Nigel Roome

Society of Toxicologic Pathology position paper: organ weight recommendations for toxicology studies.

The evaluation of organ weights in toxicology studies is an integral component in the assessment of pharmaceuticals, chemicals, and medical devices. The Society of Toxicologic Pathology (STP) has created recommendations for weighing organs in GLP general toxicology studies lasting from 7 days to 1 year. The STP recommends that liver, heart, kidneys, brain, testes, and adrenal glands be weighed in all multidose general toxicology studies. Thyroid gland and pituitary gland weights are recommended for all species except mice. Spleen and thymus should be weighed in rodent studies and may be weighed in non-rodent studies. Weighing of reproductive organs is most valuable in sexually mature animals. Variability in age, sexual maturity, and stage of cycle in non-rodents and reproductive senescence in female rodents may complicate or limit interpretation of reproductive organ weights. The STP recommends that testes of all species be weighed in multidose general toxicology studies. Epididymides and prostate should be weighed in rat studies and may be weighed on a case-by-case basis in non-rodent and mouse studies. Weighing of other organs including female reproductive organs should be considered on a case-by-case basis. Organ weights are not recommended for any carcinogenicity studies including the alternative mouse bioassays. Regardless of the study type or organs evaluated, organ weight changes must be evaluated within the context of the compound class, mechanism of action, and the entire data set for that study.

Evaluation of Organ Weights for Rodent and Non-Rodent Toxicity Studies: A Review of Regulatory Guidelines and a Survey of Current Practices

The Society of Toxicologic Pathology convened a working group to evaluate current practices regarding organ weights in toxicology studies. A survey was distributed to pharmaceutical, veterinary, chemical, food/nutritional and consumer product companies in Europe, North America, and Japan. Responses were compiled to identify organs routinely weighed for various study types in rodent and non-rodent species, compare methods of organ weighing, provide perspectives on the value of organ weights and identify the scientist(s) responsible for organ weight data interpretation. Data were evaluated as a whole as well as by industry type and geographic location. Regulatory guidance documents describing organ weighing practices are generally available, however, they differ somewhat dependent on industry type and regulatory agency. While questionnaire respondents unanimously stated that organ weights were a good screening tool to identify treatment-related effects, opinions varied as to which organ weights are most valuable. The liver, kidneys, and testes were commonly weighed and most often considered useful by most respondents. Other organs thatbreak were commonly weighed included brain, adrenal glands, ovaries, thyroid glands, uterus, heart, and spleen. Lungs, lymph nodes, and other sex organs were weighed infrequently in routine studies, but were often weighed in specialized studies such as inhalation, immunotoxicity, and reproduction studies. Organ-to-body weight ratios were commonly calculated and were considered more useful when body weights were affected. Organ to brain weight ratios were calculated by most North American companies, but rarely according to respondents representing veterinary product or European companies. Statistical analyses were generally performed by most respondents. Pathologists performed interpretation of organ weight data for the majority of the industries.

Interlaboratory Evaluation of Genomic Signatures for Predicting Carcinogenicity in the Rat

The Critical Path Institute recently established the Predictive Safety Testing Consortium, a collaboration between several companies and the U.S. Food and Drug Administration, aimed at evaluating and qualifying biomarkers for a variety of toxicological endpoints. The Carcinogenicity Working Group of the Predictive Safety Testing Consortium has concentrated on sharing data to test the predictivity of two published hepatic gene expression signatures, including the signature by Fielden et al. (2007, Toxicol. Sci. 99, 90-100) for predicting nongenotoxic hepatocarcinogens, and the signature by Nie et al. (2006, Mol. Carcinog. 45, 914-933) for predicting nongenotoxic carcinogens. Although not a rigorous prospective validation exercise, the consortium approach created an opportunity to perform a meta-analysis to evaluate microarray data from short-term rat studies on over 150 compounds. Despite significant differences in study designs and microarray platforms between laboratories, the signatures proved to be relatively robust and more accurate than expected by chance. The accuracy of the Fielden et al. signature was between 63 and 69%, whereas the accuracy of the Nie et al. signature was between 55 and 64%. As expected, the predictivity was reduced relative to internal validation estimates reported under identical test conditions. Although the signatures were not deemed suitable for use in regulatory decision making, they were deemed worthwhile in the early assessment of drugs to aid decision making in drug development. These results have prompted additional efforts to rederive and evaluate a QPCR-based signature using these samples. When combined with a standardized test procedure and prospective interlaboratory validation, the accuracy and potential utility in preclinical applications can be ascertained.

