Incidental Gliosis in the Central Nervous System of Control Nonhuman Primates and Rats.

Gliosis, including microgliosis and astrocytosis, can be challenging to interpret in nonclinical studies. Incidences of glial foci in brains and spinal cords of control rats and nonhuman primates (NHPs) were reviewed in the historical control databases from two contract research organizations, including one specializing in neuropathology. In the brain, minimal to mild (grades 1-2) microgliosis was the most common diagnosis, especially in NHPs, although occasional moderate or marked microgliosis (grades 3 and 4) was encountered in both species. Microgliosis was more common in the cerebral cortex, cerebellum, and medulla oblongata in both species and was frequent in the white matter (brain), thalamus, and basal nuclei of NHPs. Gliosis ("not otherwise specified") of minimal severity was diagnosed in similar brain sub-sites for both species and was more common in NHPs compared with rats. Astrocytosis was most prominent in the cerebellum (molecular layer) of NHPs but was otherwise uncommon. In the spinal cord, microgliosis was most common in the lateral white matter tracts in rats and NHPs, and in the dorsal white matter tracts in NHPs. These data indicate that low-grade spontaneous glial responses occur with some frequency in control animals of two common nonclinical species.

The road to virtual control groups and the importance of proper body-weight selection.

Virtual control groups (VCGs) created from historical control data (HCD) can reduce the number of concurrent control group animals needed in regulatory toxicity studies by up to 25%. This study investigates the performance of VCGs on statistical outcomes of body weight development between treatment and control groups in legacy studies. The objective is to reproduce the statistical outcomes of 28-day sub-chronic studies (legacy studies) after replacing the concurrent control group with virtual ones. In rodent toxicity studies initial body weight is used as surrogate for the age of animals. For the assessment of VCG-sampling methods three different approaches are explored: (i) sampling VCGs from the entire HCD ignoring initial body weight information of the legacy study, (ii) sampling from HCD matching the legacy study's initial body weights, and (iii) sampling from HCD with assigned statistical weights derived from legacy study initial body weight information. It is shown that the ability to reproduce statistical outcomes by virtual controls is mainly determined by the congruence between the legacy study and the HCD weight distribution: regardless of the chosen approach, the ability to reproduce statistical outcomes was well for VCGs when the legacy study's initial-body-weight distribution was similar to the HCD's. When the initial body weight range of the legacy study was at the extreme ends of the HCD's distribution, the weighted-sampling approach was superior. This article highlights the importance of proper HCD-matching by the legacy study's initial body weight and discusses required conditions to accurately reproduce body weight development.

Animal control data from past studies performed in a standardized manner can be used to create virtual control groups (VCGs) to use in new studies instead of control animals. This approach can reduce the number of study animals by up to 25%. This study assesses the performance of VCGs selected by body weight in rat studies. The objective was to reproduce the original study results as closely as possible after replacing the original control group values with VCGs from a pool of historical control values. Several methods for selecting control animal data to create VCGs were compared. Among these, assigning statistical weights to the sampling pool yielded the best performance. Ideally the body weight distributions on day 1 of the study should be similar between the VCG and the original study animals. This article shows that proper selection VCGs can yield reliable study data with fewer animals.

Historical Control Data of Spontaneous Pathological Findings in C57BL/6J Mice Used in 18-Month Dietary Carcinogenicity Assays.

A retrospective analysis in C57BL6/J mice used in dietary carcinogenicity studies was performed to determine the survival rate, causes of death and incidences of spontaneous non-tumoral and tumoral findings. Data were collected from 1600 mice from control dose groups of sixteen 18-month carcinogenicity assays performed between 2003 and 2021 at the same test facility with similar environmental conditions and experimental procedures. The survival rate was high in both sexes (81%-85%) and the causes of humane euthanasia or death were mainly non-tumoral (chronic ulcerative dermatitis, atrial thrombosis). Benign tumors were more frequent than malignant tumors and females were more affected than males. Pituitary gland adenoma in females, lymphoma, bronchioloalveolar adenoma, and harderian gland adenoma in both sexes were the most common tumors. Systemic amyloidosis, the most frequent non-tumoral lesion, was observed variably across studies without sex predilection. The analysis by cohort (3 time periods of 6 years) showed a tendency toward higher incidences of lymphoma and pituitary gland adenoma and lower incidences of amyloidosis over time. The results presented here provide for the first time a robust set of control historical data in untreated C57BL/6J mice kept for 18 months contributing to build in depth knowledge of this animal model.

