We conducted a two-year inhalation study of butyraldehyde using F344/DuCrlCrlj rats. The rats were exposed to 0, 300, 1,000 and 3,000 ppm (v/v) for 6 hr/day, 5 days/ week for 104 weeks using whole-body inhalation chambers. The incidence of squamous cell carcinoma of the nasal cavity was increased in the 3,000 ppm groups of both male and female rats, with Fisher\'s exact test and the Peto test indicating that the incidence was significant. In addition to squamous cell carcinoma in the nasal cavity, in the 3,000 ppm groups one male had an adenosquamous carcinoma, one male had a carcinosarcoma, one male had a sarcoma NOS (Not Otherwise Specified), and one female had a squamous cell papilloma in the nasal cavity. The combined incidence of squamous cell carcinoma, adenosquamous carcinoma and carcinosarcoma was significantly increased in male rats and the combined incidence of squamous cell papilloma and carcinoma was significantly increased in female. Based on these results, we conclude that there is clear evidence of butyraldehyde carcinogenicity in male and female rats.

Nonclinical safety studies are typically conducted to establish a toxicity profile of a new pharmaceutical in clinical development. Such a profile may encompass multiple differing types of animal studies, or not! Some types of animal studies may not be warranted for a specific program or may only require a limited evaluation if scientifically justified. The goal of this course was to provide a practical perspective on regulatory writing of a dossier(s) using the weight of evidence (WOE) approach for carcinogenicity, drug abuse liability and pediatric safety assessments. These assessments are typically done after some clinical data are available and are highly bespoke to the pharmaceutical being developed. This manuscript will discuss key data elements to consider and strategy options with some case studies and examples. Additionally, US FDA experience with dossier(s) including WOE arguments is discussed.

Plasticizers are necessary for the usability of various products, including food contact materials. Exposure to plasticizers is most commonly made through the oral route. Several plasticizers have been reported to have adverse effects on humans and the environment. Thus, the present study aimed to determine the long-term toxicity and carcinogenicity of a novel plasticizer called bis(2-ethylhexyl) cyclohexane-1,4-dicarboxylate (Eco-DEHCH), which is an ecofriendly and biologically less harmful replacer. Groups of 50 male and 50 female Han Wistar rats were fed Eco-DEHCH at daily doses of 1,600, 5,000, or 16,000 ppm in their diet for at least 104 weeks. The rats were regularly monitored for mortality, clinical signs, body weight, food consumption, food efficiency, and perceivable mass. All animals were subjected to complete necropsy and histopathological examination. The results indicate that the rats well tolerated chronic exposure to Eco-DEHCH at highest daily doses of 16,000 ppm, with was equivalent to 805.1 mg/kg/day in males and 1060.6 mg/kg/day in females and did not show signs of toxicity or carcinogenicity. In conclusion, Eco-DEHCH could be a safe and promising alternative plasticizer.

Introduction: The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) initiated a process in 2012 to revise the S1B Guideline \"Testing for Carcinogenicity of Pharmaceuticals\". Previous retrospective analysis indicated the importance of histopathological risk factors in chronic toxicity studies, evidence of endocrine perturbation, and positive genetic toxicology results as potentially predictive indicators of carcinogenic risk. In addition, a relationship between pharmacodynamic activity and carcinogenicity outcome in long-term rodent studies has been reported. It was postulated that these factors could be evaluated in a Weight-of-Evidence (WoE) approach to predict the outcome of a 2-year rat study. Methods: The ICH S1B(R1) Expert Working Group (EWG) conducted a Prospective Evaluation Study (PES) to determine the regulatory feasibility of this WoE approach. Drug Regulatory Authorities (DRAs) evaluated 49 Carcinogenicity Assessment Documents (CADs), which describe the WoE for submitted pharmaceutical compounds. Each compound was categorized into a carcinogenic risk category including a statement of the value of the 2-year rat study. The outcome of the completed 2-year rat studies was evaluated in relation to the prospective CAD to determine the accuracy of predictions. Results: Based on the results of the PES, the EWG concluded that the evaluation process for assessing human carcinogenic risk of pharmaceuticals described in ICH S1B could be expanded to include a WoE approach. Approximately 27% of 2-year rat studies could be avoided in cases where DRAs and sponsors unanimously agreed that such a study would not add value. Discussion: Key factors supporting a WoE assessment were identified: data that inform carcinogenic potential based on drug target biology and the primary pharmacologic mechanism of the parent compound and major human metabolites; results from secondary pharmacology screens for this compound and major human metabolites that inform carcinogenic risk; histopathology data from repeated-dose toxicity studies; evidence for hormonal perturbation; genotoxicity data; and evidence of immune modulation. The outcome of the PES indicates that a WoE approach can be used in place of conducting a 2-year rat study for some pharmaceuticals. These data were used by the ICH S1B(R1) EWG to write the R1 Addendum to the S1B Guideline published in August 2022.