Best Practices for Reporting Pathology Interpretations within GLP Toxicology Studies

Pfizer, Inc., Groton, Connecticut 06340, USA Merck Research Laboratories, West Point, Pennsylvania 19586 USA Pathology Branch, Center for Food Safety and Applied Nutrition, FDA, College Park, Maryland 20740, USA U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711, USA Charles River Laboratories, Senneville, Quebec H9X 3R3, Canada Merck and Company, Inc., West Point, Pennsylvania 19486, USA Wyeth Research, Chazy, New York 12921 USA Health Canada Ottawa, Ontario K1A 0L2, Canada Sanofi-Aventis, Porcheville 78440, France Vet Path Services, Inc., Cincinnati, Ohio 45249, USA Albert Einstein College of Medicine Bronx, New York 10461, USA Purdue University, West Lafeyette, Indiana 47907, USA

Spontaneous Iron Overload in Sprague-Dawley Rats

In a review of the toxicological studies performed in our laboratory during the period 1986-1995, we occasionally observed significant iron overloading in the liver. Liver tissue was examined by light and electron microscopy, and the results were analyzed by sex and age (7, 9, 11, 19, 31, 59, and 111 wk). The intensity of iron overload increased with age: the accumulation began in pericanalicular siderosomes of periportal hepatocytes and extended progressively to the entire lobule and also to nonhepatocytic cells (Kupffer cells in sinusoids and macrophages around bile ducts in portal tracts) and occasionally with distortion of sinusoids by sideroblastic nodules and moderate enlargment of portal tracts in the oldest animals. No significant inflammatory infiltrates, degeneration, necrosis or fibrosis were noted. Hepatocyte pigmentation alone was prominent at 9 and 11 wk. The frequency of pigmentation of parenchymal and nonhepatocytic cells increased from 9 wk for females; in males, this was seen only at 111 wk. The intensity of pigmentation of nonhepatocytic cells versus parenchymal cells increased with aging. The frequency of those different types of iron overloading was higher for females up to 111 wk. The pathology of spontaneous iron overloading in the Sprague-Dawley rat, described here in spite of differences, has some similarities to that of human hereditary hemochromatosis.

Histopathological and Molecular Changes During Apoptosis Produced by 7H-Dibenzo(c,g)-Carbazole in Mouse Liver

The topical administration of 7H-dibenzo[c,g]carbazole (7H-DBC) at very low but repeated doses causes genotoxic effects such as DNA adduct formation and produces hepatocellular apoptosis in mouse liver. The purpose of this work was to investigate the alterations in gene expression and protein levels of biomarkers associated with the p53 pathway in mouse liver after exposure to cumulative low doses of 7H-DBC by skin paint applications. The compound was administered topically at the dose of 13.35 μg per animal every 2 days to give either 6, 8, 10, or 12 applications. Animals were sacrificed 48 hours after the different treatments. The apoptotic index increased with the number of applications, with a major proportion of apoptotic cells in the periportal areas. A significant increase of Bax mRNA and protein expression was observed after the 8th application whereas the expression of mRNA levels of Fas and p53 did not show significant differences between treated and control animals. Nuclear staining of p53 was detected in hepatocyte nuclei showing the activation of this protein. Later in the apoptosis process we observed the up-regulation of TGF-β1 in parenchymal cells. In addition to the induction of the p53 apoptosis pathway in vivo by 7H-DBC, we have observed molecular changes related to cell proliferation such as the overexpression of the antiapoptotic gene Bcl-2.

