Biphenyl-2-ol

Carcinogenicity

Description of key information

Oral:

Combined chronic toxicity and carcinogenicity (OECD 453, rat, m/f): NOAEL (carcinogenicity) = 200 mg/kg bw/day

Inhalation: data not required according to Annexes VIII-IX of Regulation (EC) No 1907/2006
Dermal: data not required according to Annexes VIII-IX of Regulation (EC) No 1907/2006

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP study which meets basic scientific pinciples

Principles of method if other than guideline:

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

GLP compliance:

yes

Specific details on test material used for the study:

- 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: 9-10 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:

other: 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

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

confirmed in stability in rodent diet at room and freezer temperatures for 14 and 28 days, respectively

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily

Post exposure period:

approximately 4 weeks

Remarks:

Doses / Concentrations:
1000, 4000, and 12,500 ppm
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
54±2, 224±9, 684±22 mg/kg bw
Basis:
other: mean daily intake calculated from feed consumption, body weight, and diet analysis data

No. of animals per sex per dose:

- vehicle control group: 30 males
- 1000 ppm group: 20 males
- 4000 ppm group: 20 males
- 12,500 ppm group: 30 males

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on an published data; doses are consistent with those necessary to profile urinary bladder toxicity
- Post-exposure recovery period in satellite groups: approximately 4 weeks

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a week

BODY WEIGHT: Yes
- Time schedule for examinations: once a week

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

URINALYSIS: Yes
- Time schedule for collection of urine: in various intervals throughout the study
- Metabolism cages used for collection of urine: Yes (for overnight urine). Fresh urine samples were collected either through spontaneous micturition subsequent to bein picked up and handled by the technican, or, if necessary, urination was "coaxed" or "induced" by the handling technican through the application of a slight pressure to the abdomen of the rat.
- Parameters checked: pH, absorbance, creatinine, total protein, calcium, phosphorous, magnesium, and osmolality. Additionally, freshly voided samples collected during Weeks 4±1, 13±1, and 17±1 were prepared and submitted to scanning electron microscopic examination (SEM).

Sacrifice and pathology:

GROSS PATHOLOGY: No
HISTOPATHOLOGY: Yes (urinary bladder, stomach and kidney tissue): Histopathologic and scanning electron microscopic evaluations

Other examinations:

CELL PROLIFERATION EVALUATION
Up to 10 surviving animals of each treatment group received 100 mg/kg bw BrDU i.p. 60±5 min prior to sacrifice during Weeks 5, 14, and 18 of the study. At necropsy the urinary bladder and stomach were inflated in situ with fixation solution. Bladders were bisected, rinsed at least 5 timed with 70% EtOH, and weighed before being processed further. To validate the integrity of the BrDU injections, the mitotically-active transitional area of the stomach served as a positive control by virtue of the presence of BrDU-positive cells (a labelling index is not determined) in the samples taken. To allow for comparative micropathological analysis, insofar as the urothelium of the renal pelvis is very similar to the bladder, a kidney specimen was collected, weighed, and preserved during each animals' scheduled necropsy. However, unlike the urinary bladder and the stomach, cell proliferative data were not collected on the kidney.

Statistics:

Group means for continous data were compared either by Student's t-test (unpaired) or a one-way variance analysis (ANOVA) followed by either Dunnett's test or Duncan's Multiple Range Rest. Frequency data examined statistically were evaluated usinf the chi-square and/or Fisher exact tests. Differences with p values ≤0.05 were considered statistically significant.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

increased incidence of urine staining in mid and high dose animals

Mortality:

mortality observed, treatment-related

Description (incidence):

increased incidence of urine staining in mid and high dose animals

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

reduced body weight gain in high dose animals, statistically significant

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

hyperplasia of the urinary bladder eoithelium at 12,500 ppm

Details on results:

CLINICAL SIGNS AND MORTALITY
Increased incidence of urine staining was observed in mid (4000 ppm) and high dose (12,500 ppm) animals.

BODY WEIGHT AND WEIGHT GAIN
Body weight gain, relative to control group, was reduced in the high dose group (12,500 ppm); all other dose groups remained unchanged.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Mean dauly compound intake was 54±2, 224±9, and 684±22 mg/kg bw/day, calculated from feed consumption, body weight, and diet analysis data.

FOOD EFFICIENCY
No effects were noted throughout the study period.

URINALYSIS
No effects were noted in low (1000 ppm) and mid dose (4000 ppm) animals. The changes noted in high dose animals (12,500 ppm) are not considered to be of biological relevance and therefore not advers: the decreases in ketones, urobilinogen, leukocytes, calcium, cloride, total protein, sodium, potassium, and creatinine are considered to be due to an increased urine volume in the high dose animals as compared to the control and therefore physiologically normal.

