with 0.075 M potassium chloride aqueous solution. Fixation time was 7 mins. Afterwards, the cells were fixed with a force Lunoa solution (acetic acid: methanol; ratio 1: 3). Samples were prepared by a flame drying method and stained with Giemsa solution.

METHOD OF ANALYSIS:
Metaphase images that could be analyzed were selected and 50 were observed for each individual, with structural and numerical abnormalities of chromosomes being observed. For gaps and breaks, the unstained part along the long axis of the chromatid was defined as gaps, according to Cohen and Hirschhorn (1971). When the chromatid fragment was off the major axis, it was defined as a break.
Recording of gaps was performed as described by Cohen and Hirschhorn (1971) and the Ad Hoc Committee of the Environmental Mutagen Society and the Institute for Medical Research (1972).

Evaluation criteria:

Not specified

Statistics:

A significance test between the control group and the treatment groups was performed via Fisher's exact test method.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
No details on the test design and results of the preliminary study are reported.

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels: no significant effects occurred. Non-significant effects include 1 chromatid break observed each in the 1000 mg/kg bw and 4000 mg/kg bw single dose groups, and 1 each in the 50 mg/kg bw/day and 100 mg/kg bw/day continuous dose groups but the frequency of occurrence was less than 0.5 % and no dose-related increasing tendency was observed. Only a weak increase without dose-dependency was found in the cells of the continuous administration group in respect of the chromatid gap. For details on the results, please refer to section "any other information on results incl. tables", table 1.
- Statistical evaluation: No significant increase in the frequency of occurrence of chromosomal abnormalities in bone marrow cells was observed in either single or continuous administration of the test substance compared to the control group.

Table 1: Results of the cytogenetic analysis of bone marrow cells in 4 week-old rats treated with the test substance and positive control Mitomycin C

Chemical	Dose (mg/kg bw)	No. of injections	No. of animals	Aberrant cells		Gaps	Breaks	Exchanges and others	Aneuploid cells	Polyploid cells
				Total cells observed					Total cells observed	Total cells observed
				No.	%
Test substance	0	1	4	0/211	0.0	2	0	0	2/211	0/211
	250	1	4	0/221	0.0	0	0	0	1/221	0/221
	500	1	4	0/204	0.0	2	0	0	0/204	0/204
	1000	1	4	1/204	0.5	2	1	0	0/204	3/204
	2000	1	4	0/209	0.0	1	0	0	0/209	0/209
	4000	1	4	1/207	0.5	2	1	0	0/207	2/207
Mitomycin C	5	1	4	110/205	53.6	98	107	86	2/205	0/205
Chemical	Dose (mg/kg bw/day)	No. of injections	No. of animal	Aberrant cells		Gaps	Breaks	Exchanges and others	Aneuploid cells	Polyploid cells
				Total cells observed					Total cells observed	Total cells observed
				No.	%
Test substance	0	5	4	0/206	0.0	0	0	0	1/206	3/206
	50	5	4	1/210	0.5	1	1	0	1/210	1/210
	100	5	4	1/206	0.5	3	1	0	0/206	0/206
	200	5	4	0/209	0.0	6	0	0	1/209	0/209
	400	5	4	0/209	0.0	5	0	0	2/209	3/209
	800	5	4	0/209	0.0	3	0	0	0/209	1/209

Conclusions:

Under the conditions of this experiment, the results lead to the conclusion that the test substance is negative for inducing chromosomal aberration in rat bone marrow cells. However, as bone marrow exposure to the test substance was not demonstrated in this study, the significance of the negative outcome is considerably weakened.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

Please refer to the field „Additional information“ for further explanation.

Additional information

In vitro

Gene mutation in bacteria

A total of 12 Ames tests and a host-mediated mutation assay are available for 2-phenylphenol (OPP). 

In a key study from 2021, 2-phenylphenol was investigated for gene mutation in bacteria according to OECD guideline 471 and in compliance with GLP (Ringelstetter, 2021). Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli strain WP2uvrA were exposed to the test item, negative (aqua dest), solvent (DMSO) and appropriate positive controls in the presence and absence of metabolic activation (S9 mix) for at least 48 h at 37 °C.  Concentrations for the test were selected based on the results of a preliminary experiment with strains TA98 and TA100. Two independent experiments were performed using the plate incorporation method (experiment 1) and the pre-incubation method (experiment 2). Triplicate cultures were exposed to concentrations in the range of 3.16 – 2500 µg/plate (experiment 1) and 1.0 – 2500 µg/plate (experiment 2). After at least 48 h of incubation at 37 °C, the bacterial background lawn was inspected and the mean number of revertant colonies was counted for each plate.

Precipitation of the test item was not observed for any condition up to the highest concentrations tested.Cytotoxicity was observed in all tester strains, i.e. in experiment I at concentrations of≥100µg/plate without S9 mix and at concentrations of≥316 µg/plate with S9 mix, depending on the strain, and in experiment II at concentrations of≥316 µg/plate with/-out S9 mix.

There was no significant increase in the frequency of revertant colonies noted for any of the bacterial strains at any dose level, neither with nor without S9 mix. The number of revertant colonies induced by the negative and solvent controls were within the range of the historical control data for each strain, thus demonstrating an acceptable experimental performance. Appropriate positive control compounds showed a strong increase in the number of revertant colonies, confirming the activity of the S9 mix and the validity of the test system. Based on the experimental findings, OPP was considered not mutagenic in bacteria with and without metabolic activation.

 

The remaining 11 Ames tests were conducted pre-guideline or according to OECD guideline 471 (1983), many of them with less than 5 valid strains or including only one experiment. These tests are therefore considered as supporting information. Nine of the supporting Ames assays confirm the negative test result which was observed in the key study (San and Springfield, 1989, Pagano et al., 1988, Shirasu et al., 1978, Moriya et al., 1983, Probst et al., 1981, McMahon et al., 1979, Cline and McMahon, 1977, Ishidate et al., 1984 and NTP, 1986).

For the Ames test by Haworth et al. (1983), which was conducted in strains TA98, TA100, TA1535 and TA1537 with and without metabolic activation, a weakly positive test result was noted for strain TA1535 in the absence of S9 mix. A second positive Ames test is available (Nishioka and Ogasawara, 1978) which was conducted in strains TA98 and TA100 in the presence and absence of S9 mix. In this test a weak positive result was obtained for strain TA98. For both Ames tests showing positive test results only limited documentation is available.

In a host-mediated assay (Shirasu et al., 1978), there was no increased reverse mutation rate noted in S. typhimurium G46 bacterial cells after i.p. injection in OPP-treated animals at 200 or 600 mg/kg bw/day and subsequent retrieval of a bacterial solution from the intraperitoneal compartment. Under the conditions of the study, the host-mediated assay is concluded to be negative for gene mutation in bacteria. The study is not following a standardised protocol and considered not reliable for evaluation on mutagenicity.

 

Conclusion on gene mutation in bacteria:

Considering all available data on gene mutation in bacteria and considering the results obtained in the new guideline key study (Ringelstetter, 2021), any potentially weak positive results reported in earlier studies were considered not relevant. It is concluded that OPP has no mutagenic properties in the Ames test, neither with nor without metabolic activation.

