O-phenylphenol and its sodium and potassium salts: a toxicological assessment.

Ortho-phenylphenol (OPP) and its sodium (SOPP) and potassium (POPP) salts are used as fungicides and disinfectants. Due to the widespread use of especially OPP and SOPP, the potential for consumer exposure and some "critical" findings the toxicological database is quite extensive and complex. In experimental animals toxicity after single oral and dermal administration of these compounds is low. For the skin and mucous membranes, OPP has to be considered as irritating, and SOPP and POPP as corrosive. A large number of chronic toxicity and reproduction studies did not show any indication of oestrogen-like or other endocrine effects of OPP in the mammalian organism. No teratogenic effects were observed after the administration of OPP or SOPP in rats, mice, and rabbits. In two-generation studies in rats, OPP did not affect reproduction. The available data do not suggest a relevant potential for immunotoxic properties. The administration of high dietary concentrations of OPP to 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. Only in male mice of one study, using a strain prone to develop hepatocellular tumors at high spontaneous incidences, the incidence of hepatocellular adenomas was increased. The incidence of hepatocellular carcinomas was not affected by treatment. The urothel of the urinary bladder (at very high doses also of the renal pelvis and the papilla) is the main target tissue after the repeated oral exposure of rats. The changes initially consist of increased mitosis, followed by simple epithelial hyperplasia, developing to a papillary and/or nodular form, later on to papillomas and transitional carcinomas. Crystals or stones in the bladder do not play a decisive role in this cascade. SOPP is more effective than OPP in this respect. Male rats are much more sensitive than females. In mice, hamsters, guinea pigs, and dogs, urothelial lesions do not develop even at very high oral dose levels. The findings in rats explain why there is a large genotoxicity/mutagenicity data base not only for OPP and SOPP but also for their metabolites on nearly all kinds of endpoints/targets. The weight of evidence suggests that genotoxicity of OPP/SOPP or their metabolites does not play a decisive role for the carcinogenicity at the urothel. Among them are lack of DNA binding of OPP to the rat bladder epithelium, the differences between OPP and SOPP, between male and female rats, between rats and mice (despite roughly comparable toxicokinetics), as well as the fact that tumors develop only at dose levels inducing hyperplasias. In addition, the strong dependence of the incidence and severity of the nonneoplastic and neoplastic bladder changes on urinary pH values (modified by feeding of ammonium chloride or sodium hydrogen carbonate) is consistent with the hypothesis of a nongenotoxic mode of action. Finally, there is no correlation between the urinary concentration of OPP or its metabolites and the incidence of hyperplasias/tumors in the urinary bladder. Both tumorigenic effects in rats and male mice are considered to represent high-dose, sex- and/or species-specific phenomena, based on nongenotoxic mechanisms of action and therefore allow the conclusion that the conventional margin of safety approaches are appropriate when assessing the risk of applications of OPP and its salts.

Investigations on the mutagenicity of 1,4-dichlorobenzene and its main metabolite 2,5-dichlorophenol in vivo and in vitro.

The genotoxic potential of 1,4-dichlorobenzene (1,4-DCB) has been extensively evaluated in vitro and in vivo. The majority of the studies demonstrated the absence of a genotoxic potential for 1, 4-DCB. At variance are a bone marrow micronucleus test (MNT) after intraperitoneal (i.p.) treatment of NMRI mice [Mohtashamipur et al., Mutagenesis 2 (1987) 111-113] and a gene mutation assay on mouse lymphoma cells [McGregor et al., Environ. Mol. Mutagen. 12 (1988) 85-145]. Therefore, we investigated 1,4-DCB and its main metabolite 2,5-dichlorophenol (2,5-DCP) for both endpoints. In an MNT, male and female NMRI mice were treated orally with single doses of 2500mg/kg 1,4-DCB and 1500mg/kg 2,5-DCP, respectively. Smears were prepared 24, 48 and 72h thereafter. No induction of micronuclei was detected for both compounds. Also under the conditions of Mohtashamipur et al. (1987), intraperitoneal treatments of male and female mice with 2 x 177.5 and 2 x 355mg/kg 1,4-DCB failed to induce micronuclei. In addition, CHO/HPRT-gene mutation tests with 1,4-DCB and 2,5-DCP yielded negative results for both compounds with and without metabolic activation system. Therefore, 1,4-DCB and 2,5-DCP are considered to be non-mutagenic in these test systems.