SCANNING ELECTRON MICROSCOPY OF THE URINE:
Magnesium ammonium phosphate crystals were noted in both treated and control animals without increased incidence in any of the dose groups. Amorphous material isolated from filters during sample preparation was most likely derived from these magnesium ammonium phosphate crystals. Calcium phosphate-containing material, which is associated with the administration of various sodium salts in the rat, was noted in none of the dose groups at neither time point. Additionally, there was no evidence of other crystalline forms. In summary, the urinary sediment of all the treated rats of all dosed groups was similar with respect to precipitate and crystals as the control group.


HISTOPATHOLOGY: NON-NEOPLASTIC
- URINARY BLADDER
- Light microscopy: Urothelial hyperplasia was noted in high dose animals (12,500 ppm). Half of the animals at Week 4 and 3/10 animals of the high dose group ini Week 13 showed simple hyperplasia (= flat increase in the number of cell layers of the urothelium to 4 or more compared to the normal 3 layers). One of these animals in Week 13 had a more severe form of hyperplasia, i.e. papillary-nodular hyperplasia (= represent endophytic or exophytic proliferations, respectively, with a fibrovascular core). By week 17 (i.e. after 4 weeks of recovery phase) the bladders were all normal by histopathology, suggesting reversibility of the OPP-induced changes in cellular growth. No inflammatory change was seen and there was no evidence of calcification in bladders at any doses or times.
The bladders of all control group, low dose (1000) and mid dose (40100) animals appeared normal by histopathology.
- Scanning Electron Microscopy: The bladders of rats treated for 4 weeks with 12,500 ppm test material showed superficial necrosis in multiple foci. By Week 13, the bladder lesion had become more severe and more widespread. In addition, 13-week bladders also showed evidence of regenerative hyperplasia with pilling up of round cells. After 4 weeks on control diet (recovery period), there was a decreased effect on the bladder, but areas of necrosis and hyperplasia remained. This 4-week interval was also characterised by minor changes of a focal nature in two of the bladders collected from the mid dose (4000 ppm) animals. The significance of these changes is not clear, however, as similar observations were occasionally noted in both the control and the low dose (1000 ppm) groups, suggesting that a treatment related effect is present only in the high dose group (12,500 ppm).

- KIDNEY
Focal microcalcification and tubular proliferation were observed in a few high dose animals (12,500 ppm) at each time point. Among lower dose groups (1000 and 4000 ppm), the kidneys were considered unremarkable histopathologically.

- STOMACH
Mild squamous cell hyperplasia at the limiting junction between the forestomach and glandular stomach was observed in a few high dose animals (12,500 ppm) at each time point. No other changes were seen in the stomachs of either the controls or the treatment groups.

CELL PROLIFERATION: BrDU LABELLING INDEX
At the end of approximately 4 and 13 weeks on study, a significantly increased labelling was measured in the bladders collected from the high dose (12,500 ppm) animals; the labelling index at lower doses was not significantly elevated. After a 4-week recovery period, the proliferative response of animals of the high dose group had reverted back to normal levels.

Dose descriptor:

NOAEL

Effect level:

4 000 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: corresponding to 224±9 mg/kg bw/day

Remarks on result:

other: Effect type: carcinogenicity (migrated information)

Dose descriptor:

LOAEL

Effect level:

12 500 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: corresponding to 684±22 mg/kg bw/day hyperplasia of the urinary bladder epithelium

Remarks on result:

other: Effect type: carcinogenicity (migrated information)

Conclusions:

The of this study results suggest that the test substance acts by an mechanism involving a cytotoxic action on the urothelium, followed by regenerative hyperplasia. The origing of the test substance-induced cytotoxic response and subsequent effect on urothelial growth is not known, however, the data provided by this experiment clearly demonstrate that the effect is not mediated by either the presence of abnormal crystalluria or the formation of a calcium phosphate containing amorphous precipitate. Insofar as questions remain regarding the action of the test substance on the regulation of epithelial growth of the urinary bladder, a second mechanistic study was subsequently initiated (7.6.2., 2, Christenson et al., 1996, DNA-Adducts)

Reference 2

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

yes

Remarks:

animals were > 6 weeks of age when dosing commenced and the lowest dose is < 10% of highest dose for females

Qualifier:

according to guideline

Guideline:

other: US-EPA FIFRA Guideline 83-5

Qualifier:

according to guideline

Guideline:

other: EPA OTS 798.3320 (Combined Chronic Toxicity/Oncogenicity Studies)

Qualifier:

according to guideline

Guideline:

other: MAFF Guideline 59 NohSan No. 4200 Combined Chronic Toxicity/Oncogenicity Studies

GLP compliance:

yes

Specific details on test material used for the study:

- Name of test material (as cited in study report): ortho-phenylphenol (OPP)
- Stability: proven by purity analysis
- Storage condition of test material: under freezer conditions (approximately -23°C)

