Spontaneous Primary Pleural Mesothelioma in Fischer 344 (F344) and Other Rat Strains: A Retrospective Review.

Spontaneous primary pleural mesotheliomas in Fischer 344 (F344) or other rat strains have rarely been reported. The objectives of this retrospective study were to develop historical incidence data and better characterize the light-microscopic morphology of these naturally occurring neoplasms in a large cohort of rats of several strains. A retrospective review was performed of National Toxicology Program (NTP) studies in rats conducted between 1980 and 2019 and comprising a total of 104,029 rats (51,326 males, 52,703 females), predominantly (90%) of the F344 strain. Of the 94,062 F344 rats surveyed, there were 30 cases of primary pleural mesotheliomas (22 males, 8 females). Of the 2998 Wistar Han rats surveyed, primary pleural mesotheliomas were present in 2 male rats. No primary pleural mesotheliomas were noted in male and female rats of other strains (6669 Sprague Dawley; 300 Osborne-Mendel). All primary pleural mesotheliomas in control and treated F344 and Wistar Han rats were considered spontaneous and unrelated to treatment. Based on light-microscopic evaluation of paraffin-embedded hematoxylin and eosin stained sections, only epithelioid and biphasic histologic subtypes were observed: 18 and 12 in F344 rats, respectively, and one each in Wistar Han rats. No sarcomatoid subtype cases were noted in any strain of rat.

Assessment of the biochemical pathways for acetaminophen toxicity: Implications for its carcinogenic hazard potential.

In 2019 California's Office of Environmental Health Hazard Assessment (OEHHA) initiated a review of the carcinogenic hazard potential of acetaminophen. In parallel with this review, herein we evaluated the mechanistic data related to the steps and timing of cellular events following therapeutic recommended (≤4 g/day) and higher doses of acetaminophen that may cause hepatotoxicity to evaluate whether these changes indicate that acetaminophen is a carcinogenic hazard. At therapeutic recommended doses, acetaminophen forms limited amounts of N-acetyl-p-benzoquinone-imine (NAPQI) without adverse cellular effects. Following overdoses of acetaminophen, there is potential for more extensive formation of NAPQI and depletion of glutathione, which may result in mitochondrial dysfunction and DNA damage, but only at doses that result in cell death - thus making it implausible for acetaminophen to induce the kind of stable, genetic damage in the nucleus indicative of a genotoxic or carcinogenic hazard in humans. The collective data demonstrate a lack of a plausible mechanism related to carcinogenicity and are consistent with rodent cancer bioassays, epidemiological results reviewed in companion manuscripts in this issue, as well as conclusions of multiple international health authorities.

A critical review of the acetaminophen preclinical carcinogenicity and tumor promotion data and their implications for its carcinogenic hazard potential.

In 2019 the California Office of Environmental Health Hazard Assessment (OEHHA) initiated a review of the carcinogenic hazard potential of acetaminophen, including an assessment of the long-term rodent carcinogenicity and tumor initiation/promotion studies. The objective of the analysis herein was to inform this review process with a weight-of-evidence assessment of these studies and an assessment of the relevance of these models to humans. In most of the 14 studies, there were no increases in the incidences of tumors in any organ system. In the few studies in which an increase in tumor incidence was observed, there were factors such as absence of a dose response and a rodent-specific tumor supporting that these findings are not relevant to human hazard identification. In addition, we performed qualitative analysis and quantitative simulations of the exposures to acetaminophen and its metabolites and its toxicity profile; the data support that the rodent models are toxicologically relevant to humans. The preclinical carcinogenicity results are consistent with the broader weight of evidence assessment and evaluations of multiple international health authorities supporting that acetaminophen is not a carcinogenic hazard.

Comparative Incidences and Biological Outcomes for Thymoma in Various Rat Strains in National Toxicology Program Studies.

Thymomas from 277 Fischer 344/N (F344/N), 10 Sprague Dawley (HSD:Sprague Dawley SD) (SD), 129 Wistar Han [Crl:WI(Han)] (WH), and 4 Wistar Outbred (WO) rats were reviewed from long-term studies in the National Toxicology Program (NTP) database. The incidence of thymomas in F344/N rats was slightly higher in males than in females, while the incidences in SD and WH rats were higher in females than in males. Only male WO rats were used in NTP studies. Of the 277 thymomas in F344/N rats, 235 (84.8%) were benign and 42 (15.2%) malignant, 14 of which exhibited metastasis. Of the 10 thymomas in SD rats, 5 (50%) were benign and 5 (50%) were malignant, one of which exhibited metastasis. Of the 129 thymomas in WH rats, 126 (98%) were benign and 3 (2%) were malignant, 1 with metastasis. Of the 4 thymomas in WO rats, 3 (75%) were benign and 1 (25%) was malignant, with no metastases. Malignant thymomas in F344/N and WH rats showed a propensity to be the cause of death and to result in early mortality, whereas the benign thymomas were associated less often with decreased survival. No occurrences of this neoplasm were reported to be related to exposure to any test articles.

