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Animal bioassays for carcinogenicity are essential components of occupational health studies. Animal data that have been collected under controlled experimental conditions provide definitive information about the carcinogenic activities of individual substances or defined mixtures and their relative potencies in the test species. Such information serves as a frame of reference for clinical and epidemiologic studies, pointing to potential adverse health effects and to the types of substances that might produce them. This article alerts the occupational and environmental health communities to 20 substances that produced breast tumors, 13 substances that produced uterine tumors, and 8 substances that produced ovarian tumors in long-term National Toxicology Program animal studies. Each of the substances also produced neoplasms at other body sites. Follow-up studies of molecular measures of exposure and response in people and in animals will reduce the uncertainties of transspecies extrapolations.

Scurfy (sf), is an X-linked recessive lethal mutation that occurs spontaneously in the C3H mouse. The disease is characterized by lymphoid and hematopoietic dysfunction. Affected males are of small stature and exhibit scaliness and crusting of the eyelids, ears, tail, and feet, marked splenomegaly, moderate hepatomegaly, enlarged lymph nodes, and atrophy of the thymus. The average lifespan of the affected hemizygous males (sf/y) is 24 +/- 0.7 days. Total cellular proteins were extracted from pooled samples of thymus and spleen obtained from combined litters of mice. Tissue-specific protein profiles characteristic of either sf mutant or normal mice were analyzed by two dimensional polyacrylamide gel electrophoresis (2DPAGE) at different stages of the phenotypic expression of the sf mutation, to identify changes in protein patterns that might be associated with the progression of the disease. The resultant gels were silver stained, digitized, and analyzed, by image analysis utilizing a pipelined image processor connected to a host computer. At 14 +/- 1 days of age, protein patterns from sf mutant and normal mice control organs showed considerable homogeneity, although there were proteins identified unique to the sf mutant and to the normal controls. At 20 +/- 1 days of age, the pattern differences between the sf mutant and normal control increased markedly. Differences were expressed as the percent of proteins that were unique to either the sf mutant or the normal control from the total number of each type. The percent of proteins that increased or decreased in the three organs utilized in this study ranged between 21%-39% at 14 days and were between 25%-54% at 20 days. Differences in protein expression between the normal and sf mutant as the disorder progressed for each of the three tissues examined. In addition, thymus protein profiles from 9 day old littermates that were phenotypically normal but genotypically unknown were evaluated to determine if marker proteins could be identified for the sf mutation. Limited protein changes were noted at relative molecular weights of 66, 60, 54, 39, 37, 33, 25, 23, 27, and 11 kDa. These data suggest that the sf mutation follows a trackable pattern of protein expression and repression different than the normal control C3H mouse. Several potential marker proteins associated with the sf mutation were identified in 9 day thymus prior to the phenotypic expression of the disease. These putative biomarkers may be useful for characterizing the sf mutation and the mutant may act a possible model the Wiskott-Aldrich syndrome (WAS).

Long-term rodent bioassays with chemicals administered at maximum tolerated doses identify noncarcinogens as well as carcinogens. Thirty-one chemicals recently evaluated for carcinogenic potential by the National Toxicology Program provide unique data on the relationships between mutagenicity, toxicity, and carcinogenicity. Twenty-two substances were classified as carcinogens, and nine showed no evidence of carcinogenicity. Although cellular proliferation does play an intrinsic role in neoplastic processes, the responses associated with chronic toxicity in these studies were not always sufficient to induce neoplasia. Regardless of their mutagenic potential, 19 carcinogens induced toxic effects at sites that did not show neoplastic changes; similar toxic lesions were also seen among the mutagenic and nonmutagenic noncarcinogens. Although many nonmutagens induced neoplasia at sites that showed toxic effects, some of the same chemicals also exhibited toxicity at other sites that showed no neoplastic effect. These results suggest that for some chemicals, properties other than mutagenicity or toxicity may be responsible for their carcinogenic potential.