Species:

rat

Strain:

other: CDF[F344]/BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SASCO, Inc., Madison, WI
- Age at study initiation: approximately 8 weeks
- Fasting period before study: no
- Housing: individually in suspended stainless stell wire-mesh cages; each cage containing a feeder, a pressure-activated water nipple, and deotised cage board in the bedding tray
- Diet: Purina Mills Rodent Lab Chow 5001-4 in "etts" form, ad libitum
- Water: tap water (municipal water supply of Kansas City, MO
- Acclimation period: at least 6 days

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

Route of administration:

oral: feed

Vehicle:

other: Acetone/corn oil mixture

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly

VEHICLE
- Justification for use and choice of vehicle (if other than water): solubility properties of the test item

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

9 Analysis of the test substance in various test diets during the in-life phase of the study revealed mean analytical concentrations of 732 ppm (CV=6%), 3730 ppm (CV=8%), 7385 ppm (CV=8%), and 9510 ppm (CV=6%), with all values remaining within 10% of the corresponding nominam concentrations of 800, 4000, 8000, and 10,000 ppm, respectively.
The test substance was further shown to be stable following both room temperature and freezer storage over a period of 14 and 28 days and homogeneously distributed in the feed over a concentration range of 800-10,000 ppm.

Duration of treatment / exposure:

2 years

Frequency of treatment:

daily

Post exposure period:

none

Remarks:

Doses / Concentrations:
800, 4000, and 8000 (males) or 10,000 (females) ppm
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
39±1, 200±3, and 402±6 mg/kg bw/day (males)
Basis:
other: mean daily intake calculated from feed consumption, body weight, and diet analysis data

Remarks:

Doses / Concentrations:
49±0, 248±3, and 647±6 mg/kg bw/day (females)
Basis:
other: mean daily intake calculated from feed consumption, body weight, and diet analysis data

No. of animals per sex per dose:

- control group: a total of 70 males + 70 females, 20 per sex served for interim sacrifice after 1 year, 50 per sex were kept and sacrificed after 2 years
- 800 ppm: a total of 60 males + 60 females, 10 per sex served for interim sacrifice after 1 year, 50 per sex were kept and sacrificed after 2 years
- 4000 ppm: a total of 60 males + 60 females, 10 per sex served for interim sacrifice after 1 year, 50 per sex were kept and sacrificed after 2 years
- 8000 ppm: a total of 70 males, 20 served for interim sacrifice after 1 year, 50 per sex were kept and sacrificed after 2 years
- 10,000 ppm: a total of 70 females, 20 served for interim sacrifice after 1 year, 50 per sex were kept and sacrificed after 2 years

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on literature review and a 4-week range finding study in the rat

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (once on the weekends and holidays)
- Cage side observations included: moribundity and mortality, evaluation of external surface areas, orifices, posture, general behaviour, respiration, and excretory products

DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pre-exposure and at sacrifice
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at 3, 6, 12, 18, and 24 months
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes (overnight)
- How many animals: 20 rats/sex/dose (the same animals were used for urinalysis)
- Parameters checked: platelet count, leukocyte count, erythrocyte count, Hb, haematocrit, MCV, MCH, MCHC, Met-Hb, leukocyte differential, (atypical lymphocytes, band neutrophils, basophils, blasts, hematrak comments, eosinophils, lymphocytes, metamyelocytes, monocytes, myelocytes, nucleated RBC, plasma cells, promyelocytes, segmenteed neutrophils), erythrocyte morphology (anisocytosis, basophilic stippling, hypersegmented neutrophils, hypochromasia, macrocytosis, microcytosis, poikilocytosis, polychromasia, spherocytosis, target cells, toxic granulation), special stains (reticulocyte count, heinz bodies)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at 3, 6, 12, 18, and 24 months
- Animals fasted: Yes (overnight)
- How many animals: 20 rats/sex/dose (the same animals were used for urinalysis)
- Parameters checked: Na, K, Cl, P, Ca, urea nitrogen, glucose, creatinine, uric acid, triglyceride, cholesterol, creatine kinase, LDH, AST, ALT, GGT, AP, total bilirubin, direct bilirubin, total protein, albumin, globulin

URINALYSIS: Yes
- Time schedule for collection of urine: at 3, 6, 12, 18, and 24 months (the week prior to the week of blood collection)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (overnight)
- How many animals: 20 rats/sex/dose (the same animals were used for blood collection)
- Parameters checked: appearance, clarity, colour, specific gravity, pH, protein, glucose, ketones, bilirubin, blood, urobilinogen, nitrite, microscopic evaluation of solids

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
ORGAN WEIGHTS: Yes (adrenals, brain, heart, kidneys, liver, lungs, ovaries, spleen, testicles, thyroid)
HISTOPATHOLOGY: Yes (adrenals, aorta, bone (femur, Rib/cc jct, sternum), bone marrow, brain (cerebellum, cerebrum-midbrain, medulla/pons), caecum, cervix, clitoral gland, colon, epididymidis, oesophagus, exorbital(lacrimal gland, eyes, gross lesions, harderian gland, heart, joint fem/tib, kidneys, larynx, liver, lungs, lymph nodes (cervical and mesenteric), mammary gland, muscle, nerve (optic and sciatic), ovaries, pancreas, parathyroid, physical identifier, pituitary, prepupital gland, prostate, rectum, salivary gland, seminal vesicles, skin, skull, small intestine (duodenum, ileum, jejunum), spinal cord (cervical, lumbar, thoracic), spleen, stomach, testicles, thymus, thyroid, trachea, ureters, urinary bladder, uterus, vagina)

