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Toxicological information

Genetic toxicity: in vivo

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Administrative data

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

Data source

Reference
Reference Type:
publication
Title:
Micronuclei and cell proliferation as early biological markers of orthto-phenylphenol-induced changes in the bladder of male F344 rats
Author:
Balakrishnan, S. and Eastmond, D.A.
Year:
2006
Bibliographic source:
Food Chem. Toxicol. 44:1340-1347

Materials and methods

Test guideline
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

Test material

Constituent 1
Chemical structure
Reference substance name:
Biphenyl-2-ol
EC Number:
201-993-5
EC Name:
Biphenyl-2-ol
Cas Number:
90-43-7
Molecular formula:
C12H10O
IUPAC Name:
[1,1'-biphenyl]-2-ol
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

Test animals

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

Administration / exposure

Route of administration:
oral: feed
Vehicle:
- Vehicle used: none
Duration of treatment / exposure:
15 days
Frequency of treatment:
daily
Post exposure period:
none
Doses / concentrationsopen allclose all
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

Examinations

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.

Results and discussion

Test resultsopen allclose all
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.

Applicant's summary and conclusion

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.