Time course of chronic oral cadmium nephrotoxicity in Wistar rats: excretion of urinary enzymes.

Twelve male and female Wistar rats each received cadmium (as CdCl2) in their diet at concentrations of 0, 10, 50, and 250 ppm for 72 weeks. After 1, 4, 8, 13, 18, 26, 32, 45, 57, and 68 weeks a total of 8 enzymes from different cellular compartments of the nephron were measured. At the end of the study period, the kidneys were examined histopathologically. Concentrations up to and including 50 ppm did not induce any adverse effect. At 250 ppm, growth of male and female animals was markedly retarded. Significantly increased activities of the cytosolic phosphohexose isomerase were excreted by males and females receiving 250 ppm at all timepoints from week 13. The values of the mitochondrial glutamate dehydrogenase were mostly elevated from week 1 to 57, however, due to a wide scatter range, were only occasionally significantly different from control values. The brush border enzymes (gamma-glutamyl transferase, alkaline phosphatase and leucine arylamidase) were not changed in a relevant manner in female rats, while in 250 ppm males the excreted activity of ALP and LAP from week 1 to week 18, and that of GGT during the entire study period were significantly lower than the control values. Excretion of the lysosomal enzymes aryl sulfatase A, beta-galactosidase, and beta-N-acetyl-D-glucosaminidase was at no time influenced in a noteworthy manner. Histopathology after 72 weeks revealed chronic but also acute degenerative changes in the kidneys of 250 ppm males and females. A comparison of published data on persons having undergone high cadmium exposure with the results presented here shows remarkable differences.

Trends in mortality, body weights and tumor incidences of Wistar rats over 20 years.

Historical data of more than 8,000 Wistar rats (designation: WISW SPF Cpb) used as controls in seventy 2-year studies terminated between 1975 and 1994 were analyzed for time trends in food consumption, terminal body weight, mortality, and tumor incidences. In males there was a significant (p < 0.01) time trend towards higher terminal body weight and a tendency (p > 0.05) to lower incidences of pituitary tumors and adrenal pheochromocytomas, while mortality remained stable. Leydig cell tumors showed a significant (p = 0.0005) positive trend. In females, terminal body weight did not increase over time but pituitary and mammary tumors showed a marked and highly significant (p = 0.0001) increase, which explains a significant (p = 0.0045) positive trend in mortality. There was a significant (p = 0.0001) negative time trend for uterine adenomas/carcinomas and a slight tendency (p = 0.4135) towards a decreased incidence of endo-metrial stromal polyps. Since the average food intake data do not indicate a time trend the changes observed might probably not be related to higher caloric intake. In contrast to other authors we could not find a positive correlation between either body weight and incidences of pituitary tumors or body weight and mortality. Certain selection measures at breeding and/or a genetic drift over time might explain the time trends observed. This data does not yet indicate a need for a change in ad-libitum feeding of these animals.

Short- and intermediate-term carcinogenicity testing--a review. Part 1: the prototypes mouse skin tumour assay and rat liver focus assay.

Carcinogenicity testing is by far the most expensive and time-consuming study type of toxicology. For many years, the lifetime exposure with the maximum tolerated dose in two rodent species has been the gold standard of carcinogenicity testing of pharmaceuticals. Major change was introduced by the Fourth International Conference on Harmonization in July 1997; a chronic rodent bioassay in one species and a short-term carcinogenicity assay are regarded as sufficient for registration. Such requirements provide the opportunity to redirect the vast resources previously spent on the lifetime study in the second species. Numerous experimental protocols for short- and intermediate-term carcinogenicity testing in many target tissues have been available for years. The first part of this review describes the basic principles of short- and intermediate-term carcinogenicity testing using the examples of the widely used mouse skin tumour assay and the rat liver foci assay. In the context of these experimental models, the discrimination and quantification of initiating and promoting activity and the use of preneoplastic lesions as endpoints in carcinogenicity testing are described. The review includes the limitations of the models with regard to the extrapolation from effects observed in animal experiments to a potential exposure of humans.

Short- and intermediate-term carcinogenicity testing--a review. Part 2: available experimental models.

