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Induction of tumours of the urinary bladder in F344 rats by dietary administration of o-phenylphenol
Fd (77era. Io.\i¢. Vol. 22. No. 11. pp 865 870. 1984 Printed in Great Britain All rights reserved
0278-6915 84 S3.00+0.(R) Copyright ~ 1984 Pergamon Press Lid
I N D U C T I O N OF T U M O U R S OF THE U R I N A R Y B L A D D E R IN F344 RATS BY D I E T A R Y A D M I N I S T R A T I O N OF o - P H E N Y L P H E N O L K. HtRAC;A and T. F U l l Department of Toxicology. Tokyo Metropolitan Research Laboratory' of Public Health. 24-1. It~akunincho 3 chome, Shinjuku-ku, Tokyo 160, Japan (Receired 6 March 1984) Abstract o-Phenylphenol (OPP), a fungicide approved as a fc,od additive in Japan, was given in pelleted diets at dietary levels of 0.156. 0.313.0.625, 1.25 or 2.5", to groups of 11 or 12 F344:DuCrj rats of each sex for 13 v,k, and 0.625, 1.25 or 2.5",, to groups of 20-24 male F344:DuCrj rats for 91 wk. In the 13-wk study, transitional cell papillomas of the urinary bladder occurred in 6..12 (50",,) of the male 1.25",, group. In the 91-wk stud.,,,, urinary bladder tumours appeared in 23.24 (96",,) of the 1.25",, and 4 23 (17",) of the 2.5",, group. Among these tumours, transitional cell carcinomas were found in 20.23 (87".) or 1.25",, and 2..4 (50",,) of the 2.5",, groups. A dose-related increase in the incidence of nephritic lesions was also observed in dosed rats in both the 13- and 91-wk study.
INTRODUCTION o - P h e n y l p h e n o l (OPP) and its sodium salt, sodium o - p h e n y l p h e n a t e ( O P P - N a ) are fungicides that have been a p p r o v e d for use on citrus fruits in J a p a n since 1977. O P P was reported not to be carcinogenic for rats ot" b o t h sexes (Hodge, M a y n a r d , Blanchet et al. 1952). In contrast, we found that O P P - N a produced t u m o u r s o f the urinary tract, particularly the urinary bladder, in male F 3 4 4 . D u C r j rats (Hiraga & Fujii, 1981). Based on these results, we t h o u g h t that O P P would also be carcinogenic in rats. The purpose of the present studies was to evaluate the carcinogenicity of O P P to the urinary tract in rats using the same experimental m e t h o d s as in the previous feeding study of O P P - N a . EXPE.,~IMENTAI. •kIaterials. O P P used for the present study was Dowicide 1 (Lot No. M M 0 1 0 4 0 ) obtained from Dow Chemical Co., MI, USA, as a white crystalline powder. Its purity was more than 98".. Animals. Male and female F 3 4 4 / D u C r j rats at 4 wk of age were purchased from Charles River J a p a n Inc., K a n a g a w a , Japan. Rats were housed individually in stainless-steel cages. W 22 × D 20 × H 16cm, with wire-mesh fronts and floors. The cages were suspended from belt-type racks with an a u t o m a t i c water-supply system ( t h r o u g h a bacterial tilter). Rats were initially segregated into equal weight distribution groups, and then divided into experimental groups. Their cages were allocated r a n d o m l y in the racks. The animal room was m a i n t a i n e d at 24 25 C, and relative humidity at 50--60'~.. Incoming air was passed t h r o u g h a filter of 85". efficiency and a filter of 99.9",, efficiency ( H E P A filter). R o o m air was changed ten times per hour. Fluorescent lighting was controlled to give 1 I hr light (06.00 17.00) and 13 hr dark. Rats of b o t h dosed and control groups were not Ahhret'iation: OPP = eJ-phenylphenol.
housed in the same room as rats treated with any other chemicals. Administration Of materials. O P P was mixed with the raw materials ot" the pelleted diet CE-2 (Clea J a p a n Inc., Tokyo), at c o n c e n t r a t i o n s ot" 0.156, 0.313, I).625, 1.25 and 2.5",i before they were made into pellets. Analyses of the pelleted diets by gas chrom a t o g r a p h y showed that O P P was stable and mixed homogeneously in the diets, and its initial concentrations in each diet were not changed (Kamiya & Hiraga, 1982: Mizoiri, Hirokado, N a k a j i m a et al. 1981). The m a x i m u m c o n c e n t r a t i o n of O P P in diet in the present studies was selected as 2.5",,, based on the results of the previous studies of O P P - N a . A concentration of 4'!o O P P - N a , the m a x i m u m dose of the previous studies, is equivalent to 2.5",, O P P by weight on a m o l a r basis. Accordingly, c o n c e n t r a t i o n s of 0.156, 0.313, 0.625, 1.25 and 2.5",, of O P P are equivalent to 0.25, 0.5, I. 2 and 4",, O P P - N a respectively. G r o u p s of 11 or 12 rats of each sex were given pelleted diet c o n t a i n i n g 0.156, I).313, 0.625, 1.25 or 2.5'! o O P P t h r o u g h o u t the 13-wk study. In the 91-wk study, groups of 20 24 male rats were given diet containing 0.625, 1.25 or 2.5",, t h r o u g h o u t . The control rats in these studies were fed basal diet throughout the study. Feed and water were supplied freeb. A d m i n i s t r a t i o n of the test chemical was begun ,,,.'hen the males were 35 days old and the femalcs 34 days old in the 13-wk study, and when the males were 38 or 39 days old in the 91-wk study. Observation. All of the rats were observed twice daily and all deaths, gross t u m o u r s and clinical signs were recorded. Each rat was weighed once a week in b o t h the 13- and 91-wk study. Food intakes for all rats were m o n i t o r e d once a week in the 13-wk study, and once weekly till wk 3, biweekly from wk 4 to 9 and every 4 wk from wk 10 to the end of the study in the 91-wk study. M e a n intakes of O P P in each of the dosed groups were calculated from food intakes. M o r i b u n d rats and rats that survived to the end of the study were exsanguinated and autopsied. A u t o p 865
866
K. HIRAGA and T. FUJH
sits were also performed on all rats found dead. unless precluded in whole or part by autolysis. Pathological e.vumhlation. The pathological exammation consisted of gross and microscopic examination of major tissues, major organs and all gross lesions. The tissues were prcserved in 10",, neutral buffered l\)rmalin, embedded in paraffin, sectioned. and stained with haernatoxylin and eosin. The urinary bladders were evacuated of urine by transurethral injection through a 27-gauge needle and were distended with 0.4 1.2ml of 10",, neutral buffered fornaalin, depending on the age of the rat. After the fixation, the urinatry bladders were cut longitudinally and then examined grossly' and stereoscopicallx. .~'tati.~lh'a! wmh'.vi.~. Student's t test was used to compare both the body-weight gains and food intakes of the control groups with those of dosed groups (P < 0.05). The Fisher exact test was used to compare surxival rates, incidences of tumours and the other lesions of the control group with those of dosed groups. The Fisher exact P values are indicated in the tables for ,,alucs o1" P less than 0.05.
