Scientific and Regulatory Policy Committee Points to Consider: Review of the United States Food and Drug Administration (FDA) Guidance on Pathology Peer Review in Nonclinical Toxicology Studies.

In December 2021, the United States Food and Drug Administration (FDA) issued the final guidance for industry titled Pathology Peer Review in Nonclinical Toxicology Studies: Questions and Answers. The stated purpose of the FDA guidance is to provide information to sponsors, applicants, and nonclinical laboratory personnel regarding the management and conduct of histopathology peer review as part of nonclinical toxicology studies conducted in compliance with good laboratory practice (GLP) regulations. On behalf of and in collaboration with global societies of toxicologic pathology and the Society of Quality Assurance, the Scientific and Regulatory Policy Committee (SRPC) of the Society of Toxicologic Pathology (STP) initiated a review of this FDA guidance. The STP has previously published multiple papers related to the scientific conduct of a pathology peer review of nonclinical toxicology studies and appropriate documentation practices. The objectives of this review are to provide an in-depth analysis and summary interpretation of the FDA recommendations and share considerations for the conduct of pathology peer review in nonclinical toxicology studies that claim compliance to GLP regulations. In general, this working group is in agreement with the recommendations from the FDA guidance that has added clear expectations for pathology peer review preparation, conduct, and documentation.

Brief Synopsis: Review of Renal Tubule and Interstitial Anatomy and Physiology and Renal INHAND, SEND, and DIKI Nomenclature.

This article provides a synopsis of the first two presentations from the second scientific session of the 37th Annual Symposium of the Society of Toxicologic Pathology in Indianapolis, Indiana, on June 18, 2018; the session focused on acute kidney injury. The first presentation, given by Dr. Kevin McDorman, focused on "Fundamentals of Renal Tubule and Interstitial Anatomy and Physiology." Several common background findings from toxicity studies were additionally discussed. Lastly, factors that impact the relevance and usefulness of historical control data, such as quality and consistency of histopathology, were discussed. The second presentation, given by Dr. Torrie Crabbs, provided a review of International Harmonization of Nomenclature and Diagnostic Criteria (INHAND), Standard for Exchange of Nonclinical Data (SEND), and drug-induced kidney injury (DIKI) nomenclature. INHAND is a global collaborative project that provides internationally accepted standardized nomenclature and diagnostic criteria for proliferative and nonproliferative changes in laboratory animals in toxicity and carcinogenicity studies. SEND is currently a required standard for data submission to the Food and Drug Administration (FDA). Since the FDA has indicated its preference for INHAND nomenclature, SEND will predominately use INHAND terminology; thus, familiarity with INHAND terminology is critical for toxicologic pathologists. The diagnostic features of three common DIKI findings, in addition to several complicated INHAND terminologies, were reviewed.

Career Transition Points in Toxicologic Pathology: Management and Consulting.

In the constantly evolving field of toxicologic pathology, a pathologist's career is often characterized by multiple career transitions. However, these transitions can be challenging and/or overwhelming and may require a shift in focus, strategic approach, and acquisition of new skills and expertise. In order to provide a forum to discuss challenges associated with career transitions and skill set/competencies required to navigate career changes effectively and successfully, the Career Development and Outreach Committee of the Society of Toxicologic Pathology (STP) sponsored a career development workshop entitled "Transitions in a Pathologist's Career" in conjunction with the STP 36th annual symposium. The presentations at this workshop provided perspectives of managers from pharmaceutical companies and Contract Research Organizations as well as consultants. This article is designed to provide brief summaries of their talks in this well-received career development workshop.

Industry-contract research organization pathology interactions: a perspective of contract research organizations in producing the best quality pathology report.

This article provides observations on the features of sponsor-contract research organization communication that will achieve the best quality pathology report based on our collective experience. Information on the test article and any anticipated findings should be provided, and initial slide examination should be done with knowledge of treatment group (but may be followed by blinded review of target tissues to determine no-effect levels). Only a pathologist should write or revise the pathology report or the pathology section of the overall study report. To address concerns related to undue sponsor influence, comments by sponsors should be presented as suggestions rather than directives. Adversity should be defined for each finding by the study pathologist, but the no-observed adverse effect level should not be discussed in the pathology report. Board-certified pathologists are recommended, but are not essential. Sponsors that have a particular format or report preferences should make them known well in advance. Histologic processing "to glass" of protocol-specified tissues from all dosage groups is recommended for rapid evaluation of target tissues. Telepathology is beneficial in certain situations, but it is usually more efficient for the study pathologist and reviewing pathologist to be in the same physical location to review differences of opinion and reach a consensus.