Replacing concurrent controls with virtual control groups in rat toxicity studies.

Virtual control groups (VCGs) in nonclinical toxicity represent the concept of using appropriate historical control data for replacing concurrent control group animals. Historical control data collected from standardized studies can serve as base for constructing VCGs and legacy study reports can be used as a benchmark to evaluate the VCG performance. Replacing concurrent controls of legacy studies with VCGs should ideally reproduce the results of these studies. Based on three four-week rat oral toxicity legacy studies with varying degrees of toxicity findings we developed a concept to evaluate VCG performance on different levels: the ability of VCGs to (i) reproduce statistically significant deviations from the concurrent control, (ii) reproduce test substance-related effects, and (iii) reproduce the conclusion of the toxicity study in terms of threshold dose, target organs, toxicological biomarkers (clinical pathology) and reversibility. Although VCGs have shown a low to moderate ability to reproduce statistical results, the general study conclusions remained unchanged. Our results provide a first indication that carefully selected historical control data can be used to replace concurrent control without impairing the general study conclusion. Additionally, the developed procedures and workflows lay the foundation for the future validation of virtual controls for a use in regulatory toxicology.

In vivo alkaline comet assay: Statistical considerations on historical negative and positive control data.

The alkaline comet assay is frequently used as in vivo follow-up test within different regulatory environments to characterize the DNA-damaging potential of different test items. The corresponding OECD Test guideline 489 highlights the importance of statistical analyses and historical control data (HCD) but does not provide detailed procedures. Therefore, the working group "Statistics" of the German-speaking Society for Environmental Mutation Research (GUM) collected HCD from five laboratories and >200 comet assay studies and performed several statistical analyses. Key results included that (I) observed large inter-laboratory effects argue against the use of absolute quality thresholds, (II) > 50% zero values on a slide are considered problematic, due to their influence on slide or animal summary statistics, (III) the type of summarizing measure for single-cell data (e.g., median, arithmetic and geometric mean) may lead to extreme differences in resulting animal tail intensities and study outcome in the HCD. These summarizing values increase the reliability of analysis results by better meeting statistical model assumptions, but at the cost of information loss. Furthermore, the relation between negative and positive control groups in the data set was always satisfactorily (or sufficiently) based on ratio, difference and quantile analyses.

Decoupling power and type I error rate considerations when incorporating historical control data using a test-then-pool approach.

To accelerate a randomized controlled trial, historical control data may be used after ensuring little heterogeneity between the historical and current trials. The test-then-pool approach is a simple frequentist borrowing method that assesses the similarity between historical and current control data using a two-sided test. A limitation of the conventional test-then-pool method is the inability to control the type I error rate and power for the primary hypothesis separately and flexibly for heterogeneity between trials. This is because the two-sided test focuses on the absolute value of the mean difference between the historical and current controls. In this paper, we propose a new test-then-pool method that splits the two-sided hypothesis of the conventional method into two one-sided hypotheses. Testing each one-sided hypothesis with different significance levels allows for the separate control of the type I error rate and power for heterogeneity between trials. We also propose a significance-level selection approach based on the maximum type I error rate and the minimum power. The proposed method prevented a decrease in power even when there was heterogeneity between trials while controlling type I error at a maximum tolerable type I error rate larger than the targeted type I error rate. The application of depression trial data and hypothetical trial data further supported the usefulness of the proposed method.

Toward implementing virtual control groups in nonclinical safety studies.

