During toxicology studies, fasting animals prior to clinical pathology blood collection is believed to reduce variability in some clinical chemistry analytes. However, fasting adds stress to animals that are already stressed from the administration of potentially toxic doses of the test article. The purpose of this study was to assess the impacts of different fasting durations on cynomolgus monkeys' welfare during toxicology studies. To this end, we assessed the cynomolgus monkeys traditional and ancillary clinical pathology endpoints at different fasting times. We showed that most clinical pathology endpoints were largely comparable between different fasting times suggesting that cynomolgus monkeys could be fasted for as little as 4 hours for toxicology studies, as longer fasting times (up to 20 hours) resulted in stress, dehydration, and significant decreases in blood glucose- changes that impacts animal welfare. Shorter fasting times were associated with higher triglycerides variability among individual animals. Therefore, we propose that shorter fasting time (i.e., 4 hours) should be adequate for most toxicology studies except when: (1) parameters that could be affected by non-fasting conditions are important for safety and pharmacodynamic assessments (i.e., glucose and lipids) and (2) fasting would be needed for the bioavailability of an orally administered test article.

Respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory tract infections, and vaccines are needed to treat young children and older adults. One of GSK’s candidate vaccines for RSV contains recombinant RSVPreF3 protein maintained in the prefusion conformation. The differences in immune function of young children and older adults potentially require different vaccine approaches. For young children, anti-RSV immunity can be afforded during the first months of life by vaccinating the pregnant mother during the third trimester with unadjuvanted RSVPreF3, which results in protection of the infant due to the transplacental passage of anti-RSV maternal antibodies. For older adults with a waning immune response, the approach is to adjuvant the RSVPreF3 vaccine with AS01 to elicit a more robust immune response. The local and systemic effects of biweekly intramuscular injections of the RSVPreF3 vaccine (unadjuvanted, adjuvanted with AS01, or coadministered with a diphtheria-tetanus-acellular pertussis vaccine) was tested in a repeated dose toxicity study in rabbits. After three intramuscular doses, the only changes observed were those commonly related to a vaccine-elicited inflammatory reaction. Subsequently, the effects of unadjuvanted RSVPreF3 vaccine on female fertility, embryo-fetal, and postnatal development of offspring were evaluated in rats and rabbits. There were no effects on pregnancy, delivery, lactation, or the pre- and postnatal development of offspring. In conclusion, the RSVPreF3 vaccine was well-tolerated locally and systemically and was not associated with any adverse effects on female reproductive function or on the pre- and postnatal growth and development of offspring.

Detection of test article–related effects and the determination of the adversity of those changes are the primary goals of nonclinical safety assessment studies for drugs and chemicals in development. During these studies, variables that are not of primary interest to investigators may change and influence data interpretation. These variables, often referred to as “nuisance factors,” may influence other groups of data and result in “block or batch effects” that complicate data interpretation. Definitions of the terms “nuisance factors,” “block effects,” and “batch effects,” as they apply to nonclinical safety assessment studies, are reviewed. Multiple case examples of block and batch effects in safety assessment studies are provided, and the challenges these bring to pathology data interpretation are discussed. Methods to mitigate the occurrence of block and batch effects in safety assessment studies, including statistical blocking and utilization of study designs that minimize potential confounding variables, incorporation of adequate randomization, and use of an appropriate number of animals or repeated measurement of specific parameters for increased precision, are reviewed. [Box: see text]

We report the unique pathogenesis and presentation of a rapidly progressive B-cell lymphoma in a 3-year-old female cynomolgus monkey on day 50 of a 13-week toxicity study. Clinical pathology evaluation revealed a marked leukocytosis with bicytopenia. A serum protein electrophoresis was consistent with monoclonal gammopathy. The architecture of the lymph node, spleen, and thymus were variably effaced by neoplastic cells, which also infiltrated other tissues. Immunohistochemistry of the affected tissues confirmed a predominant population of CD20+, CD79a+, CD3-, CD68-, and CD34-neoplastic cells. The full data best support a diagnosis of Stage V lymphoma. Nextgen sequencing and negative prestudy serology results suggested a recent infection by macaque lymphocryptovirus (mLCV) with a unique transcriptional profile comparable with a rarely observed direct LCV infection model. This infection model might be associated with a temporary lack of an LCV antigen-specific cytotoxic T-cell adaptive immune response. Consistent with the established mechanisms of LCV-related lymphoproliferation, MYC and BCL2L11 gene expression were increased and decreased, respectively. While there was no overt immunosuppression, immunophenotyping revealed the index animal had a relatively low NK cell count, which further decreased by >50% on day 24 of the study. In addition to the temporary lack of adaptive immunity, the low NK cell counts were suggestive of an impaired innate immunity to control the virally-transformed cells and the subsequent unchecked lymphoproliferation. To our knowledge, this is the first report of a Stage V lymphoma with a unique pathogenesis in an otherwise immunocompetent cynomolgus monkey.