Oxidative DNA damage from potassium bromate exposure in Long-Evans rats is not enhanced by a mixture of drinking water disinfection by-products.

Public drinking water treated with chemical disinfectants contains a complex mixture of disinfection by-products (DBPs) for which the relative toxicity of the mixtures needs to be characterized to accurately assess risk. Potassium bromate (KBrO(3)) is a by-product from ozonation of high-bromide surface water for production of drinking water and is a rodent carcinogen that produces thyroid, mesothelial, and renal tumors. The proposed mechanism of KBrO(3) renal carcinogenesis involves the formation of 8-oxoguanine (8-oxoG), a promutagenic base lesion in DNA typically removed through base excision repair (BER). In this study, male Long-Evans rats were exposed via drinking water to carcinogenic concentrations of KBrO(3) (0.4 g/L), 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (0.07 g/L), chloroform (1.8 g/L), bromodichloromethane (0.7 g/L), or a mixture of all these chemicals at the same concentrations for 3 weeks. Half of one kidney was processed for microscopic examination, and the remaining kidney was frozen for isolation of genomic DNA. Levels of 8-oxoG were measured using HPLC with electrochemical detection in DNA samples incubated with formamidopyrimidine-DNA glycosylase. Aldehydic lesions (e.g. abasic sites) in DNA samples were quantitated using an aldehyde-reactive probe slot-blot assay. Treatment with KBrO(3) produced a measurable increase of 8-oxoG in the kidney, and this effect was greater than that produced by treatment with the DBP mixture. No other single chemical treatment caused measurable increases of 8-oxoG. The mixture effect on the amount of 8-oxoG observed in this study suggests an interaction between chemicals that reduced the generation of oxidative DNA damage. No increases in abasic sites were observed with treatment, but a decrease was apparent in the rats treated with the DBP mixture. These data are consistent with previous studies where chronic exposure to this chemical mixture in drinking water resulted in a less than additive carcinogenic response in Tsc2 mutant Long-Evans rats.

The carcinogenic response of Tsc2 mutant Long-Evans (Eker) rats to a mixture of drinking water disinfection by-products was less than additive.

Cancer risk assessment methods for chemical mixtures in drinking water are not well defined. Current default risk assessments for chemical mixtures assume additivity of carcinogenic effects, but this may not represent the actual biological response. A rodent model of hereditary renal cancer (Eker rat) was used to evaluate the carcinogenicity of mixtures of water disinfection by-products (DBPs). Male and female Eker rats were treated with individual DBPs or a mixture of DBPs for 4 or 10 months. Potassium bromate, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone, chloroform, and bromodichloromethane were administered in drinking water at low concentrations of 0.02, 0.005, 0.4, and 0.07 g/l, respectively, and high concentrations of 0.4, 0.07, 1.8, and 0.7 g/l, respectively. Low and high dose mixture solutions comprised all four chemicals at either the low or the high concentrations, respectively. Body weights, water consumption, and chemical concentrations in the water were measured monthly. All tissues were examined macroscopically for masses and all masses were diagnosed microscopically. Total renal lesions (adenomas and carcinomas) were quantitated microscopically in male and female rats treated for 4 or 10 months. A dose response for renal tumors was present in most treatment groups after 4 or 10 months of treatment. Treatment with the mixture produced on average no more renal, splenic, or uterine tumors than the individual compound with the greatest effect. This study suggests that the default assumption of additivity may overestimate the carcinogenic effect of chemical mixtures in drinking water.

Analysis of preneoplastic and neoplastic renal lesions in Tsc2 mutant Long-Evans (Eker) rats following exposure to a mixture of drinking water disinfection by-products.