Historical data from control groups in animal toxicity studies is currently mainly used for comparative purposes to assess validity and robustness of study results. Due to the highly controlled environment in which the studies are performed and the homogeneity of the animal collectives it has been proposed to use the historical data for building so-called virtual control groups, which could replace partly or entirely the concurrent control. This would constitute a substantial contribution to the reduction of animal use in safety studies. Before the concept can be implemented, the prerequisites regarding data collection, curation and statistical evaluation together with a validation strategy need to be identified to avoid any impairment of the study outcome and subsequent consequences for human risk assessment. To further assess and develop the concept of virtual control groups the transatlantic think tank for toxicology (t4) sponsored a workshop with stakeholders from the pharmaceutical and chemical industry, academia, FDA, pharmaceutical, contract research organizations (CROs), and non-governmental organizations in Washington, which took place in March 2023. This report summarizes the current efforts of a European initiative to share, collect and curate animal control data in a centralized database and the first approaches to identify optimal matching criteria between virtual controls and the treatment arms of a study as well as first reflections about strategies for a qualification procedure and potential pitfalls of the concept.

Animal safety studies are usually performed with three groups of animals where increasing amounts of the test chemical are given to the animals and one control group where the animals do not receive the test chemical. The design of such studies, the characteristics of the animals, and the measured parameters are often very similar from study to study. Therefore, it has been suggested that measurement data from the control groups could be reused from study to study to lower the total number of animals per study. This could reduce animal use by up to 25% for such standardized studies. A workshop was held to discuss the pros and cons of such a concept and what would have to be done to implement it without threatening the reliability of the study outcome or the resulting human risk assessment.

A Retrospective Comparison of Electrocardiographic Parameters in Ketamine and Tiletamine-Zolazepam Anesthetized Indian Rhesus Monkeys (Macaca mulatta).

Electrocardiographic evaluation is performed in rhesus monkeys to establish the cardiovascular safety of candidate molecules before progressing to clinical trials. These animals are usually immobilized chemically by ketamine (KTM) and tiletamine-zolazepam (TZ) to obtain a steady-state heart rate and to ensure adequate human safety. The present study aimed to evaluate the effect of these anesthetic regimens on different electrocardiographic parameters. Statistically significant lower HR and higher P-wave duration, RR, QRS, and QT intervals were observed in the KTM-anesthetized group in comparison to TZ-anesthetized animals. No significant changes were noticed in the PR interval and p-wave amplitude. Sex-based significance amongst these parameters was observed in male and female animals of TZ- and KTM-anesthetized groups. Regression analysis of four QTc formulas in TZ-anesthetized rhesus monkeys revealed that QTcNAK (Nakayama) better corrected the QT interval than QTcHAS (Hassimoto), QTcBZT (Bazett), and QTcFRD (Fridericia) formulas. QTcNAK exhibited the least correlation with the RR interval (slope closest to zero and r = .01) and displayed no statistical significance between male and female animals. These data will prove useful in the selection of anesthetic regimens for chemical restraint of rhesus monkeys in nonclinical safety evaluation studies.

Historical control histopathology data from amphibian metamorphosis assays and fathead minnow fish short term reproductive assays: A tool for data interpretation.