 

 

Gene mutation in mammalian cells

Mutagenicity in mammalian cells in vitro by OPP and its metabolites was investigated in mouse lymphoma assays (MLA), and hypoxantine guanine phosphoribosyl transferase (HGPRT) tests.

 

HGPRT tests

One OECD guideline study with OPP and a publication with the 2 metabolites PHQ and PBQ evaluating point mutation effects at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in mammalian cells are available.

The guideline study by Brendler (1992) was conducted according to OECD guideline 476 (1984) and in compliance with GLP and considered as key study. Duplicate cultures of CHO-WB1 cells were exposed to the test item, solvent (DMSO) or positive controls (ethylmethane sulfonate, 0.9 mg/mL for incubations –S9 mix and dimethylbenzanthracene, 20 µg/mL for incubations with S9 mix) for 5 h in the presence and absence of S9 mix. Test item concentrations were 6.25 – 100 µg/mL without S9 mix and 12.5 – 115 µg/mL with S9 mix. Following exposure, the cells were incubated for 7 days to allow expression of the mutant phenotype. The expression period was followed by a selection period, in which the cells were cultivated in 6-thioguanidine-enriched medium for 7 days.

Precipitation of the test material in medium was not observed. Cytotoxicity was observed at ≥ 75 µg/mL in the presence and absence of S9 mix.

There were some random statistical increases in mutant frequency in some doses in some of the experiments. The authors did include historical control data and they noted that while these increases in mutant frequency might be considered to be indications of a positive response, none of the values were outside of the historical control data range. It is noted that 3 trials were conducted because of the steep dose-response in cytotoxicity resulting in not achieving the desired level of cytotoxicity. Overall, despite some minor deficiencies in the study design relative to the current OECD guideline 476 (e.g., slightly fewer number of cells treated and carried through mutation expression period), this study informs that OPP is not likely to be anin vitromammalian cell mutagen. 

 

Mouse lymphoma assays

Three mouse lymphoma assays are available for OPP, all of them being considered as supporting information. The study conducted by Harbell (1989) was performed using a test procedure in principle similar to OECD 476 (1997). L5178Y mouse lymphoma cells were exposed at test item concentrations of 5 – 50 µg/mL in the presence and absence of S9 mix. Concurrent solvent (ethanol), untreated and positive controls (ethylmethane sulfonate for incubations –S9 mix and 3-methylcholanthrene for incubations +S9 mix) were included.

Cytotoxicity was observed at 100 µ/mL without S9 mix and at 50 µg/mL with S9 mix. OPP was negative for mutagenicity in the absence of S9 mix but positive for mutagenicity in the presence of S9 mix at cytotoxic concentrations.

The two NTP mouse lymphoma assays from 1985 and 1986 were both conducted with agar plates. The study from 1985 provides no experimental details but summarises results from a number of chemicals, including OPP, in tabular form. OPP was positive for mutagenicity in murine lymphoma cells, and caused sister chromatid exchanges at cytotoxic concentrations.

For the NPT study from 1986, basic data are given. L5178Y mouse lymphoma cells were exposed for 4 h in the presence and absence of metabolic activation (S9 mix) at 20 – 60 µg/mL (-S9 mix) and at 0.32 – 5 µg/mL (+S9 mix). Following exposure, the cells were incubated for 48 h to allow expression of the mutant phenotype. The expression period was followed by a selection period, in which the cells were plated out in the presence of trifluorothymidine for selection of mutants. OPP exposure was shown to cause a slightly increased mutant frequency in the presence and absence of S9 mix.

All 3 mouse lymphoma assays were conducted prior to the implementation of the previous guideline OECD 476. When compared to the current OECD test guideline 490, all assays show several deficiencies. In the study by Harbell (1989), a positive response was only observed at a cytotoxic concentration < 15% RTG. Further, none of the tests was evaluatedusing the global evaluation method, which requires a defined increase in the mutant frequency (the global evaluation factor; GEF) for the results to be considered positive. Another feature of the MLA is the presence of both small and large thymidine kinase mutant colonies that are reflective of the different types of genetic damage detected in the assay. The current OECD test guideline 490 outlines the requirements for demonstrating adequate mutant small colony recovery. Poor mutant small colony recovery can invalidate the results of an experiment and has been problematic, particularly in older MLA studies. 

Based on the study deficiencies, all 3 test results should be considered with caution.In conclusion, none of the mouse lymphoma experiments enables a final conclusion whether the test item OPP would be positive or negative in the mouse lymphoma assay.

 

 

Conclusion on gene mutation in mammalian cells in vitro:

In conclusion, the available data on mutagenicity in mammalian cells in vitro are contradictory and thus not sufficient for a final conclusion. However, mutagenicity in mammalian cells has also been addressed in in vivo studies. For details, please refer to the section below.

 

Cytogenicity in mammalian cells

Cytogenicity for OPP and its metabolites was investigated in a number of chromosome aberration tests, sister chromatid exchange assays and micronucleus tests in vitro.

 

Chromosome aberration tests and sister chromatid exchange tests

A total of 7 chromosome aberration tests in vitro are available for OPP (Tayama et al., 1989, Ishidate et al., 1984, Tayama-Nawai et al., 1984, Ishidate et al., 1988, Tayama and Nakagawa, 1991, NTP, 1985 and Shirasu et al., 1978).

For 4 of the published chromosomal aberration tests, a sister chromatid exchange assay was included and performed in parallel (Tayama et al., 1989, Tayama-Nawai et al., 1984, Tayama and Nakagawa, 1991 and NTP, 1985). All tests are considered to provide supporting information.

Three of the publications (Ishidate 1984 and 1988 and NTP 1985) revealed a negative test result with regard to chromosomal damage and/or sister chromatid exchange.

In 4 of the 7 publications, induction of structural and/or numerical chromosome aberrations was observed. However, the results obtained in the different studies are inconsistent. In two studies using CHO-K1 cells (Tayama et al., 1989 and Tayama and Nakagawa, 1991) a dose-dependent effect of OPP was observed in the presence of metabolic activation only. Clastogenicity was, however, observed only at cytotoxic concentrations. In another study in CHO-K1 cells (Tayama-Nawai, 1984), OPP was positive for clastogenicity in the absence of metabolic activation only. The damage was short-lived and could be repaired during the longer incubation time. In cultivated human foetal cells (Shirasu et al., 1978), an increase in chromosomal aberrations was observed in the absence of metabolic activation only.

All tests were conducted according to previous OECD 473 guideline versions, therefore a number of substantial changes to the conduct of the test became evident when compared to the most recent version of OECD 473 guideline (2016). The current guideline version recommends the use of the relative increase in cell count (RICC) or the relative population doubling (RPD) as a measure to assess cytotoxicity in cell lines and mitotic indices are recommended as a measure of cytotoxicity in primary cells. In addition, doses used for testing should be based on cytotoxicity measures and aim to achieve 55 ± 5% cytotoxicity. Historically, many experiments were performed without any assessment of cytotoxicity, or used measures no longer recommended. In the absence of these appropriate cytotoxicity measures, it is not possible to determine if positive results in a particular experiment resulted solely from excess cytotoxicity, or to determine if negative results were due to insufficient cytotoxicity. Thus, experiments not using appropriate cytotoxicity measures cannot be interpreted conclusively. The revision of OECD guideline 473 also included the recommendation to score at least 300 cells per concentration and there are new criteria for evaluating whether a result is positive or negative. 