Environmental and occupational exposure to polycyclic aromatic hydrocarbons (PAHs) is associated with adverse health effects in humans. Uncertainty exists regarding the causation of urinary bladder cancer by benzo[a]pyrene (B[a]P) due to a lack of sufficient data. In this work, we focused on in-vitro DNA damage and the formation of micronuclei and chromosomal aberrations as predictors of cancer risk, applying a wide range of dosages and time periods to quantify the onset, intensity, and duration of the response. We chose two urothelial cell types to compare susceptibility and the ability to increase the malignity of a pre-existing bladder cancer: a cancer cell line (T24) and a pooled sample of primary urinary bladder epithelia cells (PUBEC) from pigs. The highest level of DNA damage assessed by comet assay was observed following 24-h treatment in both cell types, whereas PUBEC cells were clearly more susceptible. Even 4-h treatment induced DNA damage in PUBEC cells with benchmark doses of 0.0027 µM B[a]P and 0.00023 µM after 4-h and 24-h exposure, respectively. Nearly no effect was observed for periods of 48 h. The frequency of micronucleus formation increased more markedly in T24 cells, particularly with 24-h treatment. In PUBEC cells, 48-h exposure notably induced the formation of nucleoplasmic bridges and nuclear buds. Even though only one biological replicate was studied due to the sophisticated study design, our results give a strong indication of the potential of B[a]P to induce and increase malignity in human-relevant cell types.

Mutagenicity data is a core component of the safety assessment data required by regulatory agencies for acceptance of new drug compounds, with the OECD-471 bacterial reverse mutation (Ames) assay most widely used as a primary screen to assess drug impurities for potential mutagenic risk. N-Nitrosamines are highly potent mutagenic carcinogens in rodent bioassays and their recent detection as impurities in pharmaceutical products has sparked increased interest in their safety assessment. Previous literature reports indicated that the Ames test might not be sensitive enough to detect the mutagenic potential of N-nitrosamines in order to accurately predict a risk of carcinogenicity. To explore this hypothesis, public Ames and rodent carcinogenicity data pertaining to the N-nitrosamine class of compounds was collated for analysis. Here we present how variations to the OECD 471-compliant Ames test, including strain, metabolic activation, solvent type and pre-incubation/plate incorporation methods, may impact the predictive performance for carcinogenicity. An understanding of optimal conditions for testing of N-nitrosamines may improve both the accuracy and confidence in the ability of the Ames test to identify potential carcinogens.

We conducted a two-year inhalation study of butyl methacrylate using F344/DuCrlCrlj rats and B6D2F1/Crl mice. Rats were exposed to 0, 30, 125 and 500 ppm (v/v) and mice were exposed to 0, 8, 30 and 125 ppm (v/v) using whole-body inhalation chambers. Non-neoplastic lesions developed in the nasal cavities of both rats and mice, but neoplastic lesions were not found. There was also a positive trend in the incidence of large granular lymphocytic (LGL) leukemia in the spleen of male rats. No changes were observed in female rats. Overall, there is some evidence of carcinogenicity in male rats, but there is no evidence of carcinogenicity in female rats. In male mice, there was a positive trend by Peto\'s test in the incidence of hepatocellular adenomas, and the incidence of hepatocellular adenomas and hepatocellular carcinomas combined was significantly increased compared to the controls by Fisher\'s exact test in the 30 ppm exposed male group. In female mice, the incidence of hemangiosarcoma in all organs combined showed a positive trend by Peto\'s test. Therefore, there is some evidence of carcinogenicity in male mice, and there is equivocal evidence of carcinogenicity in female mice.