RE.NL I.'l'S
In the 13-wk study, the mean body weights ot" the 0.156. 0.313 and 0.625°,, groups ot" each sex were comparable to those of the controls throughout the study. The mean body weights were significantly lower than those of the controls in the 1.25",, groups of each sex from wk 2 to 9 and in the 2.5",, groups of each sex throughout the study. The weight depressions relative 1o the controls were 5-9",, for 1.25",, females. 7 13",, for 1.25",, males. 14 32",, for 2.5",, females and 26 35",, t\~r 2.5"o males. In the 91-wk stud5', the mean body weight of the 0.625°i, group was comparable 1o that of the controls throughout the stud).. Mean body weights were significantly lower than that of the controls m both the 1.25",, group from v,'k 21 to the end of the study and the 2.5",, group throughout the study'. The weight depressions relative to the controls were 2 12". for the 1.250,, and 17 24",, l\n the 2.5",, groups (Nakamura. Ikeda. Iguchi & Hiraga. 1982). In the [3-wk study, the daily food intakes (g.rat) of the 2.5",, groups o1" each sex were signiticantly lower than those of the controls throughout the study in males and from wk 1 to 9 in females, but the relative lk)od retakes (g.kg.day) of these groups were higher than those of the controls throughout the study except for the male 1.250,, group of wk I. The daily food intakes of the other dosed groups were comparable 1o those oi" the controls throughout the study. In the 91-wk study, the daily food intake (g. r a t ) o f the 2.5",, group was significantly lower than that of the controls from wk I to 85 except at wk 33. but the relative food intake (g:kg:day) of this group was signiticantly higher than that of the controls throughout the study except from wk 3 to 13. The daily food intakes of the other dosed groups were approximately equal to those of the controls throughOUt the stud', (Nakamura et al. 1982). In the 13-~k stt, dy, the mean retakes of OPP of the 0. 156, 0.313.0.625, 1.25 and 2.5",, groups were 98.4,
210.3, 409.9. 815.1 and 1492.Smg. kg.day in males. and 108.5, 221.1,432.3, 888.4 and 1622.6 mg..kg..day in females, respectively. In the 91-wk stUdy,,, mean intakes of OPP of" 0.625, 1.25 and 2.5". groups were 269, 531 and 1140 mg/kg;day, respectively. In the 13-wk study 8.11 (73".) of the male 2.5",, group, l 1..12 (92".) of the female 2.5",, group and all the rats of the other groups lived to the end of the study. The survival rates at the end of the 91-wk study were 23..24 (96",,) of the controls. 18.20 (90",,) of the 0.625". group. 17.24 (71",,) of the 1.25",, group and 15."23 (65",,) of the 2.5",, g,oup. The survival rates for both the 1.25 and 2.5"0 groups in the 91-wk study were signilicantly lower ( P - 0 . 0 2 3 for 1.25",, and P = 0.009 for 2.5". groups) than that of the control. A m o n g the rats which died during the study, the incidence of urinary bladder tumours was 0.1 (0",,) of the control. 0 2 (()",,) of the 0.625",, group. 7..7 (100",,) of the 1.25°. group and 0..8 [0",,) of the 2.5",, group in the 91-wk study: none were found m the 13-wk study. Occult blood in the urine as indicated by' test tapes (Labstix"-II. Miles-Sankyo ('o. [.td. Japan) was observed from wk 15. and gross haematuria was seen from wk 52 in rats of both the 1.25 and 2.5",, groups in the 91-wk stud.',.. Proliferative lesions of the urinary bladder were found in dosed groups in both the 13-and the 91-wk study. In the 13-wk study, the lesions were seen in 12..'12 (100",,) of the male 1.25",, group, but not in any other groups. Ilalf the lesions in the male 1.25",, group were transitional cell papillomas and the other hall" transitional cell hyperplasias. The incidences of these two lesions in this group were significantly higher (P = 0.007. Fisher exact test) than m the controls. In the 91-wk study', bladder lesions appeared in 10",, of the 0.625% group. 96" 0 of the 1.25",, group and 48",, of the 2.5",, group ITable 1). tJrinar~ bladder turnouts occurred m 96',. of the 1.25",, and 17",, of the 2.5"o group. The mcidence of this tumour was signiticantb higher in the 1.25",, group than in the controls (P <0.001). Of these tumours, transitional cell carcinomas accounted for X7",, m the 1.25",, group and 50",, in the 2.5",, group. In the 1.25",, group five carcinomas invaded their own stalks or the bladder wall. The first tumour ipapilloma) appeared in a rat of the 1.25",, group that died during wk 65. and the first transitional cell carcinoma was found in a rat of the 1.25"o group that died during wk 82. Grossly, urinar.,, bladder tumours were papillary or polypoid, having a caulittov,.er-like appearance. Many' of these tumours were multiple and arose m the ventral wall of the apex of the bladder growing into the lumen (Fig. 1). tlistologically, the lesions of the urinary bladder were classified as hyperplasia, papilloma and transitional cell carcinoma. The hyperpl:tsia was characterized by an epithelium with more than four cell layers and slight nuclear abnormalities (Fig. 2). The papillomas were covered by at relatively normal epithelium composed of not more than six layers of cells and grew out into the bladder lumen with a delicate fibrovascular stroma (Fig. 3). The transitional cell carcinoma contained a small amount of connective tissue with blood vessels and was covered b', transitional epithelium. The epithelium showed increased
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Fig. I. Gross appearance of thc cut surface of the urinary bladder from male rats fed orthophenylphenol at I.~5o in the diet (or 84 wk (left) or 91 wk (right). Each division represents 1 mm.