Statistics:

Continous data were evaluated initially for equality or homogeneity of variance using Bartlett's test. Group means were further analysed by a one-way variance analysis (ANOVA) followed by Dunnett's test. In the event of unequal variances, and at the discretion of the study director, data were subject to non-parametric procedures consisting of a Kruskal-Wallis ANOVA followed by the Mann-Whitney-U test for between-group comparisons. Frequency data were initially examined for trends, data suggestive of a potential effect were then statistically evaluated using the chi-square, Fisher-exact, or chi-square and Fisher-exact tests. On a case by case basis, and at the discretion of the study director, data were subject to additional statistical procedures other than mentioned above. For the Bartlett test, a probability (p) value of ≤0.01 was considered significant; for all other tests, differences with p values ≤0.05 were considered statistically significant.

Clinical signs:

no effects observed

Description (incidence and severity):

Changes noted are toxicologically not relevant.

Mortality:

no mortality observed

Description (incidence):

Changes noted are toxicologically not relevant.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

4000 ppm males/females: 5% decrased body weight gain; 8000 ppm males and 10,000 ppm females: 11% decrased body weight gain

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Description (incidence and severity):

Changes noted are toxicologically not relevant.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Changes noted are toxicologically not relevant.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

8000 ppm males: increased incidence of blood in urine

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

4000 ppm males: urinary bladder masses; 8000 ppm males: increased incidence of ventrum wet/stained and urinary bladder masses; 10,000 ppm females: increased incidence of ventrum wet/stained and pitted zones and abnormal texture in kidney

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

8000 ppm males: urinary bladder calculi, congestions, haemorrhage, mineralization, necrosis, calculi in renal pelvis, renal cystic tubular dilatation; 10,000 ppm females: renal mineralisation, renal cystic tubular dilatation, renal infarct

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

8000 ppm males: simple urinary bladder hyperplasia, transitional cell carcinoma; 10,000 ppm females: simple urinary bladder hyperplasia, ureter dilatation and/or transitional cell hyperplasia, renal tubular hyperplasia

Details on results:

CLINICAL SIGNS AND MORTALITY
A slight increase in mortality was suggested for high dose males (8000 ppm), based on an increased number of unscheduled kills and a corresponding decrease in mean time of death (days). In contrast, a slight increase in mean time of death was observed for high dose females (10,000 ppm). Number of animals found dead on study was comparable between treated and control animals of each sex.
Test material related cage side observations were generally noted in mid and high dose animals (4000 and 8000/10,000 ppm) and included a statistically increased frequency of abnormal colour urination, urine stains, and red and/or brown stains primarily located in the perigenital area. While many interpretations regarding these observations may be plausible, the true toxicological significance, if any, remains unclear.

BODY WEIGHT AND WEIGHT GAIN
There was no test material-related effect noted in the low dose group (800 ppm) animals (both males and females). A decreased body weight of 5% was noted in both males and females treated with 4000 ppm and a decline of 11% was noted in high dose males (8000 ppm) and females (10,000 ppm).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
There was no test material-related effect noted in any of the dose groups. Compound intake was calculated from feed consumption, body weight, and diet analysis data and amounted 39±1, 200±3, and 402±6 mg/kg bw/day for males and 49±0, 248±3, and 647±6 mg/kg bw/day for females, respectively.

OPHTHALMOSCOPIC EXAMINATION
There was no test material-related effect noted in any of the dose groups.

HAEMATOLOGY
There was no test material-related effect noted in low and mid dose animals (800 and 4000 ppm) and in high dose males (8000 ppm). Decreases in haemoglobin concentration, haematocrit, MCV and MCHC were suggested in high dose females (10,000 ppm); however, these changes were within the historical control range, did not persist over time intervals, and were therefore considered equivocal.

CLINICAL CHEMISTRY
There was no test material-related effect noted in any of the dose groups.

URINALYSIS
The only urinary consideration following exposure to OPP was increased incidence of blood in the urine of high dose males (8000 ppm). This finding is associated with the bladder neoplasia noted in high dose males.

ORGAN WEIGHTS
There was no test material-related effect noted in any of the dose groups.