Numerous experimental protocols for short- and intermediate-term carcinogenicity assays have been available for many years. This paper surveys various of these test systems in rodents, fish species, non-vertebrates and avian embryos in ovo. The mouse skin tumour assay and the rat liver foci assay were used to introduce the basic concepts of short- and intermediate-term carcinogenicity testing in the previous part of the review. The focus of this second part of the review is on rodent assays for carcinogenicity testing in the lung, kidney, urinary bladder, pancreas, stomach, oral cavity, small intestine, colon, and on the possibility to combine several target organs in multi-organ models. The potential use of various fish species, non-vertebrates and hatching eggs for carcinogenicity testing is outlined and the advantages and limitations are discussed. This review also presents the problem of validation of any carcinogenicity test system and proposes a strategy for contemporary safety assessment of chemicals with regard to the detection and evaluation of carcinogenicity.

D-glucose combined chronic toxicity and carcinogenicity studies in Sprague-Dawley rats and Syrian golden hamsters.

After an initial period of 16 weeks with increasing concentrations, D-glucose was administered at 30% in the diet to 50 male and 50 female Sprague-Dawley rats from the 17th to the 112th study week. Additional 10 male and 10 female animals were treated for 14 months and then sacrificed for interim examination. Groups of 60 male and 60 female Syrian golden hamsters received D-glucose in the form of 20% solution in tap water for a period of 80 weeks. In each case, groups consisting of an equal number of untreated animals served as controls. General behavior and mortality were not affected by the treatment. The rats and hamsters treated with glucose showed significantly higher body weights of up to a maximum of 16% in male and 26% in female rats, or 15% in male and female hamsters. In rats, the increase was evident by week 14, and in the hamsters by week 10. Glucose-dosed rats displayed a slightly increased feed intake and a reduced water intake. Both parameters, however, were not influenced in hamsters. Hematological and histopathological examination showed no pertinent changes in hematopoetic tissue. Sharply increased blood glucose and renal glucose excretion values were present in rats beginning with 18 months and were indicative of the development of non-insulin-dependent diabetes mellitus (NIDDM). The insulin concentrations in peripheral blood were not appreciably affected, although there was a trend to higher values in males at all evaluation times and in females only at 3 months. Pathological evaluation did not show any compound related non-neoplastic lesions. The incidences of islet cell adenomas in the pancreas of male rats were significantly increased and the cortical adenomas in the adrenals of females were decreased. In addition, the mammary gland adenomas (in females) and the Leydig cell tumors of the testes were decreased. In hamsters, the incidence of adrenocortical adenomas were increased in the females. No other pertinent neoplastic changes were observed. In conclusion, the increases and decreases in benign neoplasms of hormone-sensitive tissues, appear to be the result of nutritionally/metabolism-induced modulation of the homeostasis in these 4 tissues in both species, and not the result of chronic glucose administration.

Subacute oral toxicity of tetraethylene glycol and ethylene glycol administered to Wistar rats.

A subacute toxicity study with administration of tetraethylene glycol in dosages of 0-220-660-2000 mg/kg body weight to male and female Wistar rats via gavage was conducted in order to characterize a possible toxic action of this compound. The structurally related compound ethylene glycol is known to cause kidney toxicity. Therefore, special attention was paid to investigating possible toxic effects of tetraethylene glycol on this organ. In order to compare possible treatment-related effects of tetraethylene glycol with those known from ethylene glycol, a group of male and female rats was treated with 2000 mg ethylene glycol/kg body weight. Daily oral application of tetraethylene glycol over 4 weeks was tolerated without toxic effects up to and including 2000 mg/kg body weight. Daily oral application of ethylene glycol over 4 weeks resulted in treatment-related effects on the kidneys. A slight decrease in the urinary excretion of potassium, calcium and phosphate (males), a diminished pH-value of the urine, and a slight increase in osmolality (females) were observed. In both sexes excretion of oxalate was significantly increased and microscopic examination of urinary sediment revealed calcium oxalate crystals. Kidney weights of males and females were slightly elevated. Histopathology revealed crystals in renal tubuli, renal pelvis, and urinary bladder; tubulopathy and epithelial hyperplasia within the renal pelvis were also observed. Therefore, the study confirmed the kidney as target for ethylene glycol toxicity and gave no indications of tetraethylene glycol-induced toxic effects.

Calcium channel blockers and the risk of cancer: a preclinical assessment.