Fig. 2. Fransitional cell hyperplasia, a suspicious prcneoplastic change, in the urinary bladder of a male rat fed orthophenylphenol at 1.25",, in the diet for 13 w k Haematoxylin and eosin × 200.
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Fig. 3. Transitional cell papilloma of the urinar~ bladder of a male rat fed orthophenylphenol at 1.25",, in the diet lor 13 wk. Ilaematoxylin and eosin x 100.
Fig.4. Transitional cell carcinoma of the urinar~ bladder of a male rat l'~d orthophenylphenol at 1.25" in the diet for 91 wk. Haematoxylin and eosin × 20().
Carcinogenicity of orthophcnylpheno]
869
Table 1. Summary' of the incidence of hyperplastic or neoplastic lesions of the urinary bladder in male F344..DuCri rats fed diet containing orthophenylphenol (OPP) for 91 wk
Dietary level of OPP ( " , , )
No. of rats examined histologically
0 (('ontrol) 0.625 1.25
24 20 24
2.5
23
No. of rats with bladder tumour 0 0 23 (P < 0.001 )§ 4
No. of rats with transitional cell... ....................... Carcinoma Hyperplasia
Papilloma
0 2 0
0 0 3
NoninvasivO0 0 15
Invasive.+ 0 0 5
7
2
2
o
tTumours showed noninvasive growth into the bladder wall. ~Tumours showed invasive growth into the bladder wall. §Figures in parentheses show the probability level for the Fisher exact test for the comparison of that dosed group with the control group when t' is less than 0.05. cellularity, irregularity of cell size. nuclear abnormalities and mitotic figures (Fig. 4). Vesical calculi about 0.1 5 mm in diameter, dark or greenish brown in colour and having rough or smooth surfaces were observed grossly or stere()scopically. Calculi were found in 2.12 (17",,) of the 1.25",, and 1.12 (8".) of the 2.5'~,, groups of males in the 13-wk study, and 17.24 (71" o) of the 1.25'1,1 and 14..23 (61".) of the 2.5". groups in the 91-wk study. A m o n g rats bearing urinary bladder tumours, rats with calculi were observed in 1..6 (17",,) of the male 1.25",, group in the 13-wk study, and 17.:23 (74,(,) of the 1.25". group and 3..4 (75",,) of the 2.51',, groups in the 91-wk study. Transitional cell hyperplasia in the pelvis and.or papilla ',','as found in 3..11 (27",0 of the male 2.5'!o group in the 13-wk study, 12..23 (52'~0) of the 2.5"o group in the 91-wk study. The incidence of hyperplasia in the ql-wk study was significantly higher (P < 0.001 ) in the 2.5". group than in the controls. In the 13-wk study, no tumours at sites other than the urinary bladder were found. In the 91-wk study, interstitial cell tumours of the testes were the tumours most frequently observed at other sites. Turnouts of the pituitary and adrenal glands were also found frequently. These tumours occurred with approximately equal frequency in dosed and control groups. Nephritic lesions attributed to the administration of OPP appeared in both the 13- and 91-wk studies. In the 13-v,'k study, slight nephritic lesions were seen in 7 I 1 (64"o) males and 6..12 (50"o) females of the 2.5"o groups. These incidences were significantly higher (P -- 0.001 for males. P = 0.009 for females) than those of the controls. A m o n g these lesions, slight pyelonephritis was observed in 3.'7 (43°.) of the male and 3.6 (50",,) of the female groups, and the other lesion was interstitial nephritis. In the 91-wk study, moderate to scvere nephritic lesions appeared in 3.24 (13".1 of the 1.25". group and 23..23 (100",,) of the 2.5"o group. The incidence of this lesion was signiticantly higher (P <0.001) in the 2.5% group than in the controls. A m o n g these lesions, moderate to sevcrc pyelonephritis with papillary destruction were found in I..3 t33"o) of the 1.250o and 15..23 (65".) of the 2.5", groups, and the other lesion was interstitial nephritis. I)IS('t',~SiON
Mean body weights of both 1.25 and 2.5". groups of each sex. particularly 2.5",, groups, were lower
than those of the controls throughout most of the study in both the 13- and 91-wk studies. Survival was affected adversely in the 2.5'~,, groups ot" each sex in the 13-wk study, and showed a dose-related decrease in the 91-wk study. Thus, OPP was toxic to rats in both 1.25 and 2.5" 0 groups of each sex, particularly in 2.5'~,i groups, as shown by effects on weight and survival. Proliferative lesions in the urinary bladder attributed to the administration ot" OPP were found in the male 1.25'!,, group in the 13-wk study, and the 0.625",,, 1.25'~i, and 2.5",, groups in the 91-wk study. Urinary bladder tumours occurred in the male 1.25". group in the 13-wk study, and in the 1.25% and 2.5"o groups in the 91-wk study. A m o n g these turnouts. transitional cell carcinomas were seen in more than half of the affected animals in the 91-wk study', and all the other tumours were papillomas. Thus, urinary bladder tumours occurred in male dosed groups. particularly in the 1.25'!. groups, and hyperplastic lesions ot" the urinary bladder and suspicious preneoplastic lesions were also found only in dosed groups in both the 13- and 91-v,k studies. These results demonstrate that OPP. free phenol of OPPNa. had the same carcinogenic