The Amphibian Metamorphosis Assay (AMA) is used to determine if a tested chemical has potential to impact the hypothalamic-pituitary-thyroid (HPT) axis of Xenopus laevis tadpoles, while the Fish Short Term Reproduction Assay (FSTRA) assesses potential effects on the hypothalamic-pituitary-gonadal (HPG) axis of fish such as the fathead minnow (Pimephales promelas). Several global regulatory programs routinely require these internationally validated tests be performed to determine the potential endocrine activity of chemicals. As such, they are conducted in accordance with standardized protocols and test criteria, which were originally developed more than a decade ago. Sizeable numbers of AMA and FSTRA studies have since been carried out, which allows for the mining of extensive historical control data (HCD). Such data are useful for investigating the existence of outlier results and aberrant control groups, identifying potential confounding variables, providing context for rare diagnoses, discriminating target from non-target effects, and for refining current testing paradigms. The present paper provides histopathology HCD from 55 AMA studies and 45 fathead minnow FSTRA studies, so that these data may become publicly available and thus aid in the interpretation of future study outcomes. Histopathology is a key endpoint in these assays, in which it is considered to be one of the most sensitive indicators of endocrine perturbation. In the current review, granular explorations of HCD data were used to identify background lesions, to assess the utility of particular diagnostic findings for distinguishing endocrine from non-endocrine effects, and to help determine if specific improvements to established regulatory guidance may be warranted. Knowledge gleaned from this investigation, supplemented by information from other recent studies, provided further context for the interpretation of AMA and FSTRA histopathology results. We recommend HCDs for the AMA and FSTRA be maintained to support the interpretation of study results.

The calculation of historical control limits in toxicology: Do's, don'ts and open issues from a statistical perspective.

For reporting toxicology studies, the presentation of historical control data and the validation of the concurrent control group with respect to historical control limits have become requirements. However, many regulatory guidelines fail to define how such limits should be calculated and what kind of target value(s) they should cover. Hence, this manuscript is aimed to give a brief review on the methods for the calculation of historical control limits that are in use as well as on their theoretical background. Furthermore, this manuscript is aimed to identify open issues for the use of historical control limits that need to be discussed by the community. It seems that, even after 40 years of discussion, more issues remain open than solved, both, with regard to the available methodology as well as its implementation in user-friendly software. Since several of these topics equally apply to several research fields, this manuscript is addressed to all relevant stakeholders who deal with historical control data obtained from toxicological studies, regardless of their background or field of research.

Can Historical Control Group Data Be Used to Replace Concurrent Controls in Animal Studies?

The availability of large amounts of high-quality control data from tightly controlled regulated animal safety data has created the idea to re-use these data beyond its classical applications of quality control, identification of treatment-related effects and assessing effect-size relevance for building virtual control groups (VCGs). While the ethical and cost-saving aspects of such a concept are immediately evident, the potential challenges need to be carefully considered to avoid any effect which could lower the sensitivity of an animal study to detect adverse events, safety thresholds, target organs, or biomarkers. In our brief communication, we summarize the current discussion regarding VCGs and propose a path forward how the replacement of concurrent control with VCGs resulting from historical data could be systematically assessed and to come to conclusions regarding the scientific value of the concept.

Characterization of patterns and variability in the dynamics of outdoor aquatic mesocosms: exploring the capabilities and challenges in data supporting aquatic system models.

Aquatic mesocosms are complex test systems used within regulatory risk assessment of plant protection products. These model ecosystems allow researchers to capture interactions of multiple species under realistic environmental conditions. They enable assessment of direct and indirect effects of stressors at all trophic levels (i.e., from primary producers to secondary consumers) and impacts on ecosystem functions. Due to the limited ability to test the multitude of potential exposure scenarios, cross-linking aquatic mesocosm studies with virtual mesocosms, i.e., aquatic system models (ASMs), can serve to meet the demand for more environmental realism and ecological relevance in risk assessment. In this study, full control data sets from seven aquatic mesocosm studies conducted at a single test facility under GLP were analysed graphically and using descriptive statistics. Thereby, not only a comprehensive data base but also an insight into the species present, their dynamics over time, and variability in unchallenged mesocosms was observed. While consistency in dynamics could be discerned for physical and chemical parameters, variability was evident for several biological endpoints. This variability points to amplification of small differences over time as well as to stochastic processes. The outline of existing gaps and uncertainties in data leads to the estimation of what can be expected to be captured and predicted by ASMs.

Hurdles and signposts on the road to virtual control groups-A case study illustrating the influence of anesthesia protocols on electrolyte levels in rats.