GROSS PATHOLOGY
An increased incidence of urinary bladder masses was suggested for high dose males (8000 ppm) at both 1-year and 2-year sacrifices and for mid dose males (4000 ppm) at the 2-year sacrifice. This observation is likely associated with norphologic changes noted microscopically in the bladder.
An icrease in the incidence of wet ventrum was noted in high dose females (10,000 ppm) at the interim sacrifice after 1 year of exposure. A similar increase in wet ventrum with staining was observed at terminal sacrifice in high dose males (8000 ppm) and females (10,000 ppm), and was simply a reiteration of the urine and the red staining of the perigenital area seen during the in-life portion of the study.
In the kidney, an increased incidence of pitted zones and abnormal texture was noted in highdose females (10,000 ppm) at terminal sacrifice.

HISTOPATHOLOGY: NON-NEOPLASTIC
Urinary bladder calculi, congestions, haemorrhage, mineralization and necrosis were noted in high dose males (8000 ppm) at terminal sacrifice after 2 years. At the interim sacrifice, high dose males showed further calculi in the renal pelvis and at terminal sacrifice renal cystic tubular dilatation was noted.
In high dose females (10,000 ppm), renal mineralisation were noted at terminal sacrifice after 2 years of exposure. Renal cystic tubular dilatation and renal infarct (wedge-shaped lesions without vascular involvement were noted in animals of the same group at both interim and terminal sacrifice.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
In high dose males (8000 ppm), simple urinary bladder hyperplasia and transitional cell carcinoma were noted at both 1- and 2-year sacrifices.
In high dose females (10,000 ppm), simple urinary bladder hyperplasia, ureter dilatation and/or transitional cell hyperplasia and renal tubular hyperplasia were noted at terminal sacrifice after 2 years of exposure.

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Remarks on result:

other: Effect type: carcinogenicity (migrated information)

Dose descriptor:

LOAEL

Effect level:

402 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: based on neoplasms (malignant and benign) in the urinary bladder of male rats

Remarks on result:

other: Effect type: carcinogenicity (migrated information)

Dose descriptor:

NOAEL

Effect level:

>= 647 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Remarks on result:

other: Effect type: carcinogenicity (migrated information)

The toxicological response to the 2 year daily intake of OPP can mainly be characterized as structural alterations in the urinary bladder and kidney. But there was no compound-related change in various parameters associated with these organs at doses of 800 and 4000 ppm in both sexes. Body weight declined slightly at doses of 4000 ppm or above. Food consumption, ophthalmology, organ weights as well as haematological and clinical chemistry values remained unaffected. Urine analysis gave an increased incidence of blood in the urine of the 8000 ppm males only. Carcinogenicity: The urinary bladder showed evidence of a compound-induced neoplasia in both 4000- and 8000 ppm male rats only. While this effect was unequivocal at 8000 ppm, it was considered border-line at 4000 ppm as there was only a marginal and non-statistical increase in both urinary bladder hyperplasia and transitional cell carcinoma when compared to controls or 800 ppm males. Evidence of a compound-induced neoplasia was not observed in female animals at any dose tested.

NOAEL systemic, males: 800 ppm (39 mg/kg bw/day)

LOAEL systemic, males: 4000 ppm (200 mg/kg bw/day), based on structural alterations in the urinary bladder

NOAEL systemic, females: 4000 ppm (248 mg/kg bw/day)

LOAEL systemic, females: 10,000 ppm (647 mg/kg bw/day), based on renal mineralisation, renal cystic tubular dilatation, renal infarct

NOAEL carcinogenicity, males: 4000 ppm (200 mg/kg bw/day)

LOAEL carcinogenicity, males: 8000 ppm (402 mg/kg bw/day), based on neoplasms (malignant and benign) in the urinary bladder

NOAEL carcinogenicity, females: ≥10,000 ppm (647 mg/kg bw/day)

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

The available data comprises adequate and reliable (Klimisch score 1) studies. The data on carcinogenicity are thus of good quality and sufficient to fulfill requirents according to Regulation (EC) No 1907/2006.

System:

hepatobiliary

Organ:

bladder

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Quality of whole database:

Reliable data from a long-term study on carcinogenicity are available via the oral route.

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Quality of whole database:

Reliable data from on carcinogenicity are available via the oral route.

Mode of Action Analysis / Human Relevance Framework

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

Justification for classification or non-classification

There is convincing evidence that the carcinogenic effects shown in rodents are threshold effects with an indirect and non-genotoxic mechanism and tumours observed in rodent species (liver tumours in mice and bladder tumours in male rats) are not predictive of carcinogenicity for humans due to proven species differences. Therefore, classification and labelling for carcinogenicity according to Regulation (EC) No 1272/2008 is not required.

Additional information

 

The carcinogenic potential of 2-phenylphenol (OPP) was extensively studied in rats and mice using long-term toxicity studies or investigating the mode of action in short- and medium-term mechanistic studies.