effect on the urinary bladder of male rats as O P P - N a showed in previous studies. Urinary bladder tumours were found in rats fed OPP in the present studies, but not observed in the feeding study' of OPP by Hodgc et al. (1952). This discrepancy may be due to differences in the test conditions used, such as the strain of rats. diets or selection of doses. In the present studies, the incidences of urinary bladder turnouts in the 2.5",, groups were lower than those in the 1.25". groups. In contrast, adverse effects on both weight and survival were more severe in the 2.500 groups than in the 1.25°. groups. Administration of OPP in the diet at 2.5",; was presumably toxic for rats. In the previous 13-wk study of OPP-Na. urinary bladder tumours were found in I.:10 of the 1",, 9.. 10 of the 2",, 1..10 of the 4",, groups of males and 2.10 of the female 4". group. In the 91-wk study of O P P - N a using male rats, urinary bladder tumot, rs occurred in 6..21 (29",,) of the 1",,. 19.21 (90",,) of the 2" o and 8:20 (40",,) of the 4"i, groups, and tumours of the renal pelvis and.or papilla were also seen in the 0.5. 1. 2 and 4",, groups. The incidence of urinary bladder tumours in the 2 ' ! 0 0 P P - N a group in the previous 91-wk study was approximately equal to
870
K. thRA(;A and T. I't:Jit
that in the 1.25",, O P P group in the present study. Moreover. the incidences in these two groups were higher than those in the o t h e r dosed groups including both 2.5",, O P P and 4",, O P P - N a groups in each stud). Thus. the trend of the incidence of urinar', bladder t u m o u r s in the previous stud)' of O P P - N a was similar to thal in the present s t u d ) of OPP. No t u m o u r s of the urinary bladder were found in the 0.625". O P P groups in either the 13- or 91-,xk studies. In contrast, one papilloma in the 13-~k study and six carcinomas m the 91-wk study were seen in the male 1",, O P P - N a groups (corresponding to the 0.625% O P P groups) in the previous studies. Bladder t u m o u r s occurred in one male group (I.25",,) m the 13-wk stud,, and two groups (1.25 and 2.5",) in the 9l-wk study of ()PP. In contrast, in the stud.', of O P P - N a the t u m o u r s appeared in two male groups I 1 and 2",,) and one female group (4",,) m the 13-wk stud.~, and three groups 11. 2 and 4",,) in the 91-wk study. These findings suggest that the carcinogenicity of O P P - N a to the urinary bladder of F344.DuCrj rats was greater than that o f O P P . O P P is a weak acid. in contrast to O P P - N a which shows alkalinity ( p t t 11.8 when dihlted 1 to 511 m water). It may be that the difference between O P P and OF'P-Na in the strength of carcinogenicity to rat urinary bhtdder was caused by this alkalinity. Vesieal calculi were seen only in dosed males in the present studies, and also found in dosed rats m the previous 91-wk study of O P P - N a . These results suggest that both O P P and O P P - N a cause stone formation in the urinary tracts of male rats. In the present 13-wk study, calculi were found in 2.12 of the male 1.25"., group, m contrast hyperplasia or papilloma of the urinary bladder occurred in all of this group. These results suggest that there is no correlation between these calculi and the proliferative lesions of the urinary bladder in rats.
Nephritic lesions attributed to the a d m i n i s t r a t i o n of O P P were not unexpected, since these lesions were reported by Hodge et al. (1952). The lesions were seen in the 2.5",, groups of each sex in the 13-wk stud.,,. The incidence of the lesions showed a positive doserelated increase in the 91-wk study. It is clear that O P P caused nephritic lesions in F344.'DuCrj rats of each sex. U n d e r the conditions of the present studies, dietar5 a d m i n i s t r a t i o n of O P P was carcinogenic in male F344.DuCrj rats. causing urinary bladder tumours. and nephrotoxic in F344..Du('rj rats of both sexes. ] h e authors are grateful to Mr S. Iguchi, Mr K. Yuzawa. MrT. lkeda, Mr H. Takahashi, Mr N. Yano and Mr A Nagasav, a for their collaboration in the conduct of these studies. Acknowledgements
REFEREN(tk%
]|iraga K. & Fu.iii T. (1981L Induction oftumours of the urinar,, system in |:344 rats by dietary administration of sodium o-phenylphenate. Fd ('osmet. lbxicol. 19, 3113. Hodge tt. C., Maynard E. A . Blancher It. J.. Jr, Spencer tt. ('. & Rowe V. K. (1952). roxicological studies of orthophenylphcnol (Dowictdc I). J. Pharmac. eats. lTwr. 104, 2¢)2. Kamiya N. & Itiraga K. (1982). l,,;niformity of test article concentration in pellet diet used feeding study. ,,[#tn. Rep. Toklo ..Wetr. Re.s. Lab. Puhl. lllth 33, 561 (in Japanesel. Mizoiri S.. Hirokado M.. Nakajima K., Nakajima [. & Endo F. (1981). Quality and determination of ophenylphenoI-Na in animal feeds. Ann. Rep. Tokyo Metr Res. Lab. Puhl. tilth 32 (2), 28 (in Japanese). Nakamura K., lkeda T., lguchi S. & ttiraga K. (1982L Toxicity of o-phenylphenol (OPP) by dietary administration to male rats for 91 weeks. Ann. Rep. Tokyo Ah'tr. Res. Lab. Puhl. filth 33, 434 (in Japanese).