The two studies by Ishidate et al. (1984 and 1988) which were both conducted in CHL cells have several methodological deficiencies when compared to the current guideline, including inappropriate exposure times and cytotoxicity measurements. No metabolic activation system was used. The studies both revealed a negative result, but due to the deficiencies it is not possible to draw any definitive conclusions based on the study result.

The studies by Tayama et al. (1989), Tayama-Nawai et al. (1984) and Tayama-Nakagawa et al. (1991) were conducted in CHO-K1 cells and also show methodological deficiencies with regard to inappropriate exposure times and cytotoxicity measurements. S9 mix was not always used. Although positive test results were obtained, the study results cannot be interpreted due to the large number of guideline deficiencies. SCEs were also evaluated in all 3 tests, but this endpoint is not appropriate for evaluating chromosome damage.

As a new, fully compliant in vitro micronucleus test was conducted with OPP (refer to section below), the available data on the potential of OPP to cause chromosome aberrations were only considered as supporting information.

 

Micronucleus tests

A micronucleus test according to the current version of OECD guideline 487 was performed in accordance with GLP (Donath, 2021) and considered to be a key study. In two independent experiments, Chinese hamster V79 cells were exposed for 4 h in the presence and absence of S9 mix (experiment 1) and for 24 h in the absence of S9 mix (experiment 2). Duplicate cultures were exposed to the test item, solvent (1% DMSO) and clastogenic as well as aneugenic positive controls (colchicine, 0.16 and 1.5 µg/mL for 24 h exposure –S9 mix, methylmethane sulfonate, 25 µg/mL for 4 h exposure –S9 mix and and cyclophosphamide, 2.5 µg/mL for 4 h exposure +S9 mix). Concentration levels were selected based on the results of a preliminary cytotoxicity test, ranging from 0.10 – 50 mM for the 4 h exposure and from 0.025 – 0.3 mM for the 24 h exposure. Following 4 h of exposure, cells were incubated for 20 h in cytochalasin B. For the long-term 24 h incubation, cytochalasin B was added 1 h after start of treatment and the cells were exposed to the test item or controls in the presence of cytochalasin for another 23 h. 24 h after start of treatment, all cells were harvested and slides were prepared. A total of at least 2000 binucleated cells were scored for the presence of micronuclei. In addition, cytotoxicity was evaluated as cytokinesis block proliferation index (CBPI).

Precipitation of the test item was not observed for any test condition. No relevant influence on osmolarity or pH was observed. Cytotoxicity was observed after 4 h of exposure at ≥ 0.3 mM –S9 mix and at ≥ 0.35 mM +S9 mix, as well as after 24 h of exposure at ≥ 0.1 mM –S9 mix.

In both experiments, in the absence and presence of S9 mix, no statistically significant or concentration-dependent increase in the number of micronucleated cells was observed. The positive controls showed distinct increases in cells with micronuclei, demonstrating the functionality of the metabolic activation system and the validity of the assay. Positive as well as solvent controls showed the expected results and fell within the laboratory’s historical control range for the number of micronuclei. Under the conditions of the test, OPP was considered negative for the induction of micronuclei in V79 cells in vitro, both, in the presence and absence of metabolic activation.

 

Cytogenicity of OPP metabolites in vitro:

Two further in vitro micronucleus tests are available for the OPP metabolites PHQ and PBQ (Eastmond et al. 1993 and Lambert et al., 1994). Both tests are considered to provide supporting information. In the liver, OPP is saturably metabolised to PHQ, typically by hepatic microsomal enzymes. PHQ can thereafter be transformed to PBQ, either in liver or elsewhere including in bladder, a metabolism step which is possibly mediated by prostaglandin H synthase (PGS) via phenylsemiquinone (PSQ), a predicted intermediate. OPP and the identified metabolites can be conjugated with glucuronide or sulfate and excreted, and all of these metabolites/conjugates (except PBQ itself) and parent OPP have been identified in rat and human urine (Smithet al., 1998; Bartelset al., 1998). Kolachana et al. (1991) have shown that the metabolisation of PHQ to the reactive metabolite PBQ by prostaglandin-H-synthase depends on the presence of arachidonic acid.In the 2 publications by Eastmond et al. (1993) and Lambert et al. (1994), the micronucleus tests in Chinese hamster V79 cells were therefore conducted with PHQ in the presence and absence of arachidonic acid and with PBQ in the absence of arachidonic acid.

The first publication by Eastmond et al. (1993) is only available as secondary literature with limited reporting. V79 cells were exposed to PHQ concentrations in the range of 31 – 187 µM with and without arachidonic acid or to 6 – 50 µM PBQ only (no metabolic activation). A modified protocol of the in vitro micronuclei test was applied using an anti-kinetochore antibody to determine whether the micronuclei contain kinetochores - a condition indicating chromosomal loss and aneuploidy. In the presence of arachidonic acid, PHQ concentrations of 125 – 187 µM induced a statistically significant and dose-related increase in micronucleated cells. This increase was not observed for PHQ in the absence of arachidonic acid or following treatment with PBQ. Immunofluorescent labeling of the kinetochores revealed that this increase in micronuclei was due entirely to an increase of kinetochore-containing micronuclei, indicating that a PHS-mediated oxidation product of PHQ was interfering with normal chromosome segregation. In the presence of prostaglandin-H-synthase inhibitors and antioxidising reagents, induction of micronuclei was inhibited. The authors concluded that the induction of micronuclei is mediated through prostaglandin-H-synthase and that free radicals are involved in this process.

 

The second publication by this group (Lambert et al., 1994) provides more details on the experimental procedure of the micronucleus test and was conducted according to a similar test design. Chinese hamster V79 cells were exposed to PHQ, PBQ or the solvent (1% DMSO) for 1 h in phosphate buffered saline (PBS). Concentrations for PHQ were in the range of 31 – 187 and for PBQ in the range of 6 – 50 µM. Treatment with PHQ was performed in the presence and absence of arachidonic acid. Following treatment, all cells were exposed for further 23 h in the presence of cytochalasin B.

Cytotoxicity was evaluated based on the nuclear division index and a colony forming assay was performed to determine effects on cell viability. A total of 1000 cells per condition were investigated for the presence of micronuclei. In addition, the frequency of CREST-positive and CREST-negative micronuclei was determined.

For PHQ a decrease in nuclear division index was observed at concentrations ≥ 93 µM. In the colony formation assay, cell survival after PHQ treatment with and without arachidonic acid decreased rapidly at concentrations ≥ 125 µM. Survival at 125 µM was approx. 85%, but decreased rapidly to 47% at 187 µM and to 21% at 250 µM. For PBQ no reduction in nuclear division index was observed up to and including the highest concentration tested. However, there was a substantial decrease in cell survival, evident by the colony formation assay, at all concentrations tested.