Induction of DNA Synthesis in Mouse Liver Following Increases of DNA Adduct Levels Elicited by Very Low Cumulative Doses of the Genotoxic Hepatocarcinogen 7H-dibenzo(c,g)carbazole

The purpose of this work was to investigate the administration of very low but repeated doses of a genotoxic carcinogen and an eventual correlation with cellular DNA synthesis. The compound 7H-dibenzo[c,g]carbazole is a genotoxic carcinogen in the mouse liver and was administered topically at the dose of 13.35 μg per animal every 2 days to give a total of 13 applications. Animals were sacrificed 48 hours after every 2 applications until the 10th treatment, then 48 hours after every treatment. Postulated genotoxic effects such as DNA adduct formation were detected by the 32P-post labeling assay. Liver sections were examined for microscopic changes and DNA synthesis. Results showed an increase of the total DNA adduct level in the liver throughout the study with a slowing down in the level after the sixth application of the compound. This change could correspond to the onset of DNA synthesis and to the moderate hepatocellular apoptosis which was observed. The DNA synthesis, which was considered to be secondary to the cytotoxicity induced by the high level of DNA adducts altering normal cellular activity, could also be the opportunity to fix the DNA adducts into heritable mutations. These results raise the question regarding the risk assessment in humans exposed regularly to very low doses of chemicals in the environment: for non-proliferating tissue, the regular accumulation of DNA adducts could remain silent until a “threshold level” is reached from which stimulation of the DNA synthesis may fix the DNA adducts into heritable mutations, eventually leading to tumors.

Biomarkers of carcinogenicity and their roles in drug discovery and development

The screening of drug candidates to assess their carcinogenic potential has long been a challenge for drug development. While genotoxic compounds can be readily detected with a battery of standard tests, including short-term in vitro and in vivo assays, predicting nongenotoxic carcinogenicity remains a major challenge. The 2-year rodent bioassay has been held as the gold standard for the assessment of carcinogenic risk to humans. However, due primarily to the continuing doubt over their relevance to human risk assessment, there has been an increased demand for more efficient and accurate approaches to predict and understand human relevant risk of carcinogenicity. Novel biomarkers have helped to shed light on our understanding of the factors that lead to and are characteristic of the carcinogenic phenotypes. Tissue biomarkers of carcinogenicity identified to be concordant with drug exposures resulting in tumor outcome may assist the drug development process by resolving ambiguities, shortening timelines and enabling earlier decisions on compounds. This information could vastly improve the efficiency with which nongenotoxic carcinogens are identified and ensure earlier insight into the relevance for humans.

Regulatory Forum Opinion Piece: Review-Toxicological Pathology Profile and Regulatory Expectations for Nonclinical Development of Insulins and Insulin Analogues.

The toxicological profile of insulins is exclusively due to exaggerated pharmacology resulting in hypoglycemic findings. Insulin analogues displaying modifications and aimed at improving pharmacokinetics do not induce different toxicity. The main target is the brain displaying neuronal necrosis. Wallerian degeneration of nerves occurs rarely after severe hypoglycemia. These findings are of potential human relevance; nevertheless, these changes are induced in normoglycemic animals whereas diabetic patients suffer from hyperglycemia. Therefore, it is usually not difficult to achieve a therapeutic window for subsequent use in patients. Based upon this and in the absence of classical toxicity, there has been no scientific need for diabetic animal models. A greater challenge is the mitogenicity already inherent with regular insulin. Thus, the focus for preclinical safety evaluation of analogues is to demonstrate that modifications in regular insulin do not result in enhanced mitogenicity. The approaches used to assess the mitogenic potential of insulin analogues have changed over time driven by scientific progression and changes within the regulatory environment. Therefore, in vitro and in vivo evaluation of cell proliferation has become common practice, and to date there has been no evidence that the mitogenic potential of insulin analogues may be increased compared to regular insulin.