0278-6915 84 S3.00+0.(R) Copyright ~ 1984 Pergamon Press Lid
I N D U C T I O N OF T U M O U R S OF THE U R I N A R Y B L A D D E R IN F344 RATS BY D I E T A R Y A D M I N I S T R A T I O N OF o - P H E N Y L P H E N O L K. HtRAC;A and T. F U l l Department of Toxicology. Tokyo Metropolitan Research Laboratory' of Public Health. 24-1. It~akunincho 3 chome, Shinjuku-ku, Tokyo 160, Japan (Receired 6 March 1984) Abstract o-Phenylphenol (OPP), a fungicide approved as a fc,od additive in Japan, was given in pelleted diets at dietary levels of 0.156. 0.313.0.625, 1.25 or 2.5", to groups of 11 or 12 F344:DuCrj rats of each sex for 13 v,k, and 0.625, 1.25 or 2.5",, to groups of 20-24 male F344:DuCrj rats for 91 wk. In the 13-wk study, transitional cell papillomas of the urinary bladder occurred in 6..12 (50",,) of the male 1.25",, group. In the 91-wk stud.,,,, urinary bladder tumours appeared in 23.24 (96",,) of the 1.25",, and 4 23 (17",) of the 2.5",, group. Among these tumours, transitional cell carcinomas were found in 20.23 (87".) or 1.25",, and 2..4 (50",,) of the 2.5",, groups. A dose-related increase in the incidence of nephritic lesions was also observed in dosed rats in both the 13- and 91-wk study.
INTRODUCTION o - P h e n y l p h e n o l (OPP) and its sodium salt, sodium o - p h e n y l p h e n a t e ( O P P - N a ) are fungicides that have been a p p r o v e d for use on citrus fruits in J a p a n since 1977. O P P was reported not to be carcinogenic for rats ot" b o t h sexes (Hodge, M a y n a r d , Blanchet et al. 1952). In contrast, we found that O P P - N a produced t u m o u r s o f the urinary tract, particularly the urinary bladder, in male F 3 4 4 . D u C r j rats (Hiraga & Fujii, 1981). Based on these results, we t h o u g h t that O P P would also be carcinogenic in rats. The purpose of the present studies was to evaluate the carcinogenicity of O P P to the urinary tract in rats using the same experimental m e t h o d s as in the previous feeding study of O P P - N a . EXPE.,~IMENTAI. •kIaterials. O P P used for the present study was Dowicide 1 (Lot No. M M 0 1 0 4 0 ) obtained from Dow Chemical Co., MI, USA, as a white crystalline powder. Its purity was more than 98".. Animals. Male and female F 3 4 4 / D u C r j rats at 4 wk of age were purchased from Charles River J a p a n Inc., K a n a g a w a , Japan. Rats were housed individually in stainless-steel cages. W 22 × D 20 × H 16cm, with wire-mesh fronts and floors. The cages were suspended from belt-type racks with an a u t o m a t i c water-supply system ( t h r o u g h a bacterial tilter). Rats were initially segregated into equal weight distribution groups, and then divided into experimental groups. Their cages were allocated r a n d o m l y in the racks. The animal room was m a i n t a i n e d at 24 25 C, and relative humidity at 50--60'~.. Incoming air was passed t h r o u g h a filter of 85". efficiency and a filter of 99.9",, efficiency ( H E P A filter). R o o m air was changed ten times per hour. Fluorescent lighting was controlled to give 1 I hr light (06.00 17.00) and 13 hr dark. Rats of b o t h dosed and control groups were not Ahhret'iation: OPP = eJ-phenylphenol.
housed in the same room as rats treated with any other chemicals. Administration Of materials. O P P was mixed with the raw materials ot" the pelleted diet CE-2 (Clea J a p a n Inc., Tokyo), at c o n c e n t r a t i o n s ot" 0.156, 0.313, I).625, 1.25 and 2.5",i before they were made into pellets. Analyses of the pelleted diets by gas chrom a t o g r a p h y showed that O P P was stable and mixed homogeneously in the diets, and its initial concentrations in each diet were not changed (Kamiya & Hiraga, 1982: Mizoiri, Hirokado, N a k a j i m a et al. 1981). The m a x i m u m c o n c e n t r a t i o n of O P P in diet in the present studies was selected as 2.5",,, based on the results of the previous studies of O P P - N a . A concentration of 4'!o O P P - N a , the m a x i m u m dose of the previous studies, is equivalent to 2.5",, O P P by weight on a m o l a r basis. Accordingly, c o n c e n t r a t i o n s of 0.156, 0.313, 0.625, 1.25 and 2.5",, of O P P are equivalent to 0.25, 0.5, I. 2 and 4",, O P P - N a respectively. G r o u p s of 11 or 12 rats of each sex were given pelleted diet c o n t a i n i n g 0.156, I).313, 0.625, 1.25 or 2.5'! o O P P t h r o u g h o u t the 13-wk study. In the 91-wk study, groups of 20 24 male rats were given diet containing 0.625, 1.25 or 2.5",, t h r o u g h o u t . The control rats in these studies were fed basal diet throughout the study. Feed and water were supplied freeb. A d m i n i s t r a t i o n of the test chemical was begun ,,,.'hen the males were 35 days old and the femalcs 34 days old in the 13-wk study, and when the males were 38 or 39 days old in the 91-wk study. Observation. All of the rats were observed twice daily and all deaths, gross t u m o u r s and clinical signs were recorded. Each rat was weighed once a week in b o t h the 13- and 91-wk study. Food intakes for all rats were m o n i t o r e d once a week in the 13-wk study, and once weekly till wk 3, biweekly from wk 4 to 9 and every 4 wk from wk 10 to the end of the study in the 91-wk study. M e a n intakes of O P P in each of the dosed groups were calculated from food intakes. M o r i b u n d rats and rats that survived to the end of the study were exsanguinated and autopsied. A u t o p 865
866
K. HIRAGA and T. FUJH
sits were also performed on all rats found dead. unless precluded in whole or part by autolysis. Pathological e.vumhlation. The pathological exammation consisted of gross and microscopic examination of major tissues, major organs and all gross lesions. The tissues were prcserved in 10",, neutral buffered l\)rmalin, embedded in paraffin, sectioned. and stained with haernatoxylin and eosin. The urinary bladders were evacuated of urine by transurethral injection through a 27-gauge needle and were distended with 0.4 1.2ml of 10",, neutral buffered fornaalin, depending on the age of the rat. After the fixation, the urinatry bladders were cut longitudinally and then examined grossly' and stereoscopicallx. .~'tati.~lh'a! wmh'.vi.~. Student's t test was used to compare both the body-weight gains and food intakes of the control groups with those of dosed groups (P < 0.05). The Fisher exact test was used to compare surxival rates, incidences of tumours and the other lesions of the control group with those of dosed groups. The Fisher exact P values are indicated in the tables for ,,alucs o1" P less than 0.05.