Reevaluation of Hepatocellular Neoplasms in CD-1 Mice from a 2-year Oral Carcinogenicity Study with Permethrin.

A 24-month oral carcinogenicity study of permethrin was conducted by feeding male and female CD-1 mice diets containing concentrations of 0, 20, 500, and 2,000 ppm of permethrin (males) or 0, 20, 2,500, and 5,000 ppm of permethrin (females). After approximately two years on study, surviving mice were sacrificed for the evaluation of chronic toxicity and/or carcinogenicity. An expert panel of pathologists was convened as a Pathology Working Group (PWG) to review coded liver histology sections from male and female mice and to classify all liver neoplasms according to current nomenclature and diagnostic criteria guidelines. The PWG results indicate that permethrin induced a significant dose-dependent increase in the incidence of hepatocellular neoplasms in treated female mice ( p < .01) as well as a nonstatistically significant increase in the incidence of hepatocellular tumors in treated male mice. Given the continuum of the diagnoses of adenoma and carcinoma, and the difficulty in distinguishing some of the lesions, it is appropriate to consider only the combined incidences of hepatocellular tumors (adenoma and/or carcinoma) for biological significance and risk assessment.

Sucralose Non-Carcinogenicity: A Review of the Scientific and Regulatory Rationale.

Regulatory authorities worldwide have found the nonnutritive sweetener, sucralose, to be noncarcinogenic, based on a range of studies. A review of these and other studies found through a comprehensive search of electronic databases, using appropriate key terms, was conducted and results of that review are reported here. An overview of the types of studies relied upon by regulatory agencies to assess carcinogenicity potential is also provided as context. Physiochemical and pharmacokinetic/toxicokinetic studies confirm stability under conditions of use and reveal no metabolites of carcinogenic potential. In vitro and in vivo assays reveal no confirmed genotoxic activity. Long-term carcinogenicity studies in animal models provide no evidence of carcinogenic potential for sucralose. In studies in healthy adults, sucralose was well-tolerated and without evidence of toxicity or other changes that might suggest a potential for carcinogenic effects. In summary, sucralose does not demonstrate carcinogenic activity even when exposure levels are several orders of magnitude greater than the range of anticipated daily ingestion levels.

Utilizing Whole Slide Images for Pathology Peer Review and Working Groups.

This article describes the results of comparisons of digitally scanned whole slide images (WSIs) and glass microscope slides for diagnosis of tissues under peer review by the National Toxicology Program. Findings in this article were developed as a result of the data collected from 6 pathology working groups (PWGs), 1 pathology peer review, and survey comments from over 25 participating pathologists. For each PWG, 6-14 pathologists examined 10-143 tissues per study from 6- and 9-month perinatal studies and 2-year carcinogenicity studies. Overall it was found that evaluation of WSIs is generally equivalent to using glass slides. Concordance of PWG consensus diagnoses based upon review of WSIs versus glass slides ranged from 74% to 100% (median 86%). The intra- and interobserver diagnostic variation did not appear to influence the conclusions of any study. Based upon user opinions collected from surveys, WSIs may be less optimal than glass slides for evaluation of subtle lesions, large complex lesions, small lesions in a large section of tissue, and foci of altered hepatocytes. These results indicate that, although there may be some limitations, the use of WSIs can effectively accomplish the objectives of a conventional glass slide review and definitely serves as a useful adjunct to the conduct of PWGs.

Immunohistochemical characterization of spontaneous and acrylonitrile-induced brain tumors in the rat.