In a combined chronic toxicity / carcinogenicity study in rats, conducted according to the OECD Test Guideline 453 and in compliance with GLP, CDF[F344]/BR rats of both sexes received OPP at dietary levels of 800, 4000, and 8000 ppm (males) (corresponding to approx. 39, 200, and 402 mg/kg bw/day) or 800, 4000, and 10000 ppm (females) (corresponding to approx. 49, 248, and 647 mg/kg bw/day) for two years (Wahle, B. S. and Christenson, W. R., 1996 and Bomhard, E.M. et al., 2002). The urinary bladder showed evidence of compound-induced neoplasia in both 4000 and 8000 ppm male rats only. While this effect was unequivocal at 8000 ppm, it was considered border-line at 4000 ppm as there was only a marginal and non-statistical increase in both urinary bladder hyperplasia and transitional cell carcinoma when compared to controls or 800 ppm males. Evidence of compound-induced neoplasia was not observed in female animals at any dose tested. The NOAEL for carcinogenicity derived from this study is therefore ≥ 10000 ppm (647 mg/kg bw/day) for females and 4000 ppm (200 mg/kg bw/day) for males. The corresponding LOAEL for males is thus 8000 ppm (402 mg/kg bw/day), based on neoplasms (malignant and benign) in the urinary bladder. A mechanistic study investigating the mode of action of the OPP-induced carcinogenicity in the urinary tract of male rats was conducted as follow up study (Christenson et al., 1996; Bomhard, E. M. et al., 2002 and Brusick, D., 2005, see Section 7.6.2). 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)[1]for 13 weeks. The results suggest that the test substance acts by a mechanism involving a cytotoxic action on the urothelium, followed by regenerative hyperplasia. In support, DNA-adducts are not formed by OPP or its metabolites and consequently genotoxicity by direct interaction with DNA is unlikely.

 

Studies in mice:

A combined chronic toxicity / carcinogenicity study is available, conducted according to the OECD Test Guideline 453 and in compliance with GLP (Quast, J. F. and McGuirk, R. J., 1995 and Bomhard, E.M. et al., 2002). Groups of 50 B6C3F1 mice of each sex (5 weeks old) were fed diets supplemented with 0, 250, 500, or 1000 mg OPP/kg bw/day for 2 years. A satellite group of 10 mice/dose/sex was maintained on the diets for 12 months after which time they were necropsied and evaluated for general chronic toxicity. Administration of OPP to B6C3F1 mice for up to 2 years induced hepatocellular changes indicative of adaptations to metabolic demands, zonal degeneration, focal hepatocellular necrosis, and/ or pigmentation of the liver. However, the incidence of hepatocellular adenomas was increased only in male mice of this study, using a strain prone to develop hepatocellular tumours at high spontaneous incidences. The incidence of hepatocellular carcinomas was not affected by treatment.

 

Summary and discussion:

In summary, the oncogenic mode of action of OPP can be characterised based on the extensive scientific data available. Two tumour types have been associated with high-level exposures to OPP; tumours of the urinary bladder in rats and tumours of the liver in mice. Rats exposed chronically to OPP in the diet at doses greater than a threshold of approximately 200 mg/kg bw/day develop transitional cell tumours in the urinary bladder. There is no clear dose-response but a clear threshold for development of urinary bladder tumours, urothelial cytotoxicity, hyperplasia,
BrdU labelling index, protein adduct formation, and urine levels of the reactive oxygen species (ROS) metabolites phenylhydroquinone and phenylbenzoquinone in male rats (Wahle, B. S. and Christenson, W. R., 1996; Christenson, W.R., Wahle, B.S. and Cohen, S.M., 1996; Brusick, D., 2005 and Bomhard, E.M. et al., 2002). The fact that these effects are linked chronologically to tumour formation provides powerful mechanistic evidence for threshold oncogenesis of OPP. Additionally, the rat seems to be more susceptible to bladder changes than other mammalian species (Cohen, S.M. and Wellwein, L.B., 1995). A series of non-genotoxic substances led in the rat, but not in other species (mouse, hamster, monkey), to hyperplasia and neoplasm of the bladder epithelium. Depending on the non-genotoxic substance used, strain and sex-specific differences were found within rats (Anderson, R.L., 1991; Garland, E.M. et al., 1994 and Uwagawa, S. et al., 1994). Anatomical differences in the urogenital tract, qualitative and quantitative differences in the protein and electrolyte composition, and the different pH values in the normal urine in man and rodents could be responsible for this difference (Cohen, S.M., 1995; DeSesso, J.M., 1995 and Hard, G.C., 1995). Even handling procedures and dietary composition are known to affect the development of urinary bladder lesions in the rat (Cohen, S.M. et al., 1991; Cohen, S.M., 1995; Cohen, S.M. et al., 1996 and Bomhard, E.M. etal., 2002). This scientific evidence on the specific susceptibility of the rat is in line with the data available with OPP. Urinary bladder tumours were observed in male rats only starting at a dose of 200 mg/kg bw/day (4000 ppm in the diet), whereas in female rats hyperplasia as precursor event and first indication of bladder toxicity, but no tumour response, was observed with 647 mg/kg bw/day (10000 ppm). In mice no bladder effects were observed up to the high dose of 1000 mg/kg bw/day. In addition, a study that specifically was designed to assess species differences of Na-OPP induction of urinary bladder effects revealed no respective effect in B6C3F1 mice, Syrian golden hamsters and Hartley guinea pigs, but a clear response in rats (microvilli and simple, papillary and nodular hyperplasia). In this study, male animals were fed a diet containing as much as 20000 ppm (2%) sodium OPP for 4, 8, 12, 24, 36 and 48 weeks (Hasegawa et al. 1990). Also in the one year dog study there was no urinary bladder toxicity observed after treatment with up to 300 mg/kg bw/day OPP (Cosse, P.F., Stebbins, K.E., Stott, W.T., Johnson, K.A., and Atkin, L. (1990) and Bomhard, E. M. et al. (2002).) Together with the available genotoxicity data and the conclusion derived from the mechanistic study conducted by Christenson, W.R. and colleagues (1996), a cytotoxic mode of action together with species and sex-specific sensitivity for urinary tract lesions is likely. Although humans may respond to chronic irritation in the bladder with tumour development, the human appears to be much less sensitive than the rat (Rodent Bladder Carcinogenesis Working Group, 1995). Although mice are not susceptible to the induction of urinary tract tumours by OPP the administration of excessive dose levels of OPP has resulted in the induction of mouse liver adenomas in a sensitive strain of mouse. The hepatocellular tumour type has a high background rate in the B6C3F1 strain of mice and the tumours were observed at dose levels that would exceed the threshold for metabolism via conjugation. ROS would be produced at those dose levels.