RE.NL I.'l'S
In the 13-wk study, the mean body weights ot" the 0.156. 0.313 and 0.625°,, groups ot" each sex were comparable to those of the controls throughout the study. The mean body weights were significantly lower than those of the controls in the 1.25",, groups of each sex from wk 2 to 9 and in the 2.5",, groups of each sex throughout the study. The weight depressions relative 1o the controls were 5-9",, for 1.25",, females. 7 13",, for 1.25",, males. 14 32",, for 2.5",, females and 26 35",, t\~r 2.5"o males. In the 91-wk stud5', the mean body weight of the 0.625°i, group was comparable 1o that of the controls throughout the stud).. Mean body weights were significantly lower than that of the controls m both the 1.25",, group from v,'k 21 to the end of the study and the 2.5",, group throughout the study'. The weight depressions relative to the controls were 2 12". for the 1.250,, and 17 24",, l\n the 2.5",, groups (Nakamura. Ikeda. Iguchi & Hiraga. 1982). In the [3-wk study, the daily food intakes (g.rat) of the 2.5",, groups o1" each sex were signiticantly lower than those of the controls throughout the study in males and from wk 1 to 9 in females, but the relative lk)od retakes (g.kg.day) of these groups were higher than those of the controls throughout the study except for the male 1.250,, group of wk I. The daily food intakes of the other dosed groups were comparable 1o those oi" the controls throughout the study. In the 91-wk study, the daily food intake (g. r a t ) o f the 2.5",, group was significantly lower than that of the controls from wk I to 85 except at wk 33. but the relative food intake (g:kg:day) of this group was signiticantly higher than that of the controls throughout the study except from wk 3 to 13. The daily food intakes of the other dosed groups were approximately equal to those of the controls throughOUt the stud', (Nakamura et al. 1982). In the 13-~k stt, dy, the mean retakes of OPP of the 0. 156, 0.313.0.625, 1.25 and 2.5",, groups were 98.4,
210.3, 409.9. 815.1 and 1492.Smg. kg.day in males. and 108.5, 221.1,432.3, 888.4 and 1622.6 mg..kg..day in females, respectively. In the 91-wk stUdy,,, mean intakes of OPP of" 0.625, 1.25 and 2.5". groups were 269, 531 and 1140 mg/kg;day, respectively. In the 13-wk study 8.11 (73".) of the male 2.5",, group, l 1..12 (92".) of the female 2.5",, group and all the rats of the other groups lived to the end of the study. The survival rates at the end of the 91-wk study were 23..24 (96",,) of the controls. 18.20 (90",,) of the 0.625". group. 17.24 (71",,) of the 1.25",, group and 15."23 (65",,) of the 2.5",, g,oup. The survival rates for both the 1.25 and 2.5"0 groups in the 91-wk study were signilicantly lower ( P - 0 . 0 2 3 for 1.25",, and P = 0.009 for 2.5". groups) than that of the control. A m o n g the rats which died during the study, the incidence of urinary bladder tumours was 0.1 (0",,) of the control. 0 2 (()",,) of the 0.625",, group. 7..7 (100",,) of the 1.25°. group and 0..8 [0",,) of the 2.5",, group in the 91-wk study: none were found m the 13-wk study. Occult blood in the urine as indicated by' test tapes (Labstix"-II. Miles-Sankyo ('o. [.td. Japan) was observed from wk 15. and gross haematuria was seen from wk 52 in rats of both the 1.25 and 2.5",, groups in the 91-wk stud.',.. Proliferative lesions of the urinary bladder were found in dosed groups in both the 13-and the 91-wk study. In the 13-wk study, the lesions were seen in 12..'12 (100",,) of the male 1.25",, group, but not in any other groups. Ilalf the lesions in the male 1.25",, group were transitional cell papillomas and the other hall" transitional cell hyperplasias. The incidences of these two lesions in this group were significantly higher (P = 0.007. Fisher exact test) than m the controls. In the 91-wk study', bladder lesions appeared in 10",, of the 0.625% group. 96" 0 of the 1.25",, group and 48",, of the 2.5",, group ITable 1). tJrinar~ bladder turnouts occurred m 96',. of the 1.25",, and 17",, of the 2.5"o group. The mcidence of this tumour was signiticantb higher in the 1.25",, group than in the controls (P <0.001). Of these tumours, transitional cell carcinomas accounted for X7",, m the 1.25",, group and 50",, in the 2.5",, group. In the 1.25",, group five carcinomas invaded their own stalks or the bladder wall. The first tumour ipapilloma) appeared in a rat of the 1.25",, group that died during wk 65. and the first transitional cell carcinoma was found in a rat of the 1.25"o group that died during wk 82. Grossly, urinar.,, bladder tumours were papillary or polypoid, having a caulittov,.er-like appearance. Many' of these tumours were multiple and arose m the ventral wall of the apex of the bladder growing into the lumen (Fig. 1). tlistologically, the lesions of the urinary bladder were classified as hyperplasia, papilloma and transitional cell carcinoma. The hyperpl:tsia was characterized by an epithelium with more than four cell layers and slight nuclear abnormalities (Fig. 2). The papillomas were covered by at relatively normal epithelium composed of not more than six layers of cells and grew out into the bladder lumen with a delicate fibrovascular stroma (Fig. 3). The transitional cell carcinoma contained a small amount of connective tissue with blood vessels and was covered b', transitional epithelium. The epithelium showed increased
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Fig. I. Gross appearance of thc cut surface of the urinary bladder from male rats fed orthophenylphenol at I.~5o in the diet (or 84 wk (left) or 91 wk (right). Each division represents 1 mm.