For PHQ, a statistically significant and dose-related increase in the frequency of micronuclei was noted at ≥ 125 µM in the presence of arachidonic acid. There was no significant change in micronuclei frequency when treated in the absence of arachidonic acid. The results suggest that prostaglandin-H-synthase-mediated activation of PHQ requires the presence of arachidonic acid as co-oxidant.

For PBQ, a weak but statistically significant increase in the formation of micronuclei was observed at the lowest (6 µM) and the highest test concentration (50 µM). All intermediate concentrations showed no induced frequency of micronuclei formation. The authors claimed that the frequency of micronuclei induced by the highest concentration, representing a cytotoxic dose, fell within the range of micronuclei observed in untreated cells and concluded that the observed weak genotoxic effect of PBQ is of less biological relevance.

The induction of micronuclei by PBQ and PHQ was explained by an increase in CREST-positive micronuclei. For both substances, there was no increase in CREST-negative micronuclei. The authors concluded that an oxidation product of PHQ interfered with the mitotic spindle, resulting in the formation of chromosome-containing micronuclei which failed to segregate properly during mitosis.

To demonstrate that the conversion of the test item to a genotoxic metabolite depends on prostaglandin-H-synthase, inhibitors of prostaglandin-H-synthase (acetylsalicyclic acid and indometacin) were included in the experiment in the presence of arachidonic acid. To evaluate the effect of antioxidants, ascorbic acid was included in the experiment in the presence of arachidonic acid. To evaluate the effect of sulfhydryl reagents, glutathion and dithiothreitol were included in the experiment in the presence of arachidonic acid. Aspirin and indomethacin, ascorbic acid, glutathione and dithiothreitol significantly inhibited micronucleus induction by PHQ in arachidonic acid-supplemented V79 cells. Indomethacin reduced the PHQ-induced frequency of micronuclei from 66 to 48 per 1000 binucleated cells, a 56% reduction as compared to the DMSO control. Treatment with aspirin, ascorbic acid, dithiothreitol or glutathione inhibited the PHQ-dependent induction of micronuclei by essentially 100%, reducing the number of micronuclei from 66 to 34 or less per 1000 cells. All of the treatments were shown to be significantly different from the PHQ-treated control. In each case the number of CREST-negative micronuclei remained within the range of control frequencies. The decrease in frequencies of micronuclei observed was due to reductions in the number of CREST-positive micronuclei.

Under the conditions of the test, PBQ is concluded to have a minor effect on the formation of micronuclei in V79 cells. Treatment with PHQ was associated with the formation of CREST-containing micronuclei in V79 cells when supplemented with arachidonic acid. No cytogenicity was observed for PHQ in the absence of arachidonic acid. Under the conditions of the test, PHQ is concluded positive for the induction of genotoxic effects in the presence of prostaglandin-H-synthase-mediated metabolic activation. The test results, however, should be treated with caution as the dose levels tested and the duration of exposure do not correspond with the recommendations of the current OECD test guideline. In addition, the number of metaphases evaluated was 1000 instead of 2000 cells/concentration.

 

Conclusion on cytogenicity in mammalian cells in vitro

Based on the available in vitro data, it can be concluded that OPP itself does not induce clastogenicity in vitro, however, its metabolite PHQ induces micronuclei in the presence ofprostaglandin-H-synthase-mediated metabolic activation.

 

 

DNA damage and repair

Unscheduled DNA synthesis (UDS test)

OPP was tested for its potential to induce unscheduled DNA synthesis in primary rat hepatocytes in vitro according to OECD guideline 482 (Probst et al., 2005,). Hepatocytes were isolated from male Fischer 344 rats and exposed to the test item at a concentration of 100 nmol/mL in the presence of³H-thymidine.

After 5 h of incubation, the cells were exposed for additional 18 – 20 h. In a repeat experiment, the cells were exposed for 20 h. After termination of the experiment, cells were washed, fixed, stained with aceto-orcein, developed and examined by oil immersion microscopy.

OPP turned out negative for induction of UDS. It has to be noted though that the OECD test guideline 482 is no longer a standard method and has been deleted in 2014.

 

 

Induction of DNA oxidative damage

Henschke et al. (2000) evaluated induction of oxidative damage by OPP and its metabolites PHQ and PBQ in Chinese hamster V79 cells. The formation of 8-OH-dG was monitored and an alkaline elution assay was performed to investigate the induction of DNA single strand breaks.

OPP itself did not cause DNA single-strand breaks or 8-OH-dG formation. The metabolites PHQ and PBQ caused a significant increase in both parameters at non-cytotoxic concentrations.

 

DNA repair in E. coli

Limited information is available on a study by Nishioka and Ogasawara (1978), who evaluated DNA repair induced by OPP in E. coli strains E. coli WP2, WP2uvrA, CM571 and WP100. The outcome was reported as positive, but this is not a standardized test.

 

Rec assay

DNA damaging activity by OPP was further investigated in a Rec assay with Bacillus subtilis strains H17 and M45 conducted by Shirasu et al. (1978). Both strains were exposed to concentrations of 2 – 1000 µg/plate. Vehicle (DMSO), negative (kanamycin, 10 µg/plate) and positive controls (methylmethanesulfonat, 0.1 µ/plate) were included. After overnight incubation at 37 °C, the length of the growth inhibition zone was measured for each strain.

No relevant growth inhibition of the bacteria strains H17 (Rec+) or M45 (Rec-) was observed under any tested condition, neither in the presence, nor in the absence of metabolic activation. Positive, negative and vehicle controls showed the expected results and demonstrated the validity of the test system. Based on the results of the present study, OPP has no DNA-damaging activity in bacteria in the presence and absence of S9 mix.

 

DNA adduct formation with OPP metabolites

A number of publications are available investigating if the OPP metabolites PHQ and PBQ form DNA adducts in vitro. This was investigated by using the 32P-postlabeling method and autoradiography.

In a study by Horvath et al. (1992), HL-60 cells were exposed for 8 h to up to 500 µM PHQ or for 2 h to up to 250 µM PBQ. Both metabolites were shown to form DNA adducts, yielding one major and 3 minor products. DNA adducts were formed in a dose-dependent manner, reaching a plateau for PBQ at ≥ 100 µM. In addition, PHQ was shown to form DNA adducts with foetal calf thymus DNA, resulting in one major adduct. Co-chromatography showed that this adduct did not correspond to the major adduct formed in HL-60 cells treated with PHQ or PBQ. The authors suggested that either the quinone is not the reactive intermediate leading to DNA adduct formation or that the cellular environment influences the adduct formed by the quinone.

Further, Pathak and Roy (1992) demonstrated that PHQ chemically reacts with deoxyguanosine-3’-phosphate, forming 4 major and several minor adducts. The chromatographic mobility of the major adducts was identical to those obtained by reaction of PHQ with DNA. The same DNA adducts were further noticed for OPP and PHQ in the presence of metabolic activation (microsomes and NADPH or cumene hydroperoxide). In the presence of known cytochrome P450 inhibitors, DNA adduct formation was distinctly decreased.

Both studies have several deficiencies, as for example positive controls or historical control data were not included in any of the assays. Despite certain evidence for DNA adduct formation in vitro, DNA adduct formation in vivo was not confirmed (refer to section below).