In this combined chronic toxicity/carcinogenicity study of gardenia blue as a natural food color additive, Sprague Dawley rats were administered 0.5%, 2.5%, or 5.0% gardenia blue via the feed or carrier diet (0.0% gardenia blue) for 12 (chronic toxicity cohort) or 24 (carcinogenicity cohort) months. No abnormal clinical, ophthalmological, neurotoxicity or clinical pathology changes were attributed to treatment, and there was no increase in mortality due to gardenia blue exposure. The only treatment-related change was grossly observed blue discoloration of the stomach, intestines, and mesenteric lymph nodes as well as reversible dark discoloration of the kidneys all without associated histopathology. The no-observed-adverse-effect level (NOAEL) for gardenia blue exposure via the diet for one or two years was determined to be 5.0% (2175.3 mg/kg body weight/day in male rats and 3075.4 mg/kg body weight/day in female rats).

alpha-Glycosyl isoquercitrin (AGIQ) is a flavonoid that possesses antioxidant and tumor suppressive capabilities and is marketed as a food additive in Japan. The aim of this study was to assess the potential for oral chronic toxicity and carcinogenicity of AGIQ in male and female Sprague Dawley rats following up to 5.0% dietary exposure. In the chronic toxicity study, rats were exposed to AGIQ or vehicle for one year with a 6-month interim termination point; for the carcinogenicity study, rats were treated for 24 months. No signs of AGIQ-related toxicity clinically or histologically were observed for up to one year except for yellow discoloration of bone. In the carcinogenicity study, a statistically significant increase in the incidence of malignant glioma of the brain or spinal cord was observed in female rats exposed to 5.0% AGIQ compared to those exposed to control feed. A Scientific Advisory Panel of experienced neuropathologists reviewed the gliomas (routine stains and glial cell markers) and concluded that the gliomas were a rare, spontaneous, rat-specific neoplasm: malignant microglial tumor. The lesions could not definitively be attributed to AGIQ exposure and have limited implications with respect to predicting human cancer risk.

Anthracene is an environmental pollutant and its adverse effects on human health have long been a concern due to its persistence and bioaccumulation properties. However, there is insufficient evidence for its chronic toxicity, especially carcinogenicity, in either humans or experimental animals. In this study, its carcinogenicity and chronic toxicity were investigated in compliance with the OECD test guideline 451 (OECD TG 451) and Good Laboratory Practice Standards. Fifty male and 50 female F344 rats and 50 female BDF1 mice were administrated 0, 8000, 20000, or 50000 ppm anthracene in the diet for 104 weeks, and 50 male BDF1 mice were fed diets containing anthracene at 0, 3200, 8000, or 20000 ppm. Anthracene treatment had no adverse effect on either the survival rate or general condition of the rats or mice during the study period. Body weights were lower or tended to be lower in the anthracene-treated groups than in the control groups. Increased incidence of hepatocellular carcinoma and hepatocellular adenoma was observed in male rats and female mice. Renal cell carcinoma and renal cell adenoma, fibroadenoma in the mammary gland, and uterine endometrial stromal sarcoma were increased in female rats. Transitional cell carcinoma and transitional cell papilloma in the urinary bladder were also increased in male and female rats. In addition, several different pre-neoplastic lesions were increased in the anthracene-treated male and female rats and female mice. These results provide clear evidence that oral administration of anthracene for 104 weeks has a carcinogenic effect in male and female rats and female mice.