Proceedings of the 2021 National Toxicology Program Satellite Symposium

The 2021 annual National Toxicology Program (NTP) Satellite Symposium, entitled “Pathology Potpourri,” was the 20th anniversary of the symposia and held virtually on June 25th, in advance of the Society of Toxicologic Pathology’s 40th annual meeting. The goal of this symposium was to present and discuss challenging diagnostic pathology and/or nomenclature issues. This article presents summaries of the speakers’ talks along with select images that were presented to the audience for voting and discussion. Various lesions and topics covered during the symposium included differentiation of canine oligodendroglioma, astrocytoma, and undefined glioma with presentation of the National Cancer Institute’s updated diagnostic terminology for canine glioma; differentiation of polycystic kidney, dilated tubules and cystic tubules with a discussion of human polycystic kidney disease; a review of various rodent nervous system background lesions in control animals from NTP studies with a focus on incidence rates and potential rat strain differences; vehicle/excipient-related renal lesions in cynomolgus monkeys with a discussion on the various cyclodextrins and their bioavailability, toxicity, and tumorigenicity; examples of rodent endometrial tumors including intestinal differentiation in an endometrial adenocarcinoma that has not previously been reported in rats; a review of various rodent adrenal cortex lesions including those that represented diagnostic challenges with multiple processes such as vacuolation, degeneration, necrosis, hyperplasia, and hypertrophy; and finally, a discussion of diagnostic criteria for uterine adenomyosis, atypical hyperplasia, and adenocarcinoma in the rat.

Enhanced communication of IARC Monograph findings to better achieve public health outcomes

IARC Monographs are globally-recognized assessments of carcinogenicity published by International Agency for Research on Cancer and directed to national authorities as a contribution to public health. Currently, though not constraining the scope of studies examined, Monographs are declared to involve only hazard identification: a scenario which then dictates that overall evaluations involve a single sentence referencing only the likelihood of carcinogenicity. Consequently, for notable evaluations, commentators explain what particular Monographs encompass, sometimes indicating which national authority might act. However, upon examination, IARC Monographs cannot be recognized as only hazard identification because they include assessment of specified circumstances of exposure, document levels of exposure to particular agents and identify organ site(s) for tumours. Comparisons with assessments of carcinogenicity made by US National Toxicology Program and by World Cancer Research Fund indicate that Monographs represent a singular methodology, not precisely aligned with hazard identification or risk assessment. Hence, all key findings relevant to public health in particular Monographs may be prominently and accessibly communicated. Where currently documented, these findings specifically include situation(s) resulting in highest exposure and known or likely tumour site(s). Correspondingly-less reliance on commentators, and greater insight by the wider community, may be further achieved by WHO, in consultation with IARC, describing the type of national authority relevant to particular evaluations, this information being complementary to relevant Monographs. No changes in the scope, structure or content of Monographs are necessitated by such proposals. Examples of key findings, and the relevant national authority, are provided in respect of several notable Monographs.

Comparison of phytochemical composition of Ginkgo biloba extracts using a combination of non-targeted and targeted analytical approaches

Ginkgo biloba extract (GbE) is a dietary supplement derived from an ethanolic extract of Ginkgo biloba leaves. Unfinished bulk GbE is used to make finished products that are sold as dietary supplements. The variable, complex composition of GbE makes it difficult to obtain consistent toxicological assessments of potential risk. The National Toxicology Program (NTP) observed hepatotoxicity in its rodent studies of a commercially available, unfinished GbE product, but the application of these results to the broader GbE supplement market is unclear. Here, we use a combination of non-targeted and targeted chromatographic and spectrophotometric methods to obtain profiles of 24 commercially available finished GbE products and unfinished standardized and unstandardized extracts with and without hydrolysis, then used principal component analysis to group unfinished products according to their similarity to each other and to National Institute of Standards and Technology (NIST) standard reference materials (SRM), and the finished products. Unfinished products were grouped into those that were characteristic and uncharacteristic of standardized GbE. Our work demonstrates that different analytical approaches produced similar classifications of characteristic and uncharacteristic products in unhydrolyzed samples, but the distinctions largely disappeared once the samples were hydrolyzed. Using our approach, the NTP GbE was most similar to two unfinished GbE products classified as characteristic, finished products, and the NIST GbE SRM. We propose that a simple analysis for the presence, absence, or amounts of compounds unique to GbE in unhydrolyzed samples could be sufficient to determine a sample’s authenticity. Graphical abstract

The Assessment of Longitudinal Sections of Rat Female Reproductive Tissues for NTP 2-Year Toxicity and Carcinogenicity Studies