A method was developed for continuously exposing tracheal epithelium to measured amounts of carcinogen. Beeswax was the vehicle for sustained release of carcinogen, and tracheas transplanted to s.c. sites were target tissues. In the experiment reported here, transplanted rat tracheas were exposed to a potent carcinogen, 7,12-di-methyl benz(a)anthracene (DMBA). The rate of release of DMBA from the beeswax carrier within the tracheal lumen approached first order when the initial concentration of carcinogen was high (3200 to 325 microng in a 24.45-mg pellet). With lower concentrations, where the carcinogen was dissolved in the beeswax, initial release was rapid, and most of the carcinogen was delivered within 4 weeks. At high DMBA dose levels, the entire tracheal epithelium was uniformly replaced by keratinizing squamous metaplasia after 1 week of exposure, and after 2 months, when from 280 to 910 microng DMBA had been delivered, all transplants had developed invasive squamous carcinomas. Sarcomas also developed in 19% of the transplants. At lower dose levels the epithelial reactions were more varied, and tumor development was more protracted. The lowest DMBA dose presently known to diduce carcinomas in this experimental model is 40 microng, which is in the dose range used for tumor initiation in skin carcinogenesis studies in mice.

Heterotopic tracheal transplants of rats were exposed in vivo to 150 or 640 microng of 7, 12-dimethylbenz(a)anthracene (DMBA) delivered over 2 weeks, and the epithelium was studied in vitro in an attempt to identify changes in growth behavior during early phases of neoplastic development. Explants were made from the exposed tracheas, as well as from a series of controls, and the rate of epithelial outgrowth from the explants was measured. Secondly, the capacity of the outgrowths to survive as primary cultures and their ability to survive multiple in vitro passages were studied. During the initial planting, the rate of outgrowth was by far the greatest from the explants preexposed to 150 microng DMBA. Outgrowth from explants preexposed to 640 microng DMBA was sparse during the first planting but increased markedly during repeated planting when insulin- and hydrocortisone-supplemented medium was used. Establishment of outgrowth during repeated planting of carcinogen-exposed tracheal pieces was clearly hormone dependent. Control explants did not exhibit this hormone dependency. Primary cultures could be established from only three of six explants preexposed to 150 microng DMBA. These explants had been initiated in insulin- and hydrocortisone-supplemented medium. The primary cultures were successfully subcultured. Primary cultures were also established from five of five explants preexposed to 640 microng DMBA and cultured in hormone-supplemented medium. At least one cell line was obtained from each of the explants. To establish and maintain primary cultures from control tracheas required an enriched medium containing amino acid supplements, sodium pyruvate, and putrescine, as well as the hormone supplements. However, such cultures could not be subcultured. The primary cultures from the carcinogen-exposed explants and the subsequently developed cell lines all exhibited morphological characteristics of keratinizing squamous epithelium. These characteristics include: epithelioid cell morphology, multilayering and sloughing of orangeophilic squamous cells, and the presence of keratohyalin granules. These experiments demonstrate a markedly increased in vitro growth capacity of tracheal epithelium after a short in vivo exposure to carcinogen.

The effects of hyperoxia on lung tumor development were examined in mice and rats. In mice, exposure to 70% O2 prevented the development of urethan- or 3-methylcholanthrene-induced lung tumors. Dietary antioxidants [butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA)] were unable to prevent the inhibition of tumor development by oxygen, although BHT retained its capability to enhance tumor development in mouse lung. In visible-size tumors, oxygen did not depress DNA synthesis. Oxygen also reduced the number of pulmonary metastatic nodules after i.v. injection of mammary gland-derived carcinoma cells, but failed to inhibit growth of murine lung carcinoma or murine melanoma-derived cell lines. Rats treated with one single intratracheal instillation of 3-methylcholanthrene developed multiple lung lesions; their growth could be prevented by exposure of the animals to 40 or 70% O2. It is concluded that hyperoxia prevents development of transformed cells in vivo in the lung and may affect adversely the growth of selected cell lines metastatic to the lung.

N-Nitrosoheptamethyleneimine (NHMI) was given by gastric intubation, at a dosage of 10 mg/kg body weight twice weekly for 5-20 weeks, to F344 rats bearing subcutaneous tracheal transplants. Groups of 5 rats were killed at 5, 10, 15, and 20 weeks, and 1 group was killed at week 33 of the experiment (13 weeks after the last NHMI dose). Sequential changes in host tracheas consisted of hyperplasia with complete loss of mucociliary epithelium, squamous metaplasia, intense mononuclear infiltration, reestablishment of mucocillary epithelium (during the course of NHMI administration) except for focal areas of hyperkeratotic squamous metaplasia or marked dystrophic changes, and finally, papillomas, polyps, and invasive squamous cell carcinomas. Lesions in tracheal transplants consisted mostly of atrophic and dystrophic changes, with only a few small foci of squamous metaplasia and no neoplastic changes.