Twenty-eight spontaneously occurring glial tumors (previously diagnosed as astrocytomas, oligodendrogliomas, and gliomas) and eleven granular cell tumors (GCTs) were selected for evaluation using a panel of immunohistochemistry (IHC) stains (Ricinus communis agglutinin type 1 [RCA-1], ionized calcium-binding adapter molecule 1 [Iba-1], OX-6/major immunohistocompatibility complex class II, oligodendrocytes transcription factor 2 [Olig2], glial fibrillary acidic protein [GFAP], S100 beta, glutamine synthetase, neurofilament, proliferating cell nuclear antigen). In addition, nine brain tumors from a 2-year drinking water study for acrylonitrile were obtained from the Acrylonitrile Group, Inc. Based on IHC staining characteristics, Olig2+ oligodendrogliomas were the most commonly diagnosed spontaneous tumor in these animals. Many of the spontaneous tumors previously diagnosed as astrocytomas were RCA-1+, Iba-1+ and negative for GFAP, S100beta, and glutamine synthetase; the diagnosis of malignant microglial tumor is proposed for these neoplasms. Three mixed tumors were identified with Olig2+ (oligodendrocytes) and Iba-1+ (macrophage/microglia) cell populations. The term mixed glioma is not recommended for these tumors, as it is generally used to refer to oligoastrocytomas, which were not observed in this study. GCT were positive for RCA-1 and Iba-1. All acrylonitrile tumors were identified as malignant microglial tumors. These results may indicate that oligodendrogliomas are more common as spontaneous tumors, while acrylonitrile-induced neoplasms are microglial/histiocytic in origin. No astrocytomas (GFAP, S100 beta, and/or glutamine synthetase-positive neoplasms) were observed.

Occurrence of spontaneous amphophilic-vacuolar renal tubule tumors in Sprague-Dawley rats from subchronic toxicity studies.

The low background incidence of tumors in rodents from subchronic toxicity studies makes it difficult to assess their relevance, especially when present only in treated animals. This report investigates the occurrence of renal tubule tumors (RTTs), specifically the amphophilic-vacuolar (AV) phenotypic variant, in young Sprague-Dawley (SD) rats from a survey of laboratories conducting subchronic toxicity studies spanning a period of 10 years (2002-2012). This survey establishes a general profile of tumor occurrence; it does not estimate overall incidence or prevalence. AV tumors are spontaneous, nontreatment-related tumors of familial origin, and morphologically distinct from conventional RTTs induced by exposure to renal carcinogens. They are composed of distinct lobules of large, round to polyhedral cells with vacuolated amphophilic to eosinophilic cytoplasm and prominent nucleoli. Data from five collaborating laboratories, representing 37 qualifying studies, are presented. In total, 58 renal tubule neoplasms were recorded in this data set. The AV tumor variant was reported more commonly than the conventional RTT (n = 45 and 13, respectively), and it was recorded in both experimental (n = 32) and control (n = 13) groups. AV tumors occurred more often in females (n = 34) than in males (n = 11); conventional RTTs were recorded more often in males (n = 9) than in females (n = 4). AV tumors often occurred in more than one rat within the same study (up to 7) and were documented to occur in rats as young as 7 to 10 weeks of age. Results from this survey indicate that AV tumors are being reported more commonly in recent years; the majority (n = 33) were reported in studies commencing since 2009. In conclusion, this study reaffirms that AV tumors are spontaneous, nontreatment-related lesions, and suggests that they may be more common than conventional RTTs in young SD rats. The authors propose that AV tumors be recorded separately from conventional RTTs in order to clearly distinguish these two renal tubule neoplasms from one another and allow for appropriate interpretation of a compound's potential carcinogenic effect in the kidney.

Spontaneous renal tumors in two rats from a thirteen week rodent feeding study with grain from molecular stacked trait lepidopteran and coleopteran resistant (DP-ØØ4114-3) maize.

A thirteen week feeding study was conducted by feeding young adult male and female Sprague Dawley [Crl:CD®(SD)] rats diets containing grain from genetically modified (GM) DP-ØØ4114-3 maize that was either untreated (4114) or treated in the field with glufosinate ammonium (4114GLU). Control rats were fed diets containing the same concentration of near isogenic, non-GM maize grain (091) or one of three types of commercially available non-GM maize grain. At the end of the in-life phase, renal tubule tumors were reported in two male rats consuming diets containing 4114 maize grain. An expert panel of pathologists was convened as a Pathology Working Group (PWG) to review coded kidney histology sections from control (091) and treated (4114 and 4114GLU) male rats. The objectives were for the panel to characterize the histopathologic findings and to interpret their relationship to consumption of the indicated diet. The PWG concluded unanimously that the kidney tumors were characteristic of amphophilic-vacuolar (AV) tumors and AV atypical tubular hyperplasia which represent a distinctive phenotype that has been reported to occur sporadically in young Sprague Dawley Rats. The PWG determined that the neoplasms and atypical tubular hyperplasias were multicentric and bilateral which typifies tumors of familial origin. Degenerative/regenerative or cytotoxic changes consistent with nephrotoxicity leading to tumor induction were not observed in these rats and thus supports the conclusion that tumors were unrelated to consumption of the test diet. It was the unanimous opinion of the PWG that the proliferative renal tubule cell lesions were spontaneous and not related to consumption of diets containing 4114 maize grain.