The National Toxicology Program (NTP) now uses an extended longitudinal sectioning protocol for the uterus to better evaluate female rodent reproductive tract toxicity for all developmental and reproductive toxicology and 2-year toxicity and carcinogenicity bioassays. The previous protocol for toxicity/carcinogenicity studies involved 1 cross section midway through each uterine horn and collection of uterine cervix and vagina only if gross lesions were present. Here we compare the histological findings of the original cross sections with the additional longitudinal sections of residual uterine tissues of 7 chronic NTP rat bioassays. The goal of this study was to determine whether there might be any advantages to examining additional uterine tissue. The longitudinal protocol allowed for 10 to 20 times more uterine tissue for evaluation. Results indicate that the potential advantages of a more complete evaluation of female reproductive tract tissue include increased detection of reproductive targets, increased detection of neoplastic and nonneoplastic lesions, improved detection of tissue origin of neoplasms, less reliance on gross identification of lesions, improved accuracy in the application of severity grades, and increased detection of preneoplastic or subtle lesions.

Degradation of Phospholipids by N, N-Dimethylformamide Induced Liver Toxicity in Male Wistar Rats

Dimethylformamide (DMF) is an industrially used solvent, prioritized by the National Toxicology Program as a potent hepatotoxic compound. The effect of DMF on liver is well documented; however its impact on hepatic phospholipids remains enigmatic. Hence, to understand the phospholipid metabolism we have developed an animal model for DMF induced hepatotoxicity. In the present study, DMF (0.5, 1.0, 1.5 g/kg body wt) was given intraperitoneally to male wistar rats and terminated after 24 and 48 h. DMF with a concentration of 1.5 g/kg body wt shows maximum toxic effect. Dosages higher than 1.5 g/kg body wt showed lethal effect, hence in this study, 1.5 g/kg body wt was used as maximum concentration. Induction of hepatotoxicity by DMF was confirmed by liver marker enzymes. DMF impairs the liver phospholipid metabolism. DMF decreased the individual phospholipid levels by altering the fatty acid composition. There was an increase in unsaturated fatty acids with a concomitant decrease in saturated fatty acid. These changes in the fatty acid may directly or indirectly affect the membrane structure and fluidity. Understanding the mechanism by which DMF induced hepatotoxicity and alteration in phospholipid metabolism is a worthwhile pursuit.

Proceedings of the 2019 National Toxicology Program Satellite Symposium

The 2019 annual National Toxicology Program Satellite Symposium, entitled “Pathology Potpourri,” was held in Raleigh, North Carolina, at the Society of Toxicologic Pathology’s 38th annual meeting. The goal of this symposium was to present and discuss challenging diagnostic pathology and/or nomenclature issues. This article presents summaries of the speakers’ talks along with select images that were used by the audience for voting and discussion. Various lesions and topics covered during the symposium included aging mouse lesions from various strains, as well as the following lesions from various rat strains: rete testis sperm granuloma/fibrosis, ovarian cystadenocarcinoma, retro-orbital schwannoma, periductal cholangiofibrosis of the liver and pancreas, pars distalis hypertrophy, chronic progressive nephropathy, and renal tubule regeneration. Other cases included polyovular follicles in young beagle dogs and a fungal blood smear contaminant. One series of cases challenged the audience to consider how immunohistochemistry may improve the diagnosis of some tumors. Interesting retinal lesions from a rhesus macaque emphasized the difficulty in determining the etiology of any particular retinal lesion due to the retina’s similar response to vascular injury. Finally, a series of lesions from the International Harmonization of Nomenclature and Diagnostic Criteria Non-Rodent Fish Working Group were presented.

National Toxicology Program Position Statement on Informed (“Nonblinded”) Analysis in Toxicologic Pathology Evaluation

The National Toxicology Program (NTP) uses histopathological evaluation of animal tissues as a key element in its toxicity and carcinogenicity studies. The initial histopathological evaluations are subjected to a rigorous peer review process involving several steps. The NTP peer review process is conducted by multiple, highly trained, and experienced toxicological pathologists employing standardized terminology. In addition, ancillary data, such as body and organ weights and clinical pathology findings, are used to corroborate the diagnoses. The NTP does employ masked analysis to confirm subtle lesions or severity scores, as needed, and during its Pathology Working Groups. The use of masked analysis can have a negative effect on histopathological evaluation because it is important for the pathologist to compare treated groups to the concurrent controls, which would not be possible in a blinded evaluation. Therefore, the NTP supports an informed approach to histopathological evaluation in its toxicity and carcinogenicity studies.

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.