Finally, the publication by Zhao et al. (2002) demonstrated that PHQ covalently binds to DNA and forms DNA adducts by reaction with 2’deoxyguanosine. In HepG2 liver cells, the formation of DNA adducts was, however, only demonstrated to occur at cytotoxic concentrations.

 

 

Conclusion on DNA damage and repair in vitro:

Contradictory results on OPP-induced DNA damage and repair were obtained in a series of non-standardized in vitro studies. Based on the available data, no final conclusion on a DNA damaging effect of OPP is possible. However, evidence for DNA adduct formation and induction of DNA oxidative damage in mammalian cells was observed with the metabolites PHQ and PBQ. The endpoint has further been addressed in a number of in vivo studies. For details, please refer to the section below.

 

In vivo

GENETIC TOXICITY IN SOMATIC CELLS

Cytogenicity in vivo

Cytogenicity in vivo was assessed by conduction of an in vivo chromosome aberration test and 3 micronucleus tests in the bone marrow and/or bladder. In addition, an in vivo study for aneuploidy in the bladder of rats was conducted.

 

Chromosome aberration test in vivo:

OPP-induced structural and numerical chromosome aberrations in the bone marrow of rats were investigated in a study by Shirasu et al. (1978). The study was conducted similar to OECD guideline 475 in Wistar rats. The test item was dissolved in gum arabic and administered by oral gavage. Groups of 4 animals each were treated at either a single dose of 250, 500, 100, 2000 and 4000 mg/kg bw OPP or with 5 daily doses at 50, 100, 200, 400 and 800 mg/kg bw/day. Similar constituted groups of animals received the vehicle or the positive control mitomycin C (5 mg/kg bw by i.p. injection).

Three hours prior to sacrifice, all animals received an intraperitoneal injection with colchicine. Bone marrow was sampled 24 h post administration for all groups. Slides were prepared and a total of 50 metaphases per animal were scored for the presence of structural chromosomal aberrations.

OPP induced no statistically significant and no dose response-related increase in structural or numerical chromosome aberrations after single or repeated exposure. However, bone marrow exposure to the test substance was not demonstrated in this study and only 50 instead of 200 metaphases per animal were investigated. The negative outcome of this study should therefore be considered with caution.

 

Micronucleus test in vivo and aneuploidy in rat bladder:

In vivo data of three micronucleus tests as well as one study on aneuploidy in rats conducted with OPP were published by the group of Balakrishnan and Eastmond (2002, 2003, 2006 and 2016). Most of the studies were conducted in the bladder of the male rat, which is the target organ for the tumours observed in experimental animals.

In the first micronucleus test by this group (Balakrishnan et al., 2002), groups of 4 male rats were administered a dietary dose of 2% OPP (corresponding to approx. 2000 mg/kg bw/day) or 2% OPP combined with 2% sodium chloride for 14 days. Similar constituted groups of control animals received either 2% sodium chloride incorporated into the diet or plain diet. The bladders of the animals were investigated for induction of micronuclei and induction of BrdU-stained cell proliferation. In addition, induction of hyperdiploidy was determined by fluorescence in situ hybridization (FISH) at a single chromosome (chromosome 4). Statistically significant increases in micronuclei and in cell proliferation of epithelial bladder cells was observed for all conditions, but no hyperdiploidy or polyploidy was observed. As an increase in micronuclei was also observed for 2% sodium chloride alone, the assay’s specificity for identifying genotoxic effects is questionable.Given that sodium chloride has a postulated mechanism of excess osmolality to induce genotoxic/mutagenic effects, it is clearly a high-dose phenomenon. Based on the results of a subchronic repeated dose toxicity study with OPP (Smithet al., 1998), neither 8000 nor 12500 ppm OPP (corresponding to approx. 800 and 1250 mg/kg bw/day)[1]affected urine osmolality, thus rendering moot this possible genotoxic/ mutagenic MOA for induction of MN in bladder epithelial cells by OPP (viaincreased urine osmolality).

 

In a follow up study, Balakrishnan and Eastmond (2003) focused on a dose-response for hyperdiploidy in the bladder of male Fischer 344 rats and tested dose levels up to 12500 ppm OPP (corresponding to approx. 1250 mg/kg bw/day)¹.To increase the sensitivity of the FISH method for detection of hyperdiploidy, the authors used BrdU deliveryviamini-pumps and collagenase digestion of the bladder to obtain an enriched fraction of actively replicating bladder epithelial cells. Replicating cells and non-replicating cells were analysedviaFISH, this time for 2 chromosomes (4 + 19) to enhance the sensitivity of FISH. They also included a positive control for hyperdiploidy, treating an 8000 ppm OPP group with vinblastine, to demonstrate that their method was able to detect this effect. A statistically significant increase in cell proliferation of bladder cells was noted, but no detectable increases in hyperdiploidy were induced by OPP alone. In the OPP + vinblastine group, both chromosome 4 and chromosome 19 showed statistically increased hyperdiploidy and polyploidy. No increases in hypodiploidy were seen in any of the treatment groups as compared to the control. In conclusion, 14-days treatment with OPP alone did not result in any increases in hyperdiploidy or hypodiploidy in male rat bladder epithelial cells.

 

In the second micronucleus test (Balakrishnan, S. and Eastmond, D.A., 2006) a similar test design was used. Fisher 344 rats were daily treated with the test item at dietary doses of 2000, 4000, 8000, or 12500 ppm, respectively, for 15 days (corresponding to ingested doses of approx. 148, 320, 644 and 1114 mg/kg bw/day). 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 12500 ppm OPP, which were doses shown to produce cytotoxic effects in the target tissue. CREST staining indicated that the micronuclei resulted from both, whole chromosome loss and breakage. At the same time, bone marrow cells of animals treated with 8000 ppm OPP did not show increased micronuclei formation. Animals treated with 2000 and 4000 ppm OPP showed neither an increase in cell proliferation nor an increase in micronuclei in the bladder epithelial cells over the control animals.

In this study, no assessment of bone marrow toxicity, e.g. depression of bone marrow proliferation, was included 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 SOPP and their metabolites, reach the bone marrow in sufficient quantities. Thus, overall, the test item was concluded to be not a primary clastogenic agent under the conditions of this test.

 

A more complex study design was used for the third study published by this group (Balakrishnan et al., 2016), which integrated in vitro as well as in vivo data. The in vitro studies evaluated cytotoxicity induced in the human-derived lymphoblastoid TK-6 cell line and the rat bladder epithelial cell-derived NBT-II cell line upon treatment with the OPP metabolite PHQ at varying pH values. Abrupt increases in cytotoxicity were noted for both cell lines at pH <7.2. Cytotoxicity > 50% was noted at pH 7.4 for concentrations > 200 µM PHQ. The increase in cytotoxicity at increasing alkaline pH was likely due to increased PBQ formation via autoxidation of PHQ.