Differences in rat models used in routine toxicity studies.

The discussion on whether the Sprague Dawley (SD), the Fischer F344, or the Hannover Wistar rat is the most appropriate model for toxicity studies in rodents is ongoing. A substantial quantity of data on these strains concerning their source, diet, and housing conditions have been published. Generally, before starting a toxicology program in rodents, it should be taken into account that oncogenicity studies will be required for the majority of compounds successfully completing development. Survival, body weight development, incidence, type, time of onset of age-dependent lesions and neoplasms, as well as some special considerations of the rat model selected may be decisive. Therefore, an understanding of the historical background data is essential. These aspects demonstrate why the use of a specific rat model should be carefully considered at the beginning of the toxicology program.

Classification of neural tumors in laboratory rodents, emphasizing the rat.

Neoplasms of the nervous system, whether spontaneous or induced, are infrequent in laboratory rodents and very rare in other laboratory animal species. The morphology of neural tumors depends on the intrinsic functions and properties of the cell type, the interactions between the neoplasm and surrounding normal tissue, and regressive changes. The incidence of neural neoplasms varies with sex, location, and age of tumor onset. Although the onset of spontaneous tumor development cannot be established in routine oncogenicity studies, calculations using the time of diagnosis (day of death) have revealed significant differences in tumor biology among different rat strains. In the central nervous system, granular cell tumors (a meningioma variant), followed by glial tumors, are the most common neoplasms in rats, whereas glial cell tumors are observed most frequently in mice. Central nervous system tumors usually affect the brain rather than the spinal cord. Other than adrenal gland pheochromocytomas, the most common neoplasms of the peripheral nervous system are schwannomas. Neural tumors may develop in the central nervous system and peripheral nervous system from other cell lineages (including extraneural elements like adipose tissue and lymphocytes), but such lesions are very rare in laboratory animals.

Pathology Working Group review and evaluation of proliferative lesions of mammary gland tissues in female rats fed ammonium perfluorooctanoate (APFO) in the diet for 2 years.

Perfluorooctanoate (PFO) is a perfluorinated carboxylate that is widely distributed in the environment. A 2-year chronic study was conducted in rats fed either 30 or 300 ppm of ammonium perfluorooctanoate (APFO). To investigate the possible relationship of APFO exposure to proliferative mammary lesions, a Pathology Working Group (PWG) review of the original slides was performed. The consensus reached by the PWG was that the incidence of mammary-gland neoplasms was not affected by chronic dietary administration of APFO. Therefore, feeding female rats up to 300 ppm of APFO resulted in no increase in proliferative lesions of the mammary tissue.

Thyroid Dysplasia in Wistar Hannover GALAS Rats.

Thyroid dysplasia was recognized in WistarHan GALAS rats and confirmed as a heritable congenital disorder. The gene or genes involved were not identified, but homozygous animals with thyroid dysplasia also exhibited stunted growth, had reduced pituitary gland growth hormone (GH) and were hypothyroid. Heterozygous animals exhibited thyroid dysplasia with normal thyroid hormonal homeostasis and no difference in the incidence of preneoplastic or neoplastic lesions in oncogenicity studies.

Histopathology of the urinary bladders of cynomolgus monkeys treated with PPAR agonists.