Heart Collection Protocol of the Laboratory Dog and Monkey

Various dissection guides and published articles both in paper format and as computer based supports exist for autopsy procedures and tissue sampling and concern a variety of species and organ systems. For staff working routinely as pathologists or necropsy room technical staff, they provide a good grounding in the basics for dissection and preparation of specimens for subsequent histological examination. They are also evidently of additional use in the case of more skilled staff as a source of reference and for consultation for those who frequently perform autopsises. The collection and preparation of histological specimens for the rodent heart (rat, mouse and hamster) is relatively simple and representative areas of the organ can easily be obtained and evaluated, although the overall number of sections to be examined and the orientation of the specimens on routine studies is still a question of some debate. For this CD-Rom, the authors (GlaxoSmithKline, EPL and NIEHS/NTP) have selected the dog and non-human primate as the subject for a guide concerning the collection of the heart. In the case of the routine pathological examination of the dog and non-human primate heart for toxicology studies, the gross examination of the heart and its’ dissection and sampling to obtain representative areas for a complete review of any possible changes both macroscopic and microscopic is more complex. There has tended to be two schools of thought with either a minimalist or maximalist approach to sampling and examination, although in view of the emphasis placed upon cardiac toxicity by the authorities at the present time, the latter approach is now certainly the more employed. In this CD-Rom, the organization of the guide is such that it will enable users to pick and choose exactly how they wish to proceed with the examination and sampling of the heart from both the dog and non-human primate. The CD-Rom itself is extremely user-friendly and after accessing the program proposes the choice between the dog and non-human primate. For both species the organization for the menu is the same. The menu itself is a series of chapters in a logical sequence starting with the gross appearance of the heart (360◦ rotation of the heart with both lateral and vertical aspects, external identification, internal identification, tissue trimming, histology, macroscopic and microscopic pathology, publications and references). Each section of the menu begins with a series of photos enabling the user to access the different subsections. For the 360◦ rotation, the lateral and vertical images are of the complete heart shown in 3 dimensions which can be rotated by the use of arrows. For the internal and external identification the important areas within the heart are identified either by selecting the area presented on a photo-representation of the organ or by selecting the area from a list provided with the image. The following section in the menu concerns tissue sampling and trimming to collect the requisitre areas of the heart which will be subsequently examined histologically. The authors are to be congratulated on the clear step by step series of images with accompanying explanations on how to remove the heart from in situ, and to subsequently orientate the heart and identify the areas for sampling. This is particularly the case for sampling of the SA node which in many cases can be a hit or miss affair. The subsequent selection of areas of left and right ventricle, interventricular septum and the SA node itself followed by the trimming details to give representative samples for blocking –up are easy to follow and well documented with both clear images and simple text instructions. Technical staff who have seen this CD-Rom found it to be of great value as both a possible training tool and reference source. The histology menu of the CD-Rom follows logically on from the tissue trimming part with representative histology slides prepared of the areas shown from trimming, including step sections on how to locate the SA node. It is possible to zoom onto selected areas again simply by selecting the relevant keys. In the macroscopic/microscopic pathology menu, the authors have selected a comprehensive series of cardiac changes for the reader to consult. These cover the majority of pathologies which may be encountered in both the species selected, although it is obviously not a complete atlas of cardiac pathology, which is not the point of this CD-Rom. In an updated version, or even a separate CD-Rom this could obviously be further extended or developed. In the non-human primate, the CD-Rom also includes a section related to morphometric analysis of the heart with an introduction to techniques which can be applied to this organ, the Cavalieri method for measurement of volume is that selected for presentation within the non-human primate menu. The actual format is easy to follow and allows the reader to gain a clear insight into the histological processes involved and the subsequent data capture. The final menu consists of a section of publications and references which are subdivided into general, pharmacology/electrophysiology, spontaneous and experimental pathology and drug or chemically induced pathology. The authors have selected a number of key articles of which many have recently been published which give a good introduction to the subject of cardiac toxicity and pathology in general. There is an added bonus on the CD-Rom with an internet link in the last section on drug induced pathology to a series of presentations given at The Toxicology Forum 30th Annual Summer Meeting in 2004. In my copy of the CD-Rom