The finding of hepatocellular tumours in a chronic study in B6C3F1 mice at 500 mg/kg bw/day should be assigned little weight in the assessment of the carcinogenic potential of OPP for the following reasons:

1. Liver tumours were not induced in other species. The carcinogenicity studies in rats provide no suggestion of liver tumours.

2. This tumour type is very common in sensitive strains of mice such as B6C3F1 strain. The mean incidence of hepatocellular adenoma/carcinoma of the mouse liver is 30% in the NTP carcinogenicity studies (Maronpot, R. et al., 1987). Regulatory agencies typically place little weight on this tumour type for chemicals acting through non-genotoxic mechanisms (Dragula, C. and Burin, G., 1994 and ECHA, 2012f).

3. Latest investigations on a mode of action for the development of liver tumours in mice show that a receptor mediated mechanism via the peroxisome-proliferator-activated-receptor-alpha (PPARa) is probable for the liver enlargement (Geter, D.R., 2009, Section 7.9.3). This mechanism is of no direct concern for humans.

 

There is an extensive database investigating the possible mode of action of OPP (and its sodium salt) in the bladder of the male rat.

One proposed mode of action for OPP induced urinary bladder tumours in the male rat is a genotoxic / mutagenic mode of action, either by direct DNA reactivity or by a possibly increased urine osmolarity.

A number of studies are available investigating genetic toxicity and DNA reactivity of OPP and its metabolites (refer to section 7.6). In the liver, OPP is metabolised by the cytochrome P450 monooxygenase to PHQ, followed by a prostaglandin H synthase-mediated formation of PHQ to PBQ in the urinary tract (Kolachana et al., 1991 and Kwonk and Eastmond, 1997).

Kolachana et al. (1991) have shown that PHQ is converted to PBQ by the peroxidase component of prostaglandin H synthase. In the absence of prostaglandin H synthase, the conversion of PHQ to PBQ is low, concluding that autoxidation of PHQ plays a minor role in the formation of PBQ.

Kwonk and Eastmond (1997) re-evaluated the question of autoxidation of PHQ leading to the formation of PBQ at a variety of pH values. They found that autoxidation of PHQ was enhanced with increased pH, but also O₂and with excess PBQ present. Hence, a change in the microenvironment may lead to increased formation of reactive metabolites – which is especially the case at high dose levels.

Based on the potential involvement of prostaglandin H synthase in the bioactivation of PHQ in the human bladder, the group of Eastmond investigated PHQ and PBQ for genetic toxicity in V79 cells in vitro (Eastmond et al., 1993 and Lambert and Eastmond, 1994). In two micronucleus tests, PHQ was concluded positive for the induction of genotoxic effects in the presence of prostaglandin-H-synthase-mediated metabolic activation. In a HGPRT test under the same experimental conditions, a negative test result was obtained.

In addition, several tests on DNA-reactivity are available for OPP. Although a number of publications have demonstrated that the OPP metabolites PHQ and PBQ form DNA adducts in vitro (Horvath et al., 1992; Pathak and Roy, 1992 and Zhao et al., 2002), the effects were mainly observed at cytotoxic concentrations. No such effects of covalent binding of OPP or its metabolites to DNA were noted after repeated exposure in vivo (Christenson et al., 1996; Bomhard et al., 2002 and Brusick, 2005; Smith et al., 1998 and Kwok et al., 1999).