The preclinical evidence for a potential influence of calcium channel blockers (CCBs) on carcinogenesis is discussed in the light of a broad database from rodent carcinogenicity studies as well as literature data. In all bioassays performed in rats and mice on the dihydropyridine CCBs--nifedipine, nimodipine, nisoldipine, and nitrendipine--no evidence was found for a carcinogenic potential of these compounds. Calcium is an essential intracellular signal for cell proliferation and apoptosis. The crucial role of increased cell proliferation in all stages of carcinogenesis is well documented. Some indirect experimental evidence also points to a role of defective apoptosis in tumor promotion. CCBs uniformly inhibit cell proliferation, whereas the influence of CCBs on apoptosis is inconsistent, resulting in an inhibition or increase in apoptosis dependent on cell type. Accordingly, antitumorigenic effects of CCBs have been reported based on their antiproliferative action. A tumor-promoting effect of CCBs based on inhibition of apoptosis, however, remains purely speculative and, in fact, can be denied based on the results of in vivo bioassays. It is therefore concluded that there is no preclinical evidence that should give rise to concern over the carcinogenic potential of dihydropyridine-type CCBs.

Serotonin (5-HT1A-receptor) agonist-induced collecting duct vacuolation and renal papillary necrosis in the rat.

General anxiety in humans is treated with azaspirodecanedions, which act through a reduction of serotonin transmission. Ipsapirone also represents a serotonin (5-HT1A) receptor agonist and was under development as an anxiolytic drug. Histopathologic evaluation of animal experiments revealed cellular swelling and/or vacuolation of renal papillary and medullary collecting duct (MCD) epithelium in rats but not in dogs or mice. The changes ensued already after 1 wk of dosing and were first localized in the inner MCDs. Longer treatment periods showed that these changes proceeded from proximal to distal, approaching the papillary collecting ducts. The changes were most likely the result of altered hemodynamics in the papillary tip. Swelling resulted in partial or total papillary necrosis in some cases. Furthermore, rats treated with ipsapirone showed a sharp and transient rise in urinary endothelin excretion. Concomitantly, urinary PGE2 levels were elevated. In contrast, no elevated levels of endothelin were detected in urine samples of patients from a volunteer study, leading to the conclusion that the human kidney is not susceptible to the ipsapirone-induced alterations seen in the collecting ducts of rats.

Trimethylphosphate: a 30-month chronic toxicity/carcinogenicity study in Wistar rats with administration in drinking water.

Trimethylphosphate (TMPO) was administered to 50 male and 50 female Wistar rats through their drinking water at doses of 0, 1, 10, or 100 mg/kg body weight up to 30 months. The dosage of 100 mg/kg was reduced to 50 mg/kg in week 54 for reasons of tolerance, and the animals were euthanized in week 100. Additional 10 animals per dose and sex were treated for 12 months and then euthanized for interim analysis. Weakness of the hind limbs, increased incidences of sunken flanks, distended abdomen, and poor general condition were observed in both sexes of the 100/50 mg/kg group beginning with week 46. Food intake was reduced in high dose males. At 10 mg/kg body weights were up to 10% (males) and at 100/50 mg/kg up to 20% (males) or 15% (females) lower than in controls. Mortality was not affected in animals receiving up to 10 mg/kg. At 100/50 mg/kg it was markedly increased, reaching about 70% at week 100. Relatively slight hematologic changes (reduced hemoglobin, hematocrit, erythrocyte counts, increased reticulocyte numbers, and thrombocyte counts as well as a shift in the differential blood count) at 100/50 mg/kg are interpreted as changes most probably secondary to the other toxic effects. Increased cholesterol concentrations in plasma, shifts in the serum protein electrophoresis (males), increased organ weights (females), and an increased incidence of necroses and lymphocytic infiltrations point to a treatment-related effect on the liver at 100/50 mg/kg. Slightly increased protein excretion, increased relative kidney weights, and an increased incidence of chronic progressive nephropathy are considered treatment-related but rather secondary effects at 100/50 mg/kg. At 100/50 mg/kg an increased incidence and severity of bilateral tubular atrophy in the testes was diagnosed. The most important toxic effect was neurotoxicity, consisting of degeneration and loss of nerve fibers in the peripheral nerves and the spinal cord, associated with myopathic changes, and occurring at 100/50 mg/kg. The no-observed-adverse-effect-level, based on the suppression of body weight gain, is 1 mg/kg in males and 10 mg/kg in females. The incidence, time of occurrence, spectrum of types, and localizations of tumors provided no indication of a tumorigenic/carcinogenic effect of the test substance. TMPO is therefore considered not to be carcinogenic in Wistar rats.

Hepatic and associated response of rats to pregnancy, lactation and simultaneous treatment with butylated hydroxytoluene.

