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Nonclinical toxicology studies that are required to support human clinical trials of new drug candidates are generally conducted in a rodent and a non-rodent species. These studies typically contain a vehicle control group and low, intermediate, and high dose test article groups. In addition, a dosing-free recovery phase is sometimes included to determine reversibility of potential toxicities observed during the dosing phase and may include additional animals in the vehicle control and one or more dose groups. Typically, reversibility is determined by comparing the test article–related changes in the dosing phase animals to concurrent recovery phase animals at the same dose level. Therefore, for interpretation of reversibility, it is not always essential to euthanize the recovery vehicle control animals. In the absence of recovery vehicle control tissues, the pathologist’s experience, historical control database, digital or glass slide repositories, or literature can be used to interpret the findings in the context of background pathology of the species/strain/age. Therefore, in most studies, the default approach could be not to euthanize recovery vehicle control animals. This article provides opinions on scenarios that may or may not necessitate euthanasia of recovery phase vehicle control animals in nonclinical toxicology studies involving dogs and nonhuman primates.

Sinusoidal obstruction syndrome (SOS) is a unique form of liver injury that occurs after exposure to chemotherapeutic drugs and toxins. The diagnosis of SOS in humans remains a challenge as the clinical criteria have low specificity and there are no reliable noninvasive biomarkers. The mechanism of injury is believed to be damage to liver endothelial cells, primarily sinusoidal endothelial cells (SECs), which leads to sinusoidal dilation, central venous fibrosis, and/or nodular regeneration. Nonclinical data suggest that this uncommon liver toxicity can be recapitulated in cynomolgus monkeys, and it is critical that pathologists are familiar with its characteristic clinicopathologic features. Elevations in liver enzymes, in particular aspartate aminotransferase, associated with isolated thrombocytopenia, should raise the suspicion of SEC injury for specific drug classes. Characterization of liver microscopic findings in monkeys benefits from the use of appropriate stains, such as reticulin stain, and VEGFR2 and CD34 immunohistochemical (IHC) stains. CD41 IHC demonstrates platelet accumulation in injured sinusoids, the likely cause of thrombocytopenia commonly reported in SOS. In conclusion, this report provides a comprehensive characterization of the pathology findings of drug-induced SOS in monkeys with the objectives of ensuring appropriate nonclinical recognition of the liability and informing clinical development strategy and monitoring.

Differences were examined between male and female Sprague-Dawley rats in basal levels of a wide range of urinary biomarkers, including 7 recently qualified biomarkers. The data were generated from urine samples collected on 3 occasions from untreated rats included in a study of the effect of gentamicin nephrotoxicity on urinary renal biomarkers, reported in a companion article in this journal (Gautier et al. 2014). The performance of multiple assays (9 singleplex assays and 2 multiplex platforms from Rules Based Medicine [RBM] and Meso Scale Discovery [MSD]) was evaluated, and normal ranges and variability estimates were derived. While variability was generally greater on the RBM platform than other assays, the more striking difference in the results from different assays was in magnitude. Where differences were observed between assays for an individual biomarker, they were seen in both sexes and consistent across samples collected at different time points. Differences of up to 15-fold were observed for some biomarker values between assays indicating that results generated using different assays should not be compared. For 8 biomarkers, there was compelling evidence for a sex difference. Baseline values in males were significantly higher than in females for total protein, β2-microglobulin, clusterin, cystatin-C, glutathione-S-transferase (GST-α), tissue inhibitor of metalloproteinases (TIMP-1), and vascular endothelial growth factor (VEGF); female values were significantly higher than that of males for albumin. The largest sex differences (male greater than female by 2- to 11-fold) were seen with β2-microglobulin, GST-α, and TIMP-1. These data add substantially to the limited body of knowledge in this area and provide a useful framework for evaluation of the potential relevance of sex differences in the diagnostic performance of these biomarkers.

Kidney is a major target organ in preclinical studies. In recent years, intense research has been undertaken to characterize novel renal toxicity biomarkers. In this context, we studied nephron segment specific antibodies against aquaporin-1 (AQP-1), α-glutathione-S-transferase (alpha-GST), Tamm-Horsfall protein (TH), calbindin-D28K (CalD), and aquaporin-2 (AQP-2), using an immunoperoxidase method on formalin-fixed paraffin-embedded kidney tissues of dogs, rats, mice, and Cynomolgus monkeys. AQP-1 was specific for proximal tubules and thin descending limbs of Henle’s loops and AQP-2 for connecting and collecting ducts in dogs, rats, mice, and Cynomolgus monkeys. Alpha-GST stained the straight part of proximal tubules in dogs and proximal convoluted tubule and straight part of proximal tubules in rats. TH was specific for thick ascending limbs of Henle’s loops in mice, rats, dogs, and Cynomolgus monkeys and stained additionally scattered cells in cortical connecting/collecting ducts of dogs. CalD was found in distal convoluted tubules and cortical connecting and collecting ducts of dogs, rats, and mice and in distal convoluted tubules, connecting ducts, and cortical and medullary collecting ducts of Cynomolgus monkey. This panel of antibodies may be a helpful tool to identify renal tubules by light microscopy in preclinical studies and to validate new biomarkers of renal toxicity.

Differences were examined between male and female Sprague-Dawley rats in the response of 16 urinary biomarkers (measured using several assay platforms) to renal injury produced by gentamicin administered subcutaneously for 10 days at a dosage of 75 mg/kg. Urinary biomarkers expressed as fold difference from contemporaneous controls and renal histopathology were assessed after 3 and 10 doses. On day 4, minimal proximal tubular changes were observed microscopically in all males but no females; on day 11, more extensive and more severe injury was observed to a similar extent in all animals of both sexes. Modest increases (maximum 5-fold) in all urinary biomarkers (except epidermal growth factor [EGF], which was decreased) on day 4 and marked elevations (maximum 271-fold) on day 11 were seen consistently in both sexes. However, the magnitude of the increases differed between the sexes. On day 4, despite the lack of tubular injury, many biomarkers were more elevated in females than males but this rarely led to statistically significant sex differences; only 2 biomarkers (β2-microglobulin and total protein) showed a greater increase in males than females in line with the histopathology. On day 11, there were many more biomarkers that showed a statistically significant difference between the sexes in fold change with treatment; in line with the results on day 4, the majority of biomarkers were more increased in females than males. It remains unresolved if sex differences in the magnitude of biomarker response at injury threshold would lead to any difference in diagnostic interpretation between the sexes. These data highlight the need for publication of more studies using animals of both sexes to fully explore the influence of sex on the diagnostic performance of the novel biomarkers.