Based on the available data on genetic toxicity, there are no reliable data to support a DNA-reactive / mutagenic mode of action for OPP-induced bladder tumours in male rats. OPP was shown to be non-mutagenic in bacteria, negative for cytogenicity in a micronucleus test and in an overall weight of evidence negative for in vitro mammalian cell gene mutation.

The negative in vitro micronucleus test, coupled with a (non-fully guideline-compliant) in vivo rat bone marrow micronucleus test conducted at dose levels above metabolic saturation of OPP suggest that OPP has no clastogenic or aneugenic potential (refer to IUCLID section 7.6).

However, OPP was shown to bind to proteins in the bladder (Smith et al., 1998, Reitz et al., 1983 and 1984), which can in turn potentially result in formation of micronuclei in vivo, due to interference of chromosomal segregation or clastogenicity through enzyme inhibition. In order to investigate a possible genotoxic / mutagenic mode of action, several in vivo micronucleus assays have been conducted by the working group of Balakrishnan and Eastmond (2002, 2006 and 2016).

Several novel, non-validated micronucleus assays have been performed in the bladder as the target organ of OPP (Balakrishnan and Eastmond, 2002, 2006 and 2016). Induction of micronuclei in vivo was reproducibly reported in bladder epithelial cells with a non-linear / threshold response.

In a combined treatment with 2% OPP (corresponding to approx. 2000 mg/kg bw/day) and 2% sodium chloride, an increase in micronuclei formation was observed for treatment with OPP as well as OPP + 2% sodium chloride. The bladder micronuclei were CREST-positive and CREST-negative, thus likely comprise both, whole chromosomes and broken chromosomes. However, micronuclei formation was not only observed for treatment with OPP and OPP + 2% sodium chloride, but also for treatment with 2% sodium chloride alone (Balakrishnan et al., 2002), raising the question about the assay’s specificity for detecting genotoxic effects.

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 a 13-wk dietary OPP study (Smith et al., 1998), neither 8000 nor 12500 ppm OPP affected urine osmolality, thus rendering moot this possible genotoxic/mutagenic MOA for induction of MN in bladder epithelial cells by OPP (via increased urine osmolality). 

In a second micronucleus assay conducted by this group, increased micronuclei formation in urinary epithelial cells of F344 rats were only observed at high dietary doses (8000 and 12500 ppm), which were shown to produce cytotoxic effects in the target tissue (Balakrishnan and Eastmond, 2006). Also in a follow up study of this group (Balakrishnan et al., 2016), micronucleus formation was observed in the bladder cells of OPP-treated animals with either neutral or alkaline urinary pH after sodium bicarbonate treatment. Urinary bladder epithelial cell proliferation and micronuclei formation were found to be pH-dependent and were completely inhibited by acidic urinary pH. Under the conditions of the test, OPP was considered a genetic toxicant in urinary bladder cells at moderate and high dietary doses under neutral and alkaline conditions. The mechanism for induction of micronuclei is likely mediated by urinary protein binding with OPP, which has been shown to occur at high doses only (above metabolic saturation). Thus, a non-linear / threshold dose-response would be expected for this effect.

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). In repeated dose studies no DNA adducts caused by OPP or its metabolites were found, therefore a direct interaction with DNA can be excluded with high probability as mode of action 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 potentially 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.

In summary, it can be assumed that carcinogenicity in the urinary bladders of rats depends on a non-genotoxic mechanism and involves protein binding. The special compartmental conditions of the bladder are believed to contribute to these effect which are only observed at high dose levels exceeding metabolic saturation.

 

Conclusion:

In conclusion, based on the criteria for classification of Regulation (EC) No 1272/2008, liver tumours in a sensitive strain of mice are not of relevance for classification. In addition, classification is not required if the mode of action for the tumour response is known and the tumours are not of relevance for man. For OPP there is convincing evidence that the carcinogenic effects shown in rodents are threshold effects with an indirect and non-genotoxic mechanism and tumours observed in rodent species (liver tumours in mice and bladder tumours in male rats) are not predictive of carcinogenicity for humans due to proven species differences. The following values may be taken as key data for the characterization of carcinogenic effects:

NOAEL (rat; carcinogenicity): 200 mg/kg bw/day (Wahle, B. S. and Christenson, W. R., 1996)
NOAEL (mouse; carcinogenicity): 250 mg/kgbw/day (Quast, J. F. and McGuirk, R. J., 1995)

This is in general agreement with the evaluations of FAO-WHO (1999), US-EPA (2005) and EU EFSA (2008) who came to a similar conclusion when deriving an ADI value using the NOAEL (rat) = 39 mg/kg bw/day for systemic toxicity (Wahle, B. S. and Christenson, W. R., 1996 and Bomhard, E.M. et al., 2002, Section 7.5) as a starting point and subsequently applying the conventional margin of safety approach, as this effect level is markedly below the threshold for species specific carcinogenic effects of OPP.

 

 

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