The subsequent in vivo micronucleus test was conducted to investigate the potential role of pH-dependent autoxidation in the formation of damage in the bladder after OPP treatment. Male Fisher F344 rats were fed a diet containing OPP and urine-modifying dietary salts for 15 days. OPP dose levels of 4000 and 8000 ppm were incorporated into the diet (corresponding to ingested doses of approx. 270-280 and 550-600 mg/kg bw/day) supplemented with either 1% ammonium chloride or 3% sodium bicarbonate. These salt treatments resulted in either acidic (ammonium chloride) or alkaline (sodium bicarbonate) urinary pH, compared to the control urinary pH. Control animals were administered plain diet or corresponding salt levels without OPP. The body weight, food and water consumption were monitored for all animals and urinary pH as well as urinary protein concentration were monitored on a regular base. Upon sacrifice, the bladder epithelial cells were evaluated for induction of cell proliferation and micronucleus formation. Further, to determine the origin of the micronuclei, the presence of centromeric anti-kinetochore proteins within the micronuclei was determined using a CREST antibody.

For animals administered diet with no salts, the urinary pH was in the range of 7.2 - 7.3. Animals administered ammonium chloride-enriched diets showed an urinary pH of 5.7 - 5.8. Administration of sodium bicarbonate in the diet increased the mean urinary pH to 7.8 - 8.0. Treatment with the test item was associated with a decrease in urinary proteins for normal and for salt-enriched diet. The decreases were seen with all treatments with the test item, except for 8000 ppm + 3% sodium bicarbonate, where the total urinary protein values were comparable to those of control levels. Although body weights were not different, food consumption was slightly higher (most likely due to crumbled, unpalatable dietary mix) and water consumption was increased for animals administered sodium bicarbonate + OPP animals.

Statistically significant increases in bladder cell proliferation as detected by BrdU incorporation and micronucleus formation were observed in the bladder cells of OPP-treated animals with either neutral or alkaline urinary pH after sodium bicarbonate treatment. Treatment with 8000 ppm OPP and 8000 ppm OPP + sodium bicarbonate induced a statistically significant increase in micronuclei formation and significantly increased cell proliferation, as demonstrated by BrdU incorporation. The mean frequencies of micronuclei in the rats treated with 4000 ppm OPP + sodium bicarbonate were also statistically significantly higher than those in the rats treated with the sodium bicarbonate-enriched control diet, and a variable induction of cell proliferation was observed. No such effects were noted in animals with acidified urine administered OPP and 1% ammonium chloride treatment in the diet. Under the conditions of the test, OPP is considered a genetic toxicant in urinary bladder cells of male rats under neutral and alkaline conditions.

 

Bomhard, E. M. et al. (2002) and Brusick, D. (2005) summarise various further studies investigating genotoxicity of OPP, its sodium salt SOPP or their metabolites in vivo. They come to the conclusion that OPP does not possess a clastogenic potential relevant under in vivo conditions. The mostly slight increases in chromosomal aberrations after treatment of different cell lines essentially under S9 mix activation in vitro occurred at concentrations that were severely toxic. On the basis of the upper cytotoxicity limit defined in current guidelines, the results of most of these studies would be estimated as unreliable. Bomhard, E. M. et al. (2002) suggest that the induction of chromosome aberrations could be inhibited by the addition of glutathione or cysteine. The various in vivo studies looking for chromosomal aberrations under different, partly long-term high-dose treatment conditions gave no indication of a clastogenic effect in the bone marrow of rats and mice. Although the bone marrow is not the target of carcinogenicity, the available toxicokinetic data allow the conclusion that OPP, its sodium salt SOPP and their metabolites have reached it in sufficient quantities. The increase in micronuclei in the urinary bladder after high-dose feeding of OPP and SOPP over 14 days does not contradict the aforementioned thesis. Under these conditions they could have developed as a secondary response to toxic changes (Bomhard, E. M. et al., 2002).

 

 

Conclusion on cytogenicity

Test results on the potential of OPP to cause clastogenic or aneugenic effects obtained from in vivo studies are contradictory. However, any cytogenic effects of OPP or its metabolites reported were observed in the target organ of tumorigenic effects in male rats only, the urinary bladder. In addition, the studies evaluating cytogenicity in the urinary bladder of rats did not follow any regulatory guidelines or GLP requirements. Methods were not validated and positive controls were not included in the experiments. However, these studies provided some useful and valuable information on the potential mode of action of the tumorigenic effects observed in the bladder in male rats.

In contrast, a negative test result was obtained in a (not fully guideline compliant) micronucleus test in rat bone marrow cells, which was conducted according to a validated protocol and represents the gold standard for evaluation of cytogenicity in vivo. The results were supported by a chromosome aberration assay similar to OECD guideline 475 with some deficiencies and a fully guideline and GLP compliant negative in vitro micronucleus test. In conclusion, the available data indicate that it is unlikely that OPP is a directly acting aneugenic or clastogenic agent in vivo.

 

 

DNA damage in vivo

Comet assay

Two Comet assays in vivo are available investigating the potential of OPP to induce DNA strand breaks, one publication (non-GLP, Sasazi et al., 1997) and a more recent follow-up GLP-compliant OECD 489 guideline study (Brendler-Schwaab, 2000). Both studies were conducted in male CD-1 mice and followed a similar test design. The test item was dissolved in olive oil and administered to groups of 4 mice. A similar constituted group of control animals received the vehicle only. The animals were observed for clinical signs of toxicity and were sacrificed 3, 8 or 24 h after treatment.

In the first study (Sasaki et al., 1997), the animals were administered 2000 mg/kg bw OPP and cell suspensions were obtained from the stomach, liver, kidney, bladder, lung, brain and bone marrow. Slides were prepared and following alkaline electrophoresis, 50 cells/animal/organ were scored for comet length and head diameter. No deaths, morbidity, or distinctive clinical signs were observed after treatment. Necropsy did not reveal any treatment-related effects in any of the organs examined. OPP yielded significant DNA damage in the stomach, liver, kidney and lung 3 h after administration. DNA damage in liver and lung returned to control levels within 8 h, whereas increased migration persisted for 8 h in stomach and kidney. Increased migration was also significant in bladder DNA at 8 h and 24 h with a peak at 8 h. No increase in DNA migration was observed in brain and bone marrow. The study is considered to provide supporting information as only limited information on purity is available and technical aspects of the study may be questioned. Only a small amount of cells was investigated, positive controls were not included and cytotoxicity measurements in the respective organs were not conducted.

 

The more recent OECD guideline study (Brendler-Schwaab, 2000) was conducted similar or equivalent to OECD guideline 489 and selected as key study. Based on the previous publication by Sasaki et al. (1997), a single dose of 250 or 2000 mg/kg bw was administered by oral gavage to groups of 4 male CD-1 mice. The pitfalls of the previous study were avoided. Vehicle (olive oil) and positive controls (ethylmethanesulphonate, 400 mg/kg bw) were included in the experiment. 3, 8 or 24 h after treatment cell suspensions were obtained from livers and kidneys only. Following slide preparation and gel electrophoresis, a total of 100 cells/animal was scored for DNA damage, which was reflected as tail length.

The animals of the high dose group showed clinical symptoms and 2/12 animals died. No cytotoxicity was observed in hepatocytes and kidney cells in any treatment group. There was further no statistically significant or biologically relevant increase in tail length for the liver as well as the kidneys at any dose level. Tail lengths of the vehicle and positive controls showed the expected results, thus demonstrating the sensitivity and validity of the test. Under the conditions of the study, OPP did not induce DNA strand breaks in mice in vivo.

