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Carcinogenicity

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Description of key information

Oral: chronic, rat (OECD 453): 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
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
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)
Endpoint conclusion
Endpoint conclusion:
no 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.

Carcinogenicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available
Quality of whole database:
Reliable data from 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.

Justification for classification or non-classification

Classification and labelling for carcinogenicity according to EU Directive 67/548/EEC or 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.

Studies in rats

In a combined chronic toxicity / carcinogenicity study, 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) or 800, 4000, and 10,000 ppm (females) for two years (Wahle, B. S. and Christenson, W. R., 1996 and Bomhard, E.M. et al., 2002). 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. The NOAEL for carcinogenicity derived from this study is therefore ≥10,000 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, W.R., Wahle, B.S. and Cohen, S.M., 1996;Bomhard, E. M. et al., 2002 and Brusick, D., 2005, see Section5.7.3). Male CDF[F-344]/BR rats were given OPP at dietary levels of 800, 4000, 8000, and 12,500 ppm for 15 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 (Section 7.6) 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 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/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. et al., 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 OPP Na 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 the study male animals were fed a diet containing as much as 20000 ppm (2%) OPP Na 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 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 byChristenson, 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/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.

Conclusion:

In conclusion, based on the criteria for classification of Regulation (EC) No 1272/2008 or EU Directive 67/548/EEC, liver tumours in 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 carcinogenetic 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 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:

NOEL (rat; carcinogenicity): 200 mg/kg/d (Wahle, B. S. and Christenson, W. R., 1996)

NOEL (mouse; carcinogenicity): 250 mg/kg/d (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 NOEL (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

References:

Anderson, R.L. (1991). Early indicators of bladder carcinogenesis produced by non-genotoxic agents. Mutat Res 248:261-270.

Cohen, S.M. et al. (1991). A proposed role for silicates and protein in the proliferative effects of saccharin on the male rat urothelium. Carcinogenesis 12: 1551–1555.

Cohen, S.M. (1995). Role of urinary physiology and chemistry in bladder carcinogenesis. Food Chem Toxicol 33:715-730.

Cohen, S.M. and Wellwein, L.B. (1995). Risk assessment based on high-dose exposure experiments. Chem Res Toxicol 5:742-748.

Cohen, S.M. et al. (1996). Extensive handling of rats leads to mild urinary bladder hyperplasia. Toxicol. Pathol. 24:251–257.

DeSesso, J.M. (1995). Anatomical relationships of urinary bladders compared: their potential role in the development of bladder tumors in humans and rats. Food Chem Toxicol 33:705-714.

Dragula, C. and Burin, G. (1994). International harmonization for the Risk Assessment of Pesticides: Results of an IPCS Survey. Regulatory Toxicol. Pharmacol. 20:337.

ECHA. (2012f). Guidance on the application of the CLP criteria. Guidance to Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures.European Chemicals Agency, Helsinki.

Garland, E.M. et al. (1994). A comparison of the effects of sodium saccharin in NBR rats and in intact and castrated male F344 rats. Cancer Lett 78:99-107.

Hard, G.C. (1995). Species comparison of the content and composition of urinary proteins. Food Chem Toxicol 33:731-746.

Maronpot, R. et al. (1987). Liver Lesions in B6C3F1 Mice: The National Toxicology Program, Experience and Position. Arch. Toxicol. Suppl., 10:10.

Rodent Bladder Carcinogenesis Working Group (1995). Urinary Bladder Carcinogenesis: Implications for Risk Assessment. Fd Chem. Toxic., 33:797.

Uwagawa, S. et al. (1994). Lack of induction of epithelial cell proliferation by sodium saccharin and sodium L-ascorbate in the urinary bladder of NCL-Black-Reiter (NBR) male rats. Toxicol Appl Pharmacol 127:182-186.


Justification for selection of carcinogenicity via oral route endpoint:
The selected study is the most adequate and reliable one based on the overall assessment of quality, and with special respect to duration and effect level.

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