Fig. 2. Fransitional cell hyperplasia, a suspicious prcneoplastic change, in the urinary bladder of a male rat fed orthophenylphenol at 1.25",, in the diet for 13 w k Haematoxylin and eosin × 200.
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Fig. 3. Transitional cell papilloma of the urinar~ bladder of a male rat fed orthophenylphenol at 1.25",, in the diet lor 13 wk. Ilaematoxylin and eosin x 100.
Fig.4. Transitional cell carcinoma of the urinar~ bladder of a male rat l'~d orthophenylphenol at 1.25" in the diet for 91 wk. Haematoxylin and eosin × 20().
Carcinogenicity of orthophcnylpheno]
869
Table 1. Summary' of the incidence of hyperplastic or neoplastic lesions of the urinary bladder in male F344..DuCri rats fed diet containing orthophenylphenol (OPP) for 91 wk
Dietary level of OPP ( " , , )
No. of rats examined histologically
0 (('ontrol) 0.625 1.25
24 20 24
2.5
23
No. of rats with bladder tumour 0 0 23 (P < 0.001 )§ 4
No. of rats with transitional cell... ....................... Carcinoma Hyperplasia
Papilloma
0 2 0
0 0 3
NoninvasivO0 0 15
Invasive.+ 0 0 5
7
2
2
o
tTumours showed noninvasive growth into the bladder wall. ~Tumours showed invasive growth into the bladder wall. §Figures in parentheses show the probability level for the Fisher exact test for the comparison of that dosed group with the control group when t' is less than 0.05. cellularity, irregularity of cell size. nuclear abnormalities and mitotic figures (Fig. 4). Vesical calculi about 0.1 5 mm in diameter, dark or greenish brown in colour and having rough or smooth surfaces were observed grossly or stere()scopically. Calculi were found in 2.12 (17",,) of the 1.25",, and 1.12 (8".) of the 2.5'~,, groups of males in the 13-wk study, and 17.24 (71" o) of the 1.25'1,1 and 14..23 (61".) of the 2.5". groups in the 91-wk study. A m o n g rats bearing urinary bladder tumours, rats with calculi were observed in 1..6 (17",,) of the male 1.25",, group in the 13-wk study, and 17.:23 (74,(,) of the 1.25". group and 3..4 (75",,) of the 2.51',, groups in the 91-wk study. Transitional cell hyperplasia in the pelvis and.or papilla ',','as found in 3..11 (27",0 of the male 2.5'!o group in the 13-wk study, 12..23 (52'~0) of the 2.5"o group in the 91-wk study. The incidence of hyperplasia in the ql-wk study was significantly higher (P < 0.001 ) in the 2.5". group than in the controls. In the 13-wk study, no tumours at sites other than the urinary bladder were found. In the 91-wk study, interstitial cell tumours of the testes were the tumours most frequently observed at other sites. Turnouts of the pituitary and adrenal glands were also found frequently. These tumours occurred with approximately equal frequency in dosed and control groups. Nephritic lesions attributed to the administration of OPP appeared in both the 13- and 91-wk studies. In the 13-v,'k study, slight nephritic lesions were seen in 7 I 1 (64"o) males and 6..12 (50"o) females of the 2.5"o groups. These incidences were significantly higher (P -- 0.001 for males. P = 0.009 for females) than those of the controls. A m o n g these lesions, slight pyelonephritis was observed in 3.'7 (43°.) of the male and 3.6 (50",,) of the female groups, and the other lesion was interstitial nephritis. In the 91-wk study, moderate to scvere nephritic lesions appeared in 3.24 (13".1 of the 1.25". group and 23..23 (100",,) of the 2.5"o group. The incidence of this lesion was signiticantly higher (P <0.001) in the 2.5% group than in the controls. A m o n g these lesions, moderate to sevcrc pyelonephritis with papillary destruction were found in I..3 t33"o) of the 1.250o and 15..23 (65".) of the 2.5", groups, and the other lesion was interstitial nephritis. I)IS('t',~SiON
Mean body weights of both 1.25 and 2.5". groups of each sex. particularly 2.5",, groups, were lower
than those of the controls throughout most of the study in both the 13- and 91-wk studies. Survival was affected adversely in the 2.5'~,, groups ot" each sex in the 13-wk study, and showed a dose-related decrease in the 91-wk study. Thus, OPP was toxic to rats in both 1.25 and 2.5" 0 groups of each sex, particularly in 2.5'~,i groups, as shown by effects on weight and survival. Proliferative lesions in the urinary bladder attributed to the administration ot" OPP were found in the male 1.25'!,, group in the 13-wk study, and the 0.625",,, 1.25'~i, and 2.5",, groups in the 91-wk study. Urinary bladder tumours occurred in the male 1.25". group in the 13-wk study, and in the 1.25% and 2.5"o groups in the 91-wk study. A m o n g these turnouts. transitional cell carcinomas were seen in more than half of the affected animals in the 91-wk study', and all the other tumours were papillomas. Thus, urinary bladder tumours occurred in male dosed groups. particularly in the 1.25'!. groups, and hyperplastic lesions ot" the urinary bladder and suspicious preneoplastic lesions were also found only in dosed groups in both the 13- and 91-v,k studies. These results demonstrate that OPP. free phenol of OPPNa. had the same carcinogenic