This paper describes changes in the livers of rats fed diets containing butylated hydroxytoluene (BHT) over two generations in two separate studies. BHT did not produce tumours when tested for carcinogenicity in several studies by the conventional way. However, when BHT was given to rats in a two-generation carcinogenicity study, a high incidence of hepatic tumours was found in males but not in female rats of the F1 generation. A sequential study has been carried out to gain an insight into this unexpected finding, paying particular attention to the perinatal period. In the dose-ranging study designed to assess the tolerance of rats to BHT, groups of male and female rats (F0 generation) were fed diets calculated to deliver 0, 500, 750 and 1000 mg/kg body weight/day. Following a loading period of 5 wk the rats were mated. The BHT content of the diet was not adjusted during pregnancy and lactation. Owing to the normal increase in food consumption during lactation, intakes peaked at double the nominal value by 21 days after the birth of pups. At this time the pups (F1) were weaned onto control diet and maintained on it for 4 wk. At birth, the body weights of pups from the BHT-treated dams were comparable to those of the controls but at weaning the body weights of the pups from all three dose levels were less than those of the controls. At the termination of the experiment (4 wk after weaning), the pups from BHT-treated dams still weighed less than those from untreated controls. In the main experiment the F0 generation were fed 0, 25, 100 and 500 mg/kg body weight/day. Their offspring (F1 generation) were weaned on diets containing the same amount of BHT as the respective parents, apart from the group given the highest dose level (500 mg/kg body weight/day). This dose level was reduced to 250 mg/kg body weight/day at weaning in order to conform with previously published findings. The pups from the dams given the highest dose level were maintained on a dietary concentration of 250 mg/kg body weight/day for the entire study. A group of age-matched non-pregnant females was also studied and the results obtained compared with those from pregnant dams. Pups from all groups were examined at day 20 of gestation, at weaning (21 days after birth), and at 4 and 22 wk post-weaning. There were no effects on fertility and no increase in foetal abnormalities at any dose of BHT. Dams receiving BHT at a nominal dose of 500 mg/kg body weight/day showed liver enlargement accompained by induction of pentoxyresorufin O-depentylase and glutathione S-transferase, and proliferation of the endoplasmic reticulum. Pups from these dams were of the same weight at birth as controls but lost weight during the lactation period. This deficit was not recovered by the time the experiment was terminated. Hence, in two independent studies, the only significant finding in rats treated with BHT in utero and during lactation was that the weight gain of pups during lactation was less than expected when dams received at least 500 mg BHT/kg body weight/day. The body weight of pups did not return to normal following a return to a control diet for 4 wk. It is postulated that the retardation in weight gain of the pups could be due to inadequate milk production.

Quantitation of alpha(2)-microglobulin after administration of structurally divergent chemical compounds.

alpha(2)-Microglobulin-induced nephropathy is a phenomenon which is exclusively found in adult male rats. Various chemicals are able to bind to alpha(2)-microglobulin thus inhibiting its proteolytic degradation in lysosomes of the P2 segment of the rat nephron. The accumulation of this protein in 'protein droplets' or 'hyaline droplets' leads to necrosis, followed by regeneration which possibly later results in the formation of tumours. Here we report the development of a monoclonal antibody which is specific for alpha(2)-microglobulin. It was utilized to measure alpha(2)-microglobulin concentrations in plasma and tissues, and to stain alpha(2)-microglobulin in fixed tissue slides. In two studies we administered to adult male Wistar rats two groups of compounds: (1) one group of structurally diverse compounds, which give an overview of chemical entities capable of inducing the accumulation of alpha(2)-microglobulin; and (2) another group of structurally closely related compounds (i.e. substituted benzene derivatives) for the purpose of elucidating possible structure-activity relationships. The degree of alpha(2)-microglobulin-induced nephropathy was determined by immunohistochemical staining of kidney sections. In addition, liver and kidney tissue and plasma concentrations of alpha(2)-microglobulin were not found to be elevated whereas kidney tissue concentrations were higher than the controls. The increase over control values ranged from 154% (1,4-dichloromethyl-benzene) to 321% [alpha-methyl-4-(1-methylethyl)-cyclohexanemethanol]. Comparing structurally related benzene derivatives, the hyaline droplet accumulating (HDA) potential was found to depend both on the type of substituent and its position at the aromatic ring. In general HDA activity increased in the order benzene approximately equal to phenol approximately equal to alkylated phenols < halogenated phenols < halogenated benzenes. Further QSAR studies are needed to provide a theoretical base for these observations.