Disinfection of surface water for human consumption results in the generation of a complex mixture of chemicals in potable water. Cancer risk assessment methodology assumes additivity of carcinogenic effects in the regulation of mixtures. A rodent model of hereditary renal cancer was used to investigate the carcinogenic response to a mixture of drinking water disinfection by-products (DBPs). Rats carrying a mutation in the Tsc2 tumor suppressor gene (Eker rats) readily develop renal preneoplastic and neoplastic lesions, and are highly susceptible to the effects of renal carcinogens. Male and female Eker rats were exposed via drinking water to individual or a mixture of DBPs for 4 or 10 months. Potassium bromate, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), chloroform, and bromodichloromethane were administered at low concentrations of 0.02, 0.005, 0.4 and 0.07 g/l, respectively, and high concentrations of 0.4, 0.07, 1.8 and 0.7 g/l, respectively. Low and high dose mixture solutions were comprised of all four chemicals at either low concentrations or high concentrations, respectively, Following necropsy, each kidney was examined microscopically for preneoplastic lesions (atypical tubules and hyperplasias) and tumors. While some of the mixture responses observed in male rats did fall within the range expected for an additive response, especially at the high dose, predominantly antagonistic effects on renal lesions were observed in response to the low dose mixture in male rats and the high dose mixture in female rats. These data suggest that current default risk assessments assuming additivity may overstate the cancer risk associated with exposure to mixtures of DBPs at low concentrations.

Use of the spontaneous Tsc2 knockout (Eker) rat model of hereditary renal cell carcinoma for the study of renal carcinogens.

The kidney is a frequent site for chemically induced cancers in rodents and among the 10 most frequent sites for cancer in human patients. Renal cell carcinoma (RCC) is the most frequent upper urinary tract cancer in humans and accounts for 80-85% of malignant renal tumors. Hereditary RCC occurs in Eker rats that are heterozygous for an insertion mutation in the Tsc2 tumor suppressor gene. The germline mutation renders heterozygous mutants highly susceptible to renal carcinogens. The utility of this model in studying potential renal carcinogens is due to an ordered progression of proliferative renal lesions that can be identified and counted microscopically. The quantitative nature of the model allows for the production of statistically powerful data to understand the relative degree and potency of chemical effects and allow analysis of genetic alterations that may be chemical specific.

Methods for measuring DNA adducts and abasic sites I: isolation, purification, and analysis of DNA adducts in intact DNA.

The major event involved in the formation of mutations and the initiation and progression of cancer is the induction of DNA damage by reactive intermediates arising from exposure to endogenous and exogenous chemicals. Many electrophilic metabolites of chemicals covalently bind to the bases of DNA causing specific DNA adducts. This unit includes protocols for preparing samples of intact DNA and adduct analysis to quantify the number of adducts that can potentially cause mutagenic or carcinogenic damage.

Induction of transitional cell hyperplasia in the urinary bladder and aberrant crypt foci in the colon of rats treated with individual and a mixture of drinking water disinfection by-products.

Cancer of the urinary bladder and colon are significant human health concerns. Epidemiological studies have suggested a correlation between these cancers and the chronic consumption of chlorinated surface water containing disinfection by-products (DBPs). The present study was designed to determine if exposure to DBPs would cause preneoplastic or neoplastic lesions in the urinary bladder and colon of rats, and what effect a mixture of DBPs would have on these lesions. Male and female Eker rats were treated via drinking water with low and high concentrations of potassium bromate, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), chloroform, or bromodichloromethane individually or in a mixture for 10 months. The urinary bladders and colons were examined for the presence of preneoplastic lesions. Cell proliferation in the urothelium was examined using immunohistochemical staining for bromodeoxyuridine. Aberrant crypt foci (ACF), as well as the number of individual crypts in each ACF, were identified and counted microscopically after staining with 0.2% methylene blue. Colon crypt cell proliferation and mitotic index were determined using immunohistochemical staining for proliferating cell nuclear antigen. Labeling indexes for the urinary bladder and colon were calculated based on the percentage of positively labeled cells. Treatment with the high dose of MX caused transitional epithelial hyperplasia and cell proliferation in the rat urinary bladder, and this effect was diminished in the high dose mixture animals. Treatment with 4 individual DBPs, as well as a mixture of them, caused the development of ACF, the putative preneoplastic lesion of colon cancer.