Introduction: Virtual Control Groups (VCGs) represent the concept of using historical control data from legacy animal studies to replace concurrent control group (CCG) animals. Based on the data curation and sharing activities of the Innovative Medicine Initiatives project eTRANSAFE (enhancing TRANSlational SAFEty Assessment through Integrative Knowledge Management) the ViCoG working group was established with the objectives of i) collecting suitable historical control data sets from preclinical toxicity studies, ii) evaluating statistical methodologies for building adequate and regulatory acceptable VCGs from historical control data, and iii) sharing those control-group data across multiple pharmaceutical companies. During the qualification process of VCGs a particular focus was put on the identification of hidden confounders in the data sets, which might impair the adequate matching of VCGs with the CCG. Methods: During our analyses we identified such a hidden confounder, namely, the choice of the anesthetic procedure used in animal experiments before blood withdrawal. Anesthesia using CO2 may elevate the levels of some electrolytes such as calcium in blood, while the use of isoflurane is known to lower these values. Identification of such hidden confounders is particularly important if the underlying experimental information (e.g., on the anesthetic procedure) is not routinely recorded in the standard raw data files, such as SEND (Standard for Exchange of Non-clinical Data). We therefore analyzed how the replacement of CCGs with VCGs would affect the reproducibility of treatment-related findings regarding electrolyte values (potassium, calcium, sodium, and phosphate). The analyses were performed using a legacy rat systemic toxicity study consisting of a control and three treatment groups conducted according to pertinent OECD guidelines. In the report of this study treatment-related hypercalcemia was reported. The rats in this study were anesthetized with isoflurane. Results: Replacing the CCGs with VCGs derived from studies comprising both anesthetics resulted in a shift of control electrolyte parameters. Instead of the originally reported hypercalcemia the use of VCG led to fallacious conclusions of no observed effect or hypocalcemia. Discussion: Our study highlights the importance of a rigorous statistical analysis including the detection and elimination of hidden confounders prior to the implementation of the VCG concept.

Assessing the quality and making appropriate use of historical negative control data: A report of the International Workshop on Genotoxicity Testing (IWGT).

Historical negative control data (HCD) have played an increasingly important role in interpreting the results of genotoxicity tests. In particular, Organisation for Economic Co-operation and Development (OECD) genetic toxicology test guidelines recommend comparing responses produced by exposure to test substances with the distribution of HCD as one of three criteria for evaluating and interpreting study results (referred to herein as "Criterion C"). Because of the potential for inconsistency in how HCD are acquired, maintained, described, and used to interpret genotoxicity testing results, a workgroup of the International Workshops for Genotoxicity Testing was convened to provide recommendations on this crucial topic. The workgroup used example data sets from four in vivo tests, the Pig-a gene mutation assay, the erythrocyte-based micronucleus test, the transgenic rodent gene mutation assay, and the in vivo alkaline comet assay to illustrate how the quality of HCD can be evaluated. In addition, recommendations are offered on appropriate methods for evaluating HCD distributions. Recommendations of the workgroup are: When concurrent negative control data fulfill study acceptability criteria, they represent the most important comparator for judging whether a particular test substance induced a genotoxic effect. HCD can provide useful context for interpreting study results, but this requires supporting evidence that (i) HCD were generated appropriately, and (ii) their quality has been assessed and deemed sufficiently high for this purpose. HCD should be visualized before any study comparisons take place; graph(s) that show the degree to which HCD are stable over time are particularly useful. Qualitative and semi-quantitative assessments of HCD should also be supplemented with quantitative evaluations. Key factors in the assessment of HCD include: (i) the stability of HCD over time, and (ii) the degree to which inter-study variation explains the total variability observed. When animal-to-animal variation is the predominant source of variability, the relationship between responses in the study and an HCD-derived interval or upper bounds value (i.e., OECD Criterion C) can be used with a strong degree of confidence in contextualizing a particular study's results. When inter-study variation is the major source of variability, comparisons between study data and the HCD bounds are less useful, and consequentially, less emphasis should be placed on using HCD to contextualize a particular study's results. The workgroup findings add additional support for the use of HCD for data interpretation; but relative to most current OECD test guidelines, we recommend a more flexible application that takes into consideration HCD quality. The workgroup considered only commonly used in vivo tests, but it anticipates that the same principles will apply to other genotoxicity tests, including many in vitro tests.