 

Alkaline elution assay

Two studies by Morimoto et al. (1987 and 1989) were conducted to investigate the relationship between urinary metabolites and DNA damage in the urinary bladder epithelium in rats. In an alkaline elution assay in rats, OPP, as well as PHQ and PBQ were administered to 5-weeks old male F344 rats by intravesical injection through the bladder wall. After 10 min of exposure, the bladders were excised and cell suspensions of the epithelial cells were obtained. The cells were lysed and single-stranded DNA was eluted with alkaline solution.

In both studies, DNA-damaging effects were observed for PBQ at 0.1 and 0.5%. No effects in the urinary bladder epithelium were observed for OPP or PHQ. The studies are considered to provide supporting information, as no guideline was followed and because the route of application is not relevant for human exposure.

 

 

DNA adducts in vivo

The potential of OPP/SOPP or the metabolite PHQ to form DNA adducts was addressed in several studies and organs.

In a publication by Pathak and Roy (1993, study summary not included within the dossier), SOPP and PHQ were shown to covalently modify DNA in the skin in female CD-1 mice. SOPP or PHQ were topically applied to groups of 6 females at dose levels of 10 or 20 mg SOPP or 5 mg PHQ. The animals were sacrificed 4 h after single exposure and the formation of DNA adducts was determined using the 32P-postlabelling method. Four distinct major and several minor adducts in skin DNA were noticed for SOPP and PHQ, but not in untreated animal skin. Pre-treatment with 10 mg alpha-naphthylisothiocyanate, an inhibitor of CYP P450, or 10 mg indomethacin, an inhibitor of prostaglandin synthase, reduced DNA adduct formation by SOPP, indicating the conversion of OPP to DNA-binding metabolites by 2 metabolic pathways, i.e. cytochrome P450 and prostaglandin synthase mediated pathways.

 

DNA adducts were further examined in urinary bladders of male rats after subchronic treatment with OPP (Christenson et al. 1996; as cited in Bomhard et al., 2002 and Brusick, 2005). Male CDF[F-344]/BR rats were given OPP at dietary levels of 800, 4000, 8000, and 12500 ppm (corresponding to approx. 80, 400, 800, and 1250 mg/kg bw/day)¹for 13 weeks. During weeks 12-13 and 13-14 of the study, urine was collected for metabolite and general urinalysis determinations, respectively. In addition, urinary bladders were collected during week 14 to perform a 32P-postlabelling analysis for the DNA adduct analysis on the urothelium, while histopathological evaluations included determination of a BrdU-labelling index as well as light and scanning electron microscopy (SEM only on 0 and 8000 ppm group). ESI-LC/MS and GC/MS analysis revealed that glucuronide and sulphate conjugates of OPP and the hydroxylated metabolite, 2,5-phenylhydroquinone (PHQ), were the major urinary metabolites. For all dose groups, the major conjugate present was the sulphate conjugate of OPP. Minute levels of free OPP and PHQ were observed in all dose groups, with free PHQ comprising 0.6-1.5% of the total metabolites measured. Only at 8000 and 12,500 ppm, signs of systemic toxicity were noted, indicated by reduced body weights without changes in food consumption. Animals of these dose groups showed simple hyperplasia of the urothelium at histopathological examination and significant bladder changes during SEM analysis. Cell proliferation of the bladder epithelium, examined by means of BrdU-labelling index, was also increased in these animals. 32P-postlabelling of rat urothelial DNA did not show any evidence for formation of OPP-DNA adducts. Thus, Bomhard et al. (2002) postulated that OPP acts by a mechanism producing cytotoxicity followed by an increase of mitotic activity and regenerative hyperplasia of the urothelium at high dose levels ≥ 8000 ppm. Sustained cell proliferation/tissue damage may act as a precursor of increased tumour incidences. However, as no DNA adducts were formed by OPP or its metabolites after repeated exposure, genetic toxicity caused by direct interaction of OPP or its metabolites with DNA is considered unlikely.

 

Conclusion on DNA damage in vivo

Based on the results of the Comet assay which serves as key study and the available information on formation of DNA adducts after repeated exposure in vivo, OPP and its metabolite PHQ are considered to have no direct genotoxic effect in vivo.

 

GENOTOXICITY IN GERM CELLS

DNA damage in vivo

A dominant lethal test in the germ cells of male SLC-C3H mice was conducted by Shirasu et al. (1978). The test was conducted similar to OECD guideline 478 prior to GLP. Groups of 5 mice were administered daily oral doses of 100 or 500 mg/kg bw/day OPP for 5 consecutive days.Each treated male was mated to 2 virgin syngeneic females for 6 consecutive weeks to investigate dominant lethality in various germ cell stages. The numbers of corpora lutea, implantations, live pups, and early and late dead embryos were counted.

There was an 8% depression in absolute body weight of males treated with 500 mg/kg bw/day OPP suggesting that this dose represented an MTD. Indeed, 500 mg/kg/day is above the level of metabolic saturation for OPP and thus can be considered a Kinetically-determined Maximum Dose (KMD) (EPA, 2019; Bartelset al.,1998, Reitzet al., 1983; Smithet al., 1998). Use of a top dose demonstrating metabolic saturation, which for OPP would be >200 mg/kg bw, is an acceptable approach according to OECD test guideline 478 (2016). There was no effect of treatment on dominant lethality in male germ cells. As it is generally accepted that dominant lethals are due to structural and numerical chromosome aberrations, OPP did not cause chromosomal aberrations in germ cells in vivo. Under the conditions of the test, OPP is not a germ cell mutagen.

 

Conclusion on germ cell mutagenicity/chromosome aberrations in germ cells in vivo

Based on the available data it can be concluded that OPP has no mutagenic properties in germ cells in vivo.

 

Overall conclusion on genetic toxicity

Overall, OPP is considered not mutagenic in bacteria. The in vitro experiments on mutagenicity in mammalian cells provided ambiguous results not suitable for drawing a conclusion. OPP itself was not found to induce clastogenicity in vitro as evidenced by a fully compliant GLP OECD 487 guideline study. However, its metabolite PHQ was found to induce micronuclei in a non-validated test in V79 cells in vitro in the presence of prostaglandin H-synthase-mediated metabolic activation. The absence of cytogenicity by OPP was also demonstrated in rat bone marrow in vivo in a not fully guideline-compliant and non-GLP micronucleus test.

A number of non-validated, non-GLP studies on cytogenicity in the target organ of OPP induced toxicity, the urinary bladder, were however associated with increased cell proliferation of bladder cells, in combination with micronuclei formation at either neutral or alkaline urinary pH. However, the results obtained in the micronucleus assays in the bladder of male rats need to be considered with caution as this test system does not represent an OECD guideline-conform, validated test system. No historical control data nor positive controls are available for the studies by Balakrishnan and Eastmond (2002, 2006 and 2016).