Calcium channel blockers and cancer: is there preclinical evidence for an association?

The preclinical evidence for a potential influence of calcium channel blockers (CCB) on carcinogenesis is discussed in the light of rodent carcinogenicity studies as well as mechanistic data. In the bioassays performed in rats and mice on the dihydropyridine CCB nifedipine, nimodipine, nisoldipine and nitrendipine, no evidence was found for a carcinogenic potential of these compounds. Moreover, the mechanistic knowledge on the influence of CCB on the fundamental processes of cell proliferation and apoptosis is not in favor of a tumor-promoting activity of these compounds. It is, therefore, concluded that there is no preclinical evidence that the therapeutic use of CCB of the dihydropyridine class is associated with an increased risk of cancer.

Urinary antigens as markers of papillary toxicity. I. Identification and characterization of rat kidney papillary antigens with monoclonal antibodies.

Monoclonal antibodies were prepared in an attempt to develop diagnostic tools for the identification of toxic damage to the rat renal papilla. One IgG and five IgM monoclonal antibodies, reacting with antigens localized in the papilla were obtained. Three of the IgM class and the IgG class monoclonal antibodies were found to be specific for antigens localized in collecting ducts, two of them staining papillary collecting ducts more intensely than cortical collecting ducts. The IgG class antibody, termed Pap X 5C10, recognizes an antigen located at high density on the luminal side of papillary collecting duct epithelial cells and at lower density in cortical collecting duct cells. One of the IgM class monoclonal antibodies reacts with an antigen localized in epithelial cells as ascending and descending loops of Henle and of connecting tubules. Another of the IgM class monoclonal antibodies reacts with an antigen localized in the interstices of the inner medulla. All these monoclonal antibodies react with their antigens in native frozen as well as in Bouin-fixed and paraffin-embedded tissue slices. Molecular properties of the Pap X 5C10 antigen have been investigated by gel permeation chromatography, SDS-PAGE, Western blotting, and isoelectric focusing. The results indicate that the antigen in both its tissue-derived and urinary form is of large (150-200 kDa) molecular size and can be separated into two molecular species with isoelectric points of pH 7.2 and 7.3 respectively. In the urine the antigens recognized by the monoclonal antibodies form large complexes with Tamm-Horsfall protein. The antigen-containing complexes can be extracted from urine by adsorption to diatomaceous earth and elution with SDS-containing buffer. Using sandwich ELISA-type assays it is possible to determine the concentration of the antigens. In preliminary experiments we were able to show that at least three of the antigens are detected in the urine following toxic insults to the kidney. The monoclonal antibodies prepared and the tests developed thus may provide direct diagnostic access to the renal papilla and allow, for the first time, early detection of papillary damage.

Accumulation of p53 protein in chemically induced oval cells during early stages of rodent hepatocarcinogenesis.

Up to now the possible involvement of p53 in rodent cancerogenesis has been based on results of the endpoint of chemically or virally induced carcinogenesis-tumors. To address the role of altered p53 expression in different stages of the multi-step process of rodent carcinogenesis in a systematic way we fed potent chemical carcinogens to male rats for 6, for 12 and for 6 weeks followed by a 6 week recovery period. Assessment of alterations of p53 expression was performed by immunoperoxidase staining with a polyclonal antiserum on frozen liver sections. Positive p53-immunostaining was localized to treatment-induced proliferating oval cells on liver sections of 21/21 2-acetylaminofluorene- (AAF) and 19/21 N-nitrosomorpholine (NNM)-treated rats irrespective of application scheme as well as to foci of hepatocytes in 1/21 NNM-treated animals and in 3/21 AAF-treated animals after 6 weeks of treatment only. The induction of oval cell proliferation by AAF was more pronounced than by NNM, and for NNM appeared to be dependent on application scheme, with a similar lower abundance of oval cells after a 6 week treatment with and without recovery as compared to a 12 week treatment. These results are discussed with respect to the role of p53 in human and rodent carcinogenesis on the one hand, and the disputed function of oval cells as facultative liver stem and tumour progenitor cells on the other.

Frequency of spontaneous tumours in Wistar rats in 2-year studies.