Peroxisome proliferator-activated receptors (PPAR) are involved in the pathogenesis of insulin resistance, diabetes, hyperlipidemias, and related complications. Consequently, a mechanistic understanding of PPAR subtypes and their activation provides promising therapeutic targets for the management of type 2 diabetes mellitus and the metabolic syndrome. Available data from rodent carcinogenicity studies, however, demonstrate that PPAR agonists can be tumorigenic in one or more species of rodents at multiple sites. Sufficient data are not yet available to explain the mode(s) of action for most of these tumor types. There has been information presented by FDA that indicates there are urothelial changes in the monkey (and possibly the dog) in addition to the rat. Outstanding questions exist regarding potency, species differences, safety margins, and other issues. In 2005, the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute (HESI) PPAR Agonist Project Committee was established to advance research on the modes of action and potential human relevance of emerging rodent tumor data. Additionally, the HESI PPAR Agonist Project Committee authorized a Pathology Working Group (PWG) to examine the urinary bladder from cynomolgus monkeys. The focus of this PWG was to establish consistent diagnostic criteria for urothelial changes and to assess the potential relationship of these changes to treatment. Specific diagnostic criteria and nomenclature were recommended for the diagnosis of urothelial granules, vacuolation, hypertrophy, and hyperplasia in studies conducted with PPARgamma and dual alpha/gamma agonists in cynomolgus monkeys, which will assist investigators performing toxicity studies to provide data in a consistent manner between studies and laboratories. In this review of selected tissues, treatment with PPAR agonists was not associated with urothelial hypertrophy or hyperplasia, but there was an increased incidence in the size and frequency of vacuoles within the superficial urothelial and adjacent intermediate cell layers.

Histopathology of hemangiosarcomas in mice and hamsters and liposarcomas/fibrosarcomas in rats associated with PPAR agonists.

Peroxisome proliferator-activated receptors (PPAR) are involved in the pathogenesis of insulin resistance, diabetes, and related complications. Consequently, the identification of PPAR subtypes and the potential for their activation provides promising therapeutic targets for the management of type 2 diabetes mellitus. Available data from rodent carcinogenicity studies, however, demonstrate that PPAR agonists can be tumorigenic in one or more species of rodents at multiple sites. In 2005, the Health and Environmental Sciences Institute (HESI) PPAR Agonist Project Committee was established by a group of pharmaceutical companies to advance research on and to understand the modes of action and human relevance of this emerging rodent tumor data for PPAR agonists. Since the most commonly observed tumor types reported in rodents are hemangiosarcomas, fibrosarcomas and liposarcomas, the PPAR Agonist Project Committee approved a Pathology Working Group (PWG) to develop consensus of morphologic criteria for tumor diagnoses and consistency of diagnoses across multiple studies for hemangiosarcomas in mice and hamsters and liposarcomas/fibrosarcomas in rats. Therefore, the focus of the PWG review was to establish consistent tumor diagnostic criteria, to assess evidence of potentially preneoplastic changes and to identify distinguishing morphologic differences which may exist between spontaneous changes present in control animals with similar changes from treated animals. Specific diagnostic criteria and nomenclature are recommended for the classification of proliferative vascular lesions which may be present in mice or hamsters and for proliferative mesenchymal changes in rats in studies that are conducted with PPAR agonists.

Transplacental carcinogenicity of 3'-azido-3'-deoxythymidine in B6C3F1 mice and F344 rats.

The prophylactic use of zidovudine (3'-azido-3'-deoxythymidine, AZT) during pregnancy greatly reduces transmission of HIV-1 from infected mothers to their infants; however, the affinity of host cell DNA polymerases for AZT also allows for its incorporation into host cell DNA, predisposing to cancer development. To expand upon previous transplacental carcinogenesis assays performed in CD-1 mice, the transplacental carcinogenicity of AZT was evaluated in a second mouse strain and a second rodent species. Date-mated female mice and rats were gavaged daily with 0, 80, 240, or 480 mg AZT/kg bw during the last 7 days of gestation. At 2 years postpartum, male and female B6C3F1 mouse and F344 rat offspring (n = 44-46 of each sex and species/treatment group) were necropsied for gross and microscopic tissue examinations. Under the conditions of these two-year studies, there was clear evidence of carcinogenic activity based upon significant dose-related trends and increases in the incidences of hemangiosarcoma in male mice and mononuclear cell leukemia in female rats. There was some evidence of carcinogenic activity in the livers of male mice based upon a positive trend and an increased incidence of hepatic carcinoma in the high-dose AZT group. The incidence of gliomas in female rats exceeded the historical background rates for gliomas in F344 rats. P53 overexpression was detected in some AZT-treated mouse neoplasms. These and other cancer-related findings confirm and extend those of previous transplacental carcinogenicity studies of AZT in mice, support the need for long-term follow-up of nucleoside reverse transcriptase inhibitor (NRTI)-exposed children, and indicate the necessity for effective protective strategies against NRTI-induced side effects.