This article reviews the available data on the carcinogenicity of formaldehyde from experimental and epidemiologic studies and makes recommendations for further research. Two definitive chronic inhalation bioassays on rodents have demonstrated that formaldehyde produces nasal cancer in rats and mice at 14 ppm and in rats at 6 ppm, which is within the domain of present permissible human exposure (8-hr time-weighted average of 3 ppm, a 5 ppm ceiling, and a 10 ppm short-term exposure limit). Biochemical and physiologic studies in rats have shown that inhaled formaldehyde can depress respiration, inhibit mucociliary clearance, stimulate cell proliferation, and crosslink DNA and protein in the nasal mucosa. No deaths from nasal cancer have been reported in epidemiologic studies of cohorts exposed to formaldehyde, but three case-control studies suggest the possibility of increased risk. Although excesses of lung cancer deaths have been observed in some studies at industrial plants with formaldehyde exposure, uncertainties in interpretation limit the evaluation of these findings. Excess cancers of the brain and of lymphatic and hematopoietic tissues have been reported in certain studies of industrial groups and in most studies of formaldehyde-exposed professionals, but whether these excesses are related to formaldehyde exposure is not known. Several properties of formaldehyde pose unique problems for future research: the mechanisms responsible for its nonlinear response; its probable mechanism of carcinogenic action as a cross-linking agent; its formation in tissues as a normal metabolite; its possible action as a promoter and/or a cocarcinogen; and the importance of glutathione as a host defense at low exposure.

Heterotopically transplanted rat tracheas were continuously exposed to measured amounts of benzo(a)pyrene over a period of 1 to 6 months. The cumulative doses ranged from 10 to 2490 microng. The morphological response of the tracheal epithelium was characterized by hyperplasis during the first 2 weeks, followed by atrophy. Squamous metaplasias did not appear until after 4 months of exposure; at 4 and 6 months numerous dysplastic lesions and noninvasive carcinomas resembling those seen in the airways of humans were found in the higher carcinogen dose groups. The first invasive carcinomas developed at 4 months in the groups given 1250 microng or more benzo(a)pyrene. The lowest dose tested that produced a carcinoma within the observation period of 22 months was 300 microng benzo(a)pyrene. The majority of the neoplasms were squamous cell carcinomas, although several adenocarcinomas and sarcomas also developed. Since a variety of metaplastic and dyplastic lesions can be induced by carcinogenic polycyclic hydrocarbons in the transplanted rat tracheas, this experimental model appears to be well suited for the study of the sequential epithelial changes that lead to respiratory tract neoplasia.

During a 4-year period 43 cases of spontaneous lymphoma occurred in macaques at the California Primate Research Center. In an attempt to determine if there might be a common viral etiology to the outbreak, lymphoma tissues from 10 rhesus monkeys were explanted into tissue culture and examined for the presence of virus. Electron microscopic studies of the cultured lymphoma cells revealed viruses morphologically characteristics of adenovirus, reovirus, foamy virus, and herpesvirus. Cell-free filtrates from tissue cultures possessing the latter three viruses produced cytopathology in virus-free indicator cells. Virus particles typical of the explant culture were demonstrable in the infected indicator cells. Type-C RNA tumor virus particles were not observed electron microscopically in any of the lymphoma tissues or lymphoma cell cultures examined.

Sequential morphological changes occurring after brief carcinogen exposures of heterotopic tracheal transplants in rats were semiquantitatively studied. Tracheas were exposed to 7,12-dimethylbenz(a)anthracene for 1, 2, or 4 weeks, during which time means of 138, 152, and 160 microgram 7,12-dimethylbenz(a)anthracene, respectively, were delivered. The first two types of exposures resulted only in generalized epithelial changes; these included hyperplasia and early metaplasia, both of which regressed rapidly, and persistent atrophic alterations. No focal epithelial lesions or tumors developed. The third type of exposure (160 microgram 7,12-dimethylbenz(a)anthracene delivered in 4 weeks) resulted in the appearance of generalized mucosal changes with long-lasting, severe inhibition of mucus production. In addition, focal metaplastic lesions reappeared at 4 to 8 months after exposure, and invasive carcinomas developed after 1 year with an incidence of 9%. Overall carcinoma incidence, including carcinoma in situ, was 15%. The studies emphasize the importance of the duration of carcinogen exposure, and they demonstrate the emergence of focal lesions when effective carcinogenic exposures are being used. The possible significance of epithelial atrophy in the pathogenesis of cancer in this experimental model is discussed.