Tumours occurring in 1270 male and 1270 female control rats of the strain Wistar (BOR:WISW (SPF Cpb), breeder: Winkelmann, Borchen), which had been employed in 22 different 2-year studies, were compiled on the basis of histopathology reports. 1261 male and 1259 female rats were evaluated. The remaining animals were unavailable due to autolysis. A total of 1485 tumours (722 in males, 763 in females) were seen in 1165 rats (570 males, 595 females), of which 333 were classified als malignant (119 in males, 214 in females). About 70% of all primary tumours were located in the pituitary, thyroid gland, adrenal glands and uterus. Average frequencies of primary tumours: pituitary 19.8% (18.0% in males, 21.5% in females), uterus (excluding polyps) 11.8%, thyroid gland 9.0% (9.3% in males, 8.7% in females), adrenal glands 8.1% (12.7% in males, 3.5% in females), testes 7.2%, ovaries 3.5%, mammary glands 3.1% (0.1% in males, 6.1% in females) and various other organs having tumour frequencies of less than 3%. Male rats exhibited strikingly more tumours of the adrenal glands, brain, skin, parathyroid glands, endocrine and exocrine pancreas, heart, spleen and lung, while tumours of the pituitary, vascular tissue and thymus were predominant in females. A comparison of these data with those from preceding 2-year studies in Wistar rats shows a certain increase in primary tumours of the mammary glands, parathyroid glands, exocrine pancreas, endocard, brain and vascular tissue.

Frequency of spontaneous tumours in NMRI mice in 21-month studies.

Tumours occurring in 900 male and 900 female mice of the strain NMRI (BOR: NMRI (SPF Han), breeder: Winkelmann, Borchen) employed as control groups in 18 different studies lasting for 21 months on average (study No. 8 for 20 months, study No. 5 for 24 months) were compiled on the basis of histopathology reports. 883 male and 874 female mice were evaluated. The remaining animals were unavailable due to autolysis. A total of 1522 tumours (689 in males, 833 in females) were seen in 1061 mice (493 males, 568 females), 830 of which were classified as malignant (318 in males, 512 in females). About 70% of all primary tumours were located in the haemolymophoreticular tissue, lungs, ovaries, adrenals and uterus. Average incidences of primary tumours found in the following organs were: haemolymphoreticular tissue 22.9%, lungs 21.6%, ovaries 12.1%, adrenals 6.9%, uterus 6.6%, liver 4.4%, Harderian gland 4.4%, pituitary 3.5%, mamma 3.3% and various other organs with a tumour frequency below 3%. Male mice exhibited markedly more tumours of the lungs, adrenals, liver and Harderian glands, while tumours of the haemolymphoreticular tissue and pituitary were predominant in females. A comparison of these data with those from preceding two-year or life-span studies using NMRI mice from other breeders revealed a certain increase in primary tumours of the adrenals, Harderian glands as well as pituitary. This result could be due to more frequent examinations.

Frequency of spontaneous tumors in Wistar rats in 30-months studies.

Tumors occurring in 450 male and 450 female control rats of the strain Wistar (BOR:WISW (SPF Cpb), breeder: Winkelmann, Borchen) employed in 9 control groups from 7 different 30-month studies were compiled on the basis of histopathology reports. 445 male and 448 female rats were evaluated. The remaining animals were unavailable due to autolysis. A total of 1211 tumors (565 in males, 646 in females) were seen in 716 rats (333 males, 383 females), 251 of which were classified as malignant (100 in males, 151 in females). About 44% of all primary tumors were located in the pituitary and adrenals alone. Average frequencies of primary tumors: pituitary 35.4%, uterus 34.0%, adrenals 26.5%, testes 15.8%, thyroid gland 11.8%, mammary glands 11.4% and 22.5% in females respectively, skin/subcutis 6.3%, ovaries 2.9%, endocrine pancreas 2.5%, hemolymphoreticular system 2.3%, liver 1.9%, heart 1.7% and various other organs showing tumor frequencies of less than 1.5%. Male animals exhibited strikingly more tumors of the adrenals, skin, endocrine pancreas, heart and brain, while pituitary tumors were predominant in females. A comparison of these data with those from 24-month studies in Wistar rats from the same breeder shows a considerable increase above all in the number of primary tumors, the number of tumor bearers as well as the number of tumors in the pituitary, adrenals, testes, mammary glands and heart. An extension of carcinogenicity studies to 30 months probably makes it harder to recognise tumorigenic effects of test substances, at least in some organs, than in two-year studies because of the high and variable spontaneous tumor rates.