Historical Control Background Incidence of Spontaneous Neoplastic Lesions of Sprague-Dawley Rats in 104-Week Toxicity Studies.

Data collected from approximately 1800 male and 1800 female Sprague-Dawley (SD) rats used in 104-week carcinogenicity studies were archived in a historical control database at Labcorp Early Development, Inc, and the neoplastic microscopic observation data from these rats were retrospectively evaluated. Historical control data can provide useful information on the range and incidence of spontaneously occurring background neoplasms in the species and strain of the test animal used in different types of toxicity studies, including studies of differing lengths, delivery of test article, and test animal. Some of the most common malignant findings noted included fibrosarcoma of skin/subcutis and thyroid C-cell carcinoma in males (2.1% each) while mammary gland carcinoma and pituitary carcinoma (25% and 2.6%) were most common in females. Pituitary adenoma of pars distalis was found to be the most prevalent benign neoplasm in both males and females (56.4% and 77.1%). Fibroadenoma of mammary gland (35.6%) and thyroid C-cell adenoma (8.5%) were the second and third most common benign tumors in female SD rats. In males, the thyroid C-cell adenoma (10.9%) and benign pheochromocytoma (8.9%) were the second and third most common tumors.

A Comparison of Historical Control Data From Cynomolgus Macaques (Macaca Fascicularis) of Chinese, Cambodian, and Vietnamese Origin.

Cynomolgus macaques, the most commonly utilized nonhuman primate in nonclinical toxicology studies, are acquired from purpose-bred colonies across various geographic locations, including China, Cambodia, and Vietnam. Importation challenges and limited availability have restricted animals suitable for inclusion in nonclinical studies. The coronavirus disease 2019 (COVID-19) outbreak further stressed supply chains, reducing the ability to source animals from a singular location to complete a drug development program. These challenges raised concerns of increased variability in study endpoints due to heterogeneity of animals and that this could subsequently impact historical control data and toxicology study interpretation. To investigate the impact of Chinese, Vietnamese, or Cambodian geographic origin on standard nonclinical toxicology study endpoints, historical control data from studies conducted at a single facility from 2005 to 2020 were compiled and evaluated for the following: clinical observations, body weight, ophthalmoscopic examinations, and clinical and anatomic pathology data. Study populations consisted of 2- to 5-year-old cynomolgus macaques sourced from China (n = 750 males/741 females), Cambodia (n = 282 males/271 females), and Vietnam (n = 122 males/120 females). Interpretation of the various data demonstrated no notable differences in standard toxicology study endpoints or background findings among cynomolgus macaques originating from China, Cambodia, or Vietnam.

Immunohistochemical Characterization of Proliferative Lesions in the Thymus of Aging CD-1 Mice From Two Studies on the RITA Database, With Special Reference to the Perivascular Space.

Thymic lymphoid hyperplasia is a common age-related finding, which occurs particularly in female CD-1 mice. The main differential diagnoses are malignant lymphoma and thymoma. A systematic investigation of control groups from two carcinogenicity studies was performed including measurements of thymic size, and the immunohistochemistry (IHC) markers pan-Cytokeratin (pan-CK) for thymic epithelial cells; CD3 and CD45R/B220 for T and B lymphocytes, respectively; CD31 for endothelial cells; and F4/80 for macrophages. Thymoma can be differentiated by increased numbers of proliferating epithelial cells demonstrated by pan-CK IHC staining. Differentiation between lymphoid hyperplasia and lymphoma is more challenging as a mixture of B and T lymphocytes can be present in both findings. The present investigation showed that the thymic perivascular space is the compartment where the increased numbers of lymphocytes in hyperplasia are localized and not the medulla, as previously thought. The lymphoepithelial compartment is atrophic to the same extent in thymi diagnosed with age-related involution or lymphoid hyperplasia. Both diagnoses are thus related to variations in lymphoid cellularity of the nonepithelial perivascular space, which is continuous with the perithymic tissue. Likewise, lymphomas have a predilection to colonize the perivascular space and to spare the lymphoepithelial compartment.