Supplemental Material, sj-docx-1-tpx-10.1177_0192623320906385 for Scientific Regulatory Policy Committee Points to Consider*: Nuisance Factors, Block Effects, and Batch Effects in Nonclinical Safety Assessment Studies by Albert Eric Schultze, Bindu Bennet, Jessica Caverly Rae, Alan Y. Chiang, Kendall Frazier, Paula Katavolos, LuAnn McKinney, Daniel J. Patrick and Niraj Tripathi in Toxicologic Pathology

The design and execution of toxicology studies supporting vaccine development have some unique considerations relative to those supporting traditional small molecules and biologics. A working group of the Society of Toxicologic Pathology Scientific and Regulatory Policy Committee conducted a review of the scientific, technical, and regulatory considerations for veterinary pathologists and toxicologists related to the design and evaluation of regulatory toxicology studies supporting vaccine clinical trials. Much of the information in this document focuses on the development of prophylactic vaccines for infectious agents. Many of these considerations also apply to therapeutic vaccine development (such as vaccines directed against cancer epitopes); important differences will be identified in various sections as appropriate. The topics addressed in this Points to Consider article include regulatory guidelines for nonclinical vaccine studies, study design (including species selection), technical considerations in dosing and injection site collection, study end point evaluation, and data interpretation. The intent of this publication is to share learnings related to nonclinical studies to support vaccine development to help others as they move into this therapeutic area. [Box: see text].

The interpretation of clinical pathology results from nonclinical safety studies is a fundamental component in hazard identification of new drug candidates. The ever-increasing complexity of nonclinical safety studies and sophistication of modern analytical methods have made the interpretation of clinical pathology information by a highly trained subject matter expert imperative. Certain interpretive techniques are particularly effective in the identification and characterization of clinical pathology effects. The purpose of this manuscript is to provide an overview of contemporary interpretive practices for clinical pathology results and to provide nonbinding recommendations aimed at improving consistency, quality, and overall value of clinical pathology interpretations generated in support of nonclinical toxicology studies.

Pathologists are trained medical professionals with special expertise in diagnostics, research, and pathophysiology. In these roles, pathologists are well qualified and positioned to engage in conversations about animal use replacement, reduction, and refinement (3Rs), thereby championing the guiding principles of the 3Rs. In particular, toxicology or nonclinical safety assessment is an important area where the discipline of toxicologic pathology can have a critical role in adopting 3Rs principles. As such, a working group of the Society of Toxicologic Pathology Scientific and Regulatory Policy Committee was formed to investigate and summarize some of the areas where veterinary pathologists working in the field of toxicology can increase involvement and impact on 3Rs. This "Points to Consider" publication provides an overview of areas within toxicology where the veterinary pathologist's perspective may maximize animal value, including refinement of study design, optimizing sample collection, the development of 3Rs focused regulatory policy, and humane end point determination.[Box: see text].

There is limited direction in the literature or regulatory guidance on determination of adversity for clinical pathology (CP) biomarkers in preclinical safety studies. Toxicologic clinical pathologists representing the American Society for Veterinary Clinical PathologyRegulatory Affairs Committee and Society of Toxicologic PathologyClinical Pathology Interest Group identified principles, overall approach, and unique considerations for assessing adversity in CP data interpretation to provide a consensus opinion. Emphasized is the need for pathophysiologic context and a weight-of-evidence approach. Most CP biomarkers do not have the potential to be adverse in isolation, regardless of magnitude of change. Rather, they quantify or describe the impact of effects, provide adjunct or supportive information regarding a process or pathogenesis, and provide translational biomarkers of effect. Most often, CP changes are part of a constellation of findings that collectively are adverse. Thus, most CP changes must be interpreted in conjunction with other study findings and require contextual and integrative interpretation. Exceptions include critical CP changes without correlates that indicate a health risk in the tested species. Overall, CP changes should not be interpreted in isolation and their adversity is best addressed with an integrated approach.