With regard to DNA damage and effects on DNA repair mechanisms, OPP turned out to be negative in vitro as well as in vivo, based on a weight of evidence approach. In a number of in vitro tests following out-dated testing protocols, OPP was negative for unscheduled DNA synthesis, induction of DNA single strand breaks, induction of DNA oxidative damage and for DNA-damaging activity in Bacillus subtilis strains. Positive effects were observed for induction of DNA repair mechanisms in E. coli strains. In addition, the metabolites PHQ and PBQ were found to cause DNA single-strand breaks as well as 8-OH-dG formation. As the tests do not represent standardised methods and as the inclusion of controls was not reported, the results have to be considered with caution. However, DNA damage in vivo, assessed by a Comet assay performed in liver and kidney and by the investigation of DNA adducts in urinary bladders in male rats after subchronic treatment, was concluded negative for OPP.

At cytotoxic concentrations, OPP and its metabolites PHQ and PBQ were found to form DNA adducts in vitro, but these results were not confirmed in in vivo studies. As no DNA adducts were formed by OPP in bladder epithelial cells in vivo after repeated exposure,genetic toxicity caused by direct interaction of OPP or its metabolites with DNA in vivo is unlikely. In addition, in a reliable Comet assay in liver and kidney cells in vivo, no DNA-damaging properties for OPP became evident.

Overall, there is significant weight of evidence that a DNA-reactive/mutagenic mode of action is not operational with OPP. The proposed mode of action with regard to formation of genotoxic metabolites (PHQ and PBQ) in bladder epithelial cells under neutral and alkaline conditions in vivo is not reflected in changes of osmolality and pH in urine in repeated dose toxicity studies.

With no reliable evidence of direct DNA reactivity in bladder epithelial cells, and no validated data supporting a genotoxic mode of action, there is significant weight of evidence that a genotoxic/mutagenic mode of action is also not operational for OPP. Finally, bladder represents an isolated compartment, one where special conditions exist that appear to contribute to the effects noted in bladder epithelial cells, including presence of high levels of prostaglandin H synthase, possibly more alkaline pH, and a long residence time (intermittent urination), all of which contribute to potential increased formation of reactive metabolites from OPP. This is demonstrated by the data showing that manipulation of urinary pH significantly affects cell proliferation in bladder and induction of micronuclei in bladder epithelial cells (Balakrishnanet al., 2016), where low pH blocks these effects, while high pH augments them. Such conditions of high pH are only of limited relevance to humans, where low urinary pH (~6) represents the normal state. However, the American Association for Clinical Chemistry provides a normal urine range between 4.5 and 8. Therefore, a potential effect of a slightly alkaline urine on induction of micronuclei by OPP cannot be completely disregarded. The mechanism for micronuclei induction in the novel, non-validated bladder micronucleus assay is likely mediated by the urinary protein binding with OPP, which was demonstrated to occur only at high dose levels (above metabolic saturation). Thus, a non-linear/threshold dose-response would be expected for this effect.

Finally, no evidence for mutagenicity or DNA damaging activity by OPP was found in germ cells, as demonstrated ina not fully guideline compliant dominant lethal test inmale SLC-C3H mice. OPP is therefore considered unlikely to be a germ cell mutagen and does hence not require classification as a germ cell mutagen.

 

Assessments by national authorities

The German MAK commission (2016) evaluated the existing numerous genotoxicity and mutagenicity studies performed with OPP and SOPP and concluded that the clastogenic effects are dose-dependent occurring at high dose levels only, accompanied by overt cytotoxic effects in the target tissue. Thus, the observed genetic toxicity is regarded to be not a primary effect, but considered as secondary consequence of cell damage and the proliferation in the epithelium of the urinary bladder. Moreover, the MAK commission discussed the impact of the pH in the urine on the micronucleus formation after treatment with OPP for the clastogenic effects, which was proven by an additional study by Balakrishnan und Eastmond (2002) cited in MAK (2016).

Overall, the weight of evidence from the combined database indicates that OPP/SOPP-induced DNA damage is a threshold-dependent response associated with target tissue toxicity, most likely induced by their breakdown products phenylhydroquinone and phenylbenzoquinone. It can be assumed that this threshold-dependent clastogenicity contribute to the carcinogenic mode of action for OPP or SOPP in male rats (Brusick, D., 2005). Moreover, it can be concluded, that OPP does not interact as primary mutagen and therefore a genotoxic potential was excluded (MAK, 2016).

This view is in general agreement with the evaluations of FAO-WHO (1999), US-EPA (2006) and EU EFSA (2008), which come to the overall conclusion that OPP is not a genetic toxicant.

 

References:

Bartels M.J., McNett D.A., Timchalk C., Mendrala A.L., Christenson W.R., Sangha G.K., Brzak K.A., Shabrang S.N. 1998. Comparative metabolism of ortho-phenylphenol in mouse, rat and man. Xenobiotica. 28(6):579-594.

 

EU-EFSA (2008): Conclusion on pesticide peer review: Peer review of the pesticide risk assessment of the active substance 2-phenylphenol. EFSA Scientific Report 217, 1-67.

 

FAO-WHO (1999).2-phenylphenol. In: Pesticide residues in food–2002. Report of the 1999 Joint Meeting of the FAO Panel of Experts on Pesticide Residues in Food and the Environment and the WHO Core Assessment Group on Pesticide Residues.

 

Kolachana P., Subrahmanyam V.V., Eastmond D.A., Smith M.T., 1991.Metabolism of phenylhydroquinone by prostaglandin (H) synthase: possible implications in o-phenylphenol carcinogenesis.Carcinogenesis. 12(1):145-49

 

MAK (2016).ortho-Phenylphenol (OPP) und ortho-Phenylphenol-Natrium (OPP-Na). MAK Value Documentation in German language.The MAK Collection for Occupational Health and Safety, 1(2):1067-1110.

 

Pathak, D.N. and Roy, D., 1993. In vivo genotoxicity of sodium ortho-phenylphenol: phenylbenzoquinone is one of the DNA-binding metabolite(s) of sodium ortho-phenylphenol. Mutation Research, 286: 309 - 319

 

Reitz, R.H., Tox, T.R., Quast, J.F., Hermann, E.A and Watanabe, P.G., 1983. Molecular mechanisms involved in the toxicity of othophenylphenol and its sodium salts.Chem Biol Interact. 43(1):99-119

 

Smith R.A., Christenson W.R., Bartels M.J., Arnold L.L., St John M.K., Cano M., Garland E.M., Lake S.G., Wahle B.S., McNett D.A., Cohen S.M., 1998. Urinary physiologic and chemical metabolic effects on the urothelial cytotoxicity and potential DNA adducts of o-phenylphenol in male rats. Toxicol Appl Pharmacol. 150(2):402-413.

 

US-EPA (2006): Reregistration Eligibility Decision for 2-phenylphenol and Salts (Orthophenylphenol or OPP). 739-R-06-004

 

[1]Conversion of ppm in mg/kg bw/day based on Table 28 of Derelanko, The Toxicologist’s Pocket handbook, 2^ndedition, 2008

Justification for classification or non-classification

The available data on genetic toxicity in vitro and in vivo do not meet the criteria for classification according to Regulation (EC) No. 1272/2008 and are therefore conclusive but not sufficient for classification.