Using historical control data in bioassays for regulatory toxicology.

Historical control data (HCD) consist of pooled control group responses from bioassays. These data must be collected and are often used or reported in regulatory toxicology studies for multiple purposes: as quality assurance for the test system, to help identify toxicological effects and their effect-size relevance and to address the statistical multiple comparison problem. The current manuscript reviews the various classical and potential new approaches for using HCD. Issues in current practice are identified and recommendations for improved use and discussion are provided. Furthermore, stakeholders are invited to discuss whether it is necessary to consider uncertainty when using HCD formally and statistically in toxicological discussions and whether binary inclusion/exclusion criteria for HCD should be revised to a tiered information contribution to assessments. Overall, the critical value of HCD in toxicological bioassays is highlighted when used in a weight-of-evidence assessment.

An examination of historical control histopathology metadata from 51 Amphibian Metamorphosis Assays.

The Amphibian Metamorphosis Assay (AMA) is used to identify substances that potentially interfere with the normal function of the hypothalamic-pituitary-thyroid (HPT) axis. Although numerous AMA studies have been performed since the establishment of this assay a decade earlier, a comprehensive, large-scale examination of histopathology data obtained from control larvae has not been performed. The current investigation reviewed 51 AMA experiments conducted at 7 different laboratories in Europe and North America. Dilution water control and/or solvent control specimens from each study (1,335 animals total) had been evaluated microscopically by one of eight anatomic pathologists. In order of descending frequency, the most common findings in prometamorphic Xenopus laevis controls were the core criteria of follicular cell (FC) hypertrophy, FC hyperplasia, thyroid hypertrophy, and thyroid atrophy, respectively. Less frequently recorded were non-core and ad hoc diagnoses, the toxicological relevance and utility of which were in some cases uncertain. As anticipated, the prevalence of FC hypertrophy and FC hyperplasia diagnoses were at least partially dependent on the Nieuwkoop and Faber (NF) stage at sacrifice. The recorded frequencies of each of the four core diagnoses also differed according to pathologist, which suggests that pathologist diagnostic interpretation is a potential source of variability across AMA study outcomes. Based on the current examination of the AMA historical data, and further hands-on experience with this assay, diagnostic approaches to evaluating the histopathology endpoint are discussed, and several recommendations are proposed for the refinement of core diagnostic criteria assessment.

Multisite Analysis of Lesions in the Respiratory Tract of the Rat and Nonhuman Primate (Cynomolgus Monkey) Exposed to Air, Vehicle, and Inhaled Small Molecule Compounds.

This paper presents a review of the nature, range, and incidences of background pathology findings in the respiratory tract of cynomolgus monkeys and rats. Data were collected from 81 inhalation studies and 133 non-inhalation studies evaluated at 3 geographically distinct contract research organization facilities. The inhalation studies were comprised of 44 different small molecule pharmaceuticals or chemicals which were also analyzed in order to understand the patterns of induced changes within the respiratory tract. The lung was the most frequently affected organ in both species, with increased alveolar macrophages being the most common background and test article-related finding. In the upper respiratory tract (URT), inflammatory cell infiltrates were the most common background findings in the nasal cavity in monkeys. Induced URT findings were more frequent in rats than monkeys, with squamous metaplasia in the larynx, and goblet cell hyperplasia in the nasal cavity being the most common. Overall, the data revealed a limited pattern of response to inhaled molecules in the respiratory tract, with background and test article-related findings often occurring in the same regions. It is hoped that these data will assist in the interpretation of findings in the respiratory tract induced by novel inhaled small molecule entities.