effect on the urinary bladder of male rats as O P P - N a showed in previous studies. Urinary bladder tumours were found in rats fed OPP in the present studies, but not observed in the feeding study' of OPP by Hodgc et al. (1952). This discrepancy may be due to differences in the test conditions used, such as the strain of rats. diets or selection of doses. In the present studies, the incidences of urinary bladder turnouts in the 2.5",, groups were lower than those in the 1.25". groups. In contrast, adverse effects on both weight and survival were more severe in the 2.500 groups than in the 1.25°. groups. Administration of OPP in the diet at 2.5",; was presumably toxic for rats. In the previous 13-wk study of OPP-Na. urinary bladder tumours were found in I.:10 of the 1",, 9.. 10 of the 2",, 1..10 of the 4",, groups of males and 2.10 of the female 4". group. In the 91-wk study of O P P - N a using male rats, urinary bladder tumot, rs occurred in 6..21 (29",,) of the 1",,. 19.21 (90",,) of the 2" o and 8:20 (40",,) of the 4"i, groups, and tumours of the renal pelvis and.or papilla were also seen in the 0.5. 1. 2 and 4",, groups. The incidence of urinary bladder tumours in the 2 ' ! 0 0 P P - N a group in the previous 91-wk study was approximately equal to
870
K. thRA(;A and T. I't:Jit
that in the 1.25",, O P P group in the present study. Moreover. the incidences in these two groups were higher than those in the o t h e r dosed groups including both 2.5",, O P P and 4",, O P P - N a groups in each stud). Thus. the trend of the incidence of urinar', bladder t u m o u r s in the previous stud)' of O P P - N a was similar to thal in the present s t u d ) of OPP. No t u m o u r s of the urinary bladder were found in the 0.625". O P P groups in either the 13- or 91-,xk studies. In contrast, one papilloma in the 13-~k study and six carcinomas m the 91-wk study were seen in the male 1",, O P P - N a groups (corresponding to the 0.625% O P P groups) in the previous studies. Bladder t u m o u r s occurred in one male group (I.25",,) m the 13-wk stud,, and two groups (1.25 and 2.5",) in the 9l-wk study of ()PP. In contrast, in the stud.', of O P P - N a the t u m o u r s appeared in two male groups I 1 and 2",,) and one female group (4",,) m the 13-wk stud.~, and three groups 11. 2 and 4",,) in the 91-wk study. These findings suggest that the carcinogenicity of O P P - N a to the urinary bladder of F344.DuCrj rats was greater than that o f O P P . O P P is a weak acid. in contrast to O P P - N a which shows alkalinity ( p t t 11.8 when dihlted 1 to 511 m water). It may be that the difference between O P P and OF'P-Na in the strength of carcinogenicity to rat urinary bhtdder was caused by this alkalinity. Vesieal calculi were seen only in dosed males in the present studies, and also found in dosed rats m the previous 91-wk study of O P P - N a . These results suggest that both O P P and O P P - N a cause stone formation in the urinary tracts of male rats. In the present 13-wk study, calculi were found in 2.12 of the male 1.25"., group, m contrast hyperplasia or papilloma of the urinary bladder occurred in all of this group. These results suggest that there is no correlation between these calculi and the proliferative lesions of the urinary bladder in rats.
Nephritic lesions attributed to the a d m i n i s t r a t i o n of O P P were not unexpected, since these lesions were reported by Hodge et al. (1952). The lesions were seen in the 2.5",, groups of each sex in the 13-wk stud.,,. The incidence of the lesions showed a positive doserelated increase in the 91-wk study. It is clear that O P P caused nephritic lesions in F344.'DuCrj rats of each sex. U n d e r the conditions of the present studies, dietar5 a d m i n i s t r a t i o n of O P P was carcinogenic in male F344.DuCrj rats. causing urinary bladder tumours. and nephrotoxic in F344..Du('rj rats of both sexes. ] h e authors are grateful to Mr S. Iguchi, Mr K. Yuzawa. MrT. lkeda, Mr H. Takahashi, Mr N. Yano and Mr A Nagasav, a for their collaboration in the conduct of these studies. Acknowledgements
REFEREN(tk%
]|iraga K. & Fu.iii T. (1981L Induction oftumours of the urinar,, system in |:344 rats by dietary administration of sodium o-phenylphenate. Fd ('osmet. lbxicol. 19, 3113. Hodge tt. C., Maynard E. A . Blancher It. J.. Jr, Spencer tt. ('. & Rowe V. K. (1952). roxicological studies of orthophenylphcnol (Dowictdc I). J. Pharmac. eats. lTwr. 104, 2¢)2. Kamiya N. & Itiraga K. (1982). l,,;niformity of test article concentration in pellet diet used feeding study. ,,[#tn. Rep. Toklo ..Wetr. Re.s. Lab. Puhl. lllth 33, 561 (in Japanesel. Mizoiri S.. Hirokado M.. Nakajima K., Nakajima [. & Endo F. (1981). Quality and determination of ophenylphenoI-Na in animal feeds. Ann. Rep. Tokyo Metr Res. Lab. Puhl. tilth 32 (2), 28 (in Japanese). Nakamura K., lkeda T., lguchi S. & ttiraga K. (1982L Toxicity of o-phenylphenol (OPP) by dietary administration to male rats for 91 weeks. Ann. Rep. Tokyo Ah'tr. Res. Lab. Puhl. filth 33, 434 (in Japanese).