Defining a noncarcinogenic dose of recombinant human parathyroid hormone 1-84 in a 2-year study in Fischer 344 rats.

The carcinogenic potential of human parathyroid hormone 1-84 (PTH) was assessed by daily subcutaneous injection (0, 10, 50, 150 microg/kg/day) for 2 years in Fischer 344 rats. Histopathological analyses were conducted on the standard set of soft tissues, tissues with macroscopic abnormalities, selected bones, and bones with abnormalities identified radiographically. All PTH doses caused widespread osteosclerosis and significant, dose-dependent increases in femoral and vertebral bone mineral content and density. In the mid-and high-dose groups, proliferative changes in bone increased with dose. Osteosarcoma was the most common change, followed by focal osteoblast hyperplasia, osteoblastoma, osteoma and skeletal fibrosarcoma. The incidence of bone neoplasms was comparable in control and low-dose groups providing a noncarcinogenic dose for PTH of 10 microg/kg/day at a systemic exposure to PTH that is 4.6-fold higher than for a 100 microg dose in humans. The ability of PTH to interact with and balance the effects of both the PTH-1 receptor and the putative C-terminal PTH receptor, may lead to the lower carcinogenic potential observed with PTH than reported previously for teriparatide.

Propylene glycol monomethyl ether (PGME): inhalation toxicity and carcinogenicity in Fischer 344 rats and B6C3F1 mice.

A series of inhalation studies with propylene glycol monomethyl ether (PGME) vapor were undertaken to characterize its subchronic toxicity in mice and chronic toxicity/oncogenicity in rats and mice. Groups of male and female Fischer 344 rats and B6C3F1 mice were exposed to 0, 300, 1,000, or 3,000 ppm vapor from 1 week to 2 years. Primary treatment-related effects included: initial sedation of animals exposed to 3,000 ppm and its subsequent resolution correlating with induction of hepatic mixed function oxidase activity and S-phase DNA synthesis; elevated mortality in high-exposure male rats and mice (chronic study); elevated deposition of alpha2u-globulin (alpha2U-G) and associated nephropathy and S-phase DNA synthesis in male rat kidneys; accelerated atrophy of the adrenal gland X-zone in female mice (subchronic study only); and increased occurrence and/or severity of eosinophilic foci of altered hepatocytes in male rats. No toxicologically relevant statistically significant increases in neoplasia occurred in either species. A numerical increase in the incidence of kidney adenomas occurred in intermediate-exposure male rats; however, the association with alpha2U-G nephropathy, a male rat specific effect, indicated a lack of relevance for human risk assessment.

Comparative prevalence, multiplicity, and progression of spontaneous and vinyl carbamate-induced liver lesions in five strains of male mice.

The overall and age-specific prevalences and multiplicities of spontaneous and chemically induced hepatocellular neoplasia were compared among male B6D2F1, B6C3F1, C3H (C3H/HeNCr1 MTV-), B6CF1, and C57BL/6 (C57BL/6NCr1) mice following a single intraperitoneal injection of 0.03 microM vinyl carbamate (VC)/g body weight or vehicle alone at 15 days of age. Additional groups of B6C3F1, C3H, and C57BL/6 males received 0.15 microM VC/g body weight at 15 days of age. For male B6D2F1, B6C3F1, C3H, B6CF1, and C57BL/6 mice, the estimated overall prevalences (and multiplicities) of hepatocellular adenomas or carcinomas in vehicle controls were 14.1% (0.19), 12.3% (0.15), 8.2% (0.10), 7.2% (0.09), and 2.4% (0.02), respectively. The analogous estimates in the low-dose group were 59.2% (1.19), 72.9% (4.07), 48.6% (1.99), 22.8% (0.29), and 43.9% (0.82). Analogous estimates for B6C3F1, C3H, and C57BL/6 mice in the high-dose group were 45.3% (4.29), 59.7% (6.63), and 46.8% (1.74), respectively. Age-specific multiplicity estimates suggested a progression from altered hepatocellular foci (AHF) to hepatocellular neoplasms. Further evidence of progression was provided by the temporal occurrence of hepatocellular adenomas before carcinomas, and the apparent origination of carcinomas within adenomas. Pulmonary metastases were observed in many of the mice with hepatocellular carcinomas. These findings confirm previous observations of strain differences in liver neoplasm response, suggest a progressive development from AHF to adenomas, and ultimately to carcinomas, and show sensitivity to VC-induced hepatocarcinogenesis in all 5 strains.