A classification scheme is proposed for degrees of experimental evidence for the carcinogenicity of chemicals for animals. The classification stems from the suggestions of an IARC Working Group that the evaluation of bioassays include consideration of whether an increase in malignant tumours occurred and whether it occurred to an unusual degree or in multiple experiments. We extended the evaluative process to chemical experiments with no evidence of carcinogenicity and gave increased emphasis to results from more than one animal species. Although the proposed classification was developed for a group of NCI bioassays which were similar in design and conduct, it may provide a general framework for the evaluation of carcinogenesis bioassays and stimulate development and application of evaluative methods.

Hyperplastic-metaplastic lesions were produced in transplanted rat tracheas by exposure to 310 or 75 mug 7,12-dimethylbenz(a)anthracene or to 650 mug benzo(a)pyrene delivered over a 2-week exposure period. Tissue recovery was studied for up to 16 weeks. After cessation of carcinogen exposure, most of the hyperplastic-metaplastic epithelium was rapidly replaced by mucociliary epithelium. In the 7,12-dimethylbenz(a)anthracene-exposed tracheas, a few mataplastic foci remained at 4 and 8 weeks. In all 3 exposure groups, portions of the mucosa were occupied by atrophic-pleomorphic epithelium that persisted throughout the observation period. The significance of these lesions in the evolution of neoplasia is as yet unknown. In vitro studies with epithelial cells derived from 7,12-dimethylbenz(a)anthracene-exposed tracheal transplants revealed that some cells have acquired a growth potential that is markedly different from that of normal mucociliary epithelium. This altered in vitro growth potential of epithelial cells may be an early indicator of an important event in the evolution of epithelial transformation in vivo.

A large scale national carcinogenesis testing program requires quality assurance procedures for each scientific discipline, including pathology. The NCl Carcinogenesis Testing Program's pathology procedures involve the necropsy examination, histology, histopathologic diagnosis, data recording, and report writing. Each phase of the program requires specific quality assurance procedures, some of which are clerical, others scientific. In the latter case, a consensus from a group of pathologists serves to resolve differences of opinion. Only with the implementation of quality assurance procedures will the confidence in these tests be assured and quality maintained. Subsequently, modifications to established protocols may be discussed and accurately evaluated.

Intravenous inoculation of pregnant cats with feline herpesvirus produced minimal illness but resulted in abortion, intrauterine fetal death and congenital fetal infection. Placental lesions included multiple infarcts in the placental labyrinth, thrombosis of maternal vessels in the endometrium and placenta, and multifocal necrosis of the giant-cell trophoblast and endometrial epithelium in the junctional zone of the placenta associated with eosinophilic intranuclear inclusion bodies. The virus was isolated from all the placentas and uteri but from none of the fetuses aborted 6-9 days after maternal intravenous inoculation. Viral antigen was demonstrated in the uterine vessels and in the junctional zone of the placenta at this time. On postinoculation day 26, viral antigen was demonstrated in the chorioallantoic membrane on the fetal side of the placenta and in the liver of a congenitally infected fetus. Although all 4 pregnant cats inoculated intranasally with feline herpesvirus aborted, neither virus, viral antigen nor significant lesions were detected in the uteri, placentas or fetuses. Abortion after intranasal inoculation was interpreted as a nonspecific reaction secondary to the severe, debilitating upper respiratory disease that occurred.

The procoagulant activity of rabbit peritoneal leukocytes significantly increased when the leukocytes were incubated in suspension cultures at 37 C for 24 hours. Intravenous infusions of Iysates of 232 x 10(6) rabbit leukocytes which had been incubated in cultures at 37 C for 24 hours produced disseminated intravascular coagulation and vasculitis involving the pulmonary arteries in normal rabbits. Intraaortic infusions of lysates of 230 x 10(6) similarly incubated leukocytes produced renal thrombosis and renal cortical necrosis in normal rabbits. These observations suggest that the procoagulant of granulocytic leukocytes could play a role in the generalized Shwartzman reaction and other syndromes of disseminated intravascular coagulation.

Dynamic aerosols of adenovirus 12 were generated in the same Henderson apparatus under conditions of high, medium, and low relative humidity. High relative humidities resulted in more recovery of adenovirus 12 from aerosols and lungs of newborn Syrian hamsters. At 89, 51, and 32% relative humidity, the total infectious virus recovered from a 20-min aerosol was 10(6.7), 10(6.0), and 10(4.3) TCD(50), respectively. Hamsters exposed to these 20-min aerosols retained measured lung doses of 10(3.0), 10(2.4), and 10(1.0) TCD(50), respectively. The measured retained lung doses were compared to calculated inhaled lung doses based on both total virus aerosolized and total virus recovery from the aerosols.