To test the diagnostic approach described in part 1 of this article, 2 exercises were completed by pathologists from multiple companies/agencies. Pathologist's examination of whole slide image (WSI) heart sections from rats using personal diagnostic approaches (exercise #1) corroborated conclusions from study #1. Using the diagnostic approach described in part 1, these pathologists examined the same WSI heart sections (exercise #2) to determine whether that approach increased consistency of diagnosis of rodent progressive cardiomyopathy (PCM) lesions. In exercise #2, there was improved consistency of categorization of small borderline morphologies and mild lesions, but a decrement in consistency of categorizing minimal lesions. Exercises 1 and 2 suggest the described diagnostic approach is representative of that in use by the majority of toxicologic pathologists across companies/agencies and that application by all may improve diagnostic consistency of PCM/like lesions. Additionally, a criterion of approximately 5% heart section involvement is suggested for separating mild from moderate or greater severity. While evidence is not absolute, until further investigation shows otherwise, microscopic changes resembling PCM, but located in the epicardial and subepicardial region of the right ventricle, may be considered as part of the spectrum of PCM.

Basaglar (R)/Abasaglar (R) (Lilly insulin glargine [LY IGlar]) is a long-acting human insulin analogue drug product granted marketing authorisation as a biosimilar to Lantus (R) (Sanofi insulin glargine [SA IGlar]) by the European Medicines Agency. We assessed the similarity of LY IGlar to the reference drug product, European Union-sourced SA IGlar (EU-SA IGlar), using nonclinical in vitro and in vivo studies. No biologically relevant differences were observed for receptor binding affinity at either the insulin or insulin-like growth factor-1 (IGF-1) receptors, or in assays of functional or de novo lipogenic activity. The mitogenic potential of LY IGlar and EU-SA IGlar was similar when tested in both insulin- and IGF-1 receptor dominant cell systems. Repeated subcutaneous daily dosing of rats for 4 weeks with 0, 0.3, 1.0, or 2.0 mg/kg LY IGlar and EU-SA IGlar produced mortalities and clinical signs consistent with severe hypoglycaemia. Glucodynamic profiles of LY IGlar and EU-SA IGlar in satellite animals showed comparable dose-related hypoglycaemia. Severe hypoglycaemia was associated with axonal degeneration of the sciatic nerve; the incidence and severity were low and did not differ between LY IGlar and EU-SA IGlar. These results demonstrated no biologically relevant differences in toxicity between LY IGlar and EU-SA IGlar. (C) 2017 Elsevier Inc. All rights reserved.

Clinical pathology testing is routinely performed in target animal safety studies in order to identify potential toxicity associated with administration of an investigational veterinary pharmaceutical product. Regulatory and other testing guidelines that address such studies provide recommendations for clinical pathology testing but occasionally contain outdated analytes and do not take into account interspecies physiologic differences that affect the practical selection of appropriate clinical pathology tests. Additionally, strong emphasis is often placed on statistical analysis and use of reference intervals for interpretation of test article-related clinical pathology changes, with limited attention given to the critical scientific review of clinically, toxicologically, or biologically relevant changes. The purpose of this communication from the Regulatory Affairs Committee of the American Society for Veterinary Clinical Pathology is to provide current recommendations for clinical pathology testing and data interpretation in target animal safety studies and thereby enhance the value of clinical pathology testing in these studies.

Clinical pathology endpoints are evaluated during the course of toxicity studies to monitor structural and functional changes in organs and tissues in response to administration or withdrawal of a test item. A variety of assays can be performed in a clinical pathology laboratory with body fluids using either clinical pathology analyzers or other instruments. Information from the basic assays in clinical pathology, including hematology, clinical chemistry, urinalysis and coagulation are used with other safety assessment endpoints to evaluate immunotoxicity. Non-routine clinical pathology variables, such as acute phase proteins, cytokines, complement, hormones, autoantibodies, and bone marrow cytology are also included in preclinical toxicity studies as supplementary assays to ensure adequate interpretation of test item-related effects on the immune system. This chapter will describe the clinical pathology assays that are most useful to assess immunotoxicity.

The Society of Toxicologic Pathology formed a working group in collaboration with the American Society for Veterinary Clinical Pathology to provide recommendations for the appropriate inclusion of clinical pathology evaluation in recovery arms of nonclinical toxicity studies but not on when to perform recovery studies. Evaluation of the recovery of clinical pathology findings is not required routinely but provides useful information on risk assessment in nonclinical toxicity studies and is recommended when the ability of the organ to recover is uncertain. The study design generally requires inclusion of concurrent controls to separate procedure-related changes from test article-related changes, but return of clinical pathology values toward baseline may be sufficient in some cases. Evaluation of either a select or full panel of standard hematology, coagulation, and serum and urine chemistry biomarkers can be scientifically justified. It is also acceptable to redesignate dosing phase animals to the recovery phase or vice versa to optimize data interpretation. Assessment of delayed toxicity during the recovery phase is not required but may be appropriate in development programs with unique concerns. Evaluation of the recovery of clinical pathology data for vaccine development is required and, for efficacy markers, is recommended if it furthers pharmacologic understanding.

The objectives of this session were to explore causes of variability in clinical pathology data due to preanalytical and analytical variables as well as study design and other procedures that occur in toxicity testing studies. The presenters highlighted challenges associated with such variability in differentiating test article-related effects from the effects of experimental procedures and its impact on overall data interpretation. These presentations focused on preanalytical and analytical variables and study design-related factors and their influence on clinical pathology data, and the importance of various factors that influence data interpretation including statistical analysis and reference intervals. Overall, these presentations touched upon potential effect of many variables on clinical pathology parameters, including animal physiology, sample collection process, specimen handling and analysis, study design, and some discussion points on how to manage those variables to ensure accurate interpretation of clinical pathology data in toxicity studies. This article is a brief synopsis of presentations given in a session entitled "Deciphering Sources of Variability in Clinical Pathology-It's Not Just about the Numbers'' that occurred at the 35th Annual Symposium of the Society of Toxicologic Pathology in San Diego, California.

A number of factors related to study design have the potential to impact clinical pathology test results during the conduct of nonclinical safety studies. A thorough understanding of these factors is paramount in drawing accurate conclusions from clinical pathology data generated during such studies, particularly when attempting to make the distinction between test article and nontest article–related effects. Study design and conduct variables with potential to impact clinical pathology data discussed in this overview include those related to species and test system, animal age, animal care and husbandry practices, fasting, acclimatization periods, effects of transportation and stressors, route of administration, effects of in-life and surgical procedures, influence of study length, timing of blood collections, impact of vehicle/formulation composition, and some general concepts related to drug class. The material presented here is a summary based on information presented at the 35th Annual Symposium of the Society of Toxicologic Pathology (June 2016), during Symposium Session 2 titled “Deciphering Sources of Variability in Clinical Pathology—It’s Not Just about the Numbers.”

In preclinical safety assessment, blood volume requirements for various endpoints pose a major challenge. The goal of this working group was to review current practices for clinical pathology (CP) testing in preclinical toxicologic studies, and to discuss advantages and disadvantages of methods for reducing blood volume requirements. An industry-wide survey was conducted to gather information on CP instrumentation and blood collection practices for hematology, clinical biochemistry, and coagulation evaluation in laboratory animals involved in preclinical studies. Based on the survey results and collective experience of the authors, the working group proposes the following "points to consider" for CP testing: (1) For most commercial analyzers, 0.5 mL and 0.8 mL of whole blood are sufficient for hematology and biochemistry evaluation, respectively. (2) Small analyzers with low volume requirements and low throughput have limited utility in preclinical studies. (3) Sample pooling or dilution is inappropriate for many CP methods. (4) Appropriate collection sites should be determined based on blood volume requirements and technical expertise. (5) Microsampling does not provide sufficient volume given current analyzer and quality assurance requirements. (6) Study design considerations include: the use of older/larger animals (rodents), collection of CP samples before toxicokinetic samples, use of separate subsets of mice for hematology and clinical biochemistry testing, use of a priority list for clinical biochemistry, and when possible, eliminating coagulation testing.

BACKGROUND Urinalysis data in preclinical toxicology studies can be influenced by preanalytic and analytic factors which have the potential to confound interpretation. There is a paucity of information regarding positive reagent strip urinary blood reactions in healthy nonhuman primates (NHP) and Beagle dogs used in preclinical toxicology studies. OBJECTIVES The objectives were (1) to establish historical control data for reagent strip urinary blood reactions in healthy NHP and Beagle dogs, (2) to determine the incidence of positive urinary blood reactions during predose and dosing phases, and (3) to determine if collection practice was a relevant parameter. METHODS Historical control data from 2 institutions in the biopharmaceutical industry were retrospectively analyzed for reagent strip urinary blood reactions in healthy NHP and Beagles. The incidence of positive results between the 2 institutions with different urine collection practices and between males and females was compared. RESULTS The incidence of positive urinary blood reactions in NHP was comparable between institutions (≤ 14% in males; ≤ 33% in females), while the incidence of positive urinary blood reactions in Beagles was more variable (≤ 77% in males; ≤ 69% in females), and higher in females during the dosing phase. CONCLUSIONS Positive urinary blood results that could potentially be misinterpreted as toxicologically relevant were identified in healthy NHP and Beagles during predose and dosing phases. Different incidences of positive results between the 2 institutions were likely related to collection practices. Strategies to reduce feces and food contamination of collected urine samples should help minimize false-positive urinary blood reactions.