Key Challenges and Mitigation Strategies for Animal Pregnancy in Non-clinical Reproductive Toxicity Testing of Drugs

doi:10.12300/j.issn.1674-5817.2024.177

Abstract

Abstract:

Non-clinical reproductive toxicity studies typically employ mammals like rats, rabbits, and cynomolgus monkeys, with animal pregnancy being a key challenge in such testing. This article focuses on the difficulties encountered in the animal pregnancy process and potential countermeasures. Rats can be used for fertility and early embryonic development toxicity studies (Segment Ⅰ), embryo-fetal development toxicity studies (Segment Ⅱ), and perinatal toxicity studies (Segment Ⅲ). The estrous cycle of female rats can be determined by vaginal smear, and mating behavior is confirmed through copulatory plug checks the following day after pairing one female with one male in the same cage. Rabbits are commonly used in embryo-fetal development toxicity studies (Segment Ⅱ). Mating behavior between male rabbits and estrous females is observed to determine the time of conception. However, challenges such as atypical estrus of female rabbits, large variations in estrus between batches, and mating failure often occur in reproductive toxicity testing, which may be addressed through prolonged light exposure, increased protein supplementation, optimized mating strategies, and environmental modifications like female and male rabbits are raised adjacent to each other. Non-human primates (NHPs) are typically employed in perinatal toxicity studies (Segment Ⅲ), where one of the key challenges lies in accurately determining sexual maturity in males - a critical factor for reproductive toxicity testing, which can be assessed through comprehensive evaluation of age, body weight, and testicular volume. Generally, male macaques are considered sexually mature when they meet the following criteria: age >4.5 years, body weight >4.5 kg, single testis volume >10 mL, and combined testicular volume >20 mL. For pregnancy confirmation, ultrasound examination demonstrating visible gestational sacs is required, though this necessitates experienced veterinary clinicians to establish standardized ultrasound examination protocols. In conclusion, reproductive toxicity studies should employ species-appropriate detection methods and evaluation criteria based on anatomical characteristics of the reproductive system to ensure successful mating and proper study execution.

Key words: Non-clinical reproductive toxicity testing, Animal pregnancy, Estrus and mating in animals

CLC Number:

Q95-33

LIU Kun,LAN Qing,YI Bing,et al. Key Challenges and Mitigation Strategies for Animal Pregnancy in Non-clinical Reproductive Toxicity Testing of Drugs[J]. Laboratory Animal and Comparative Medicine, 2025, 45(4): 449-456. DOI: 10.12300/j.issn.1674-5817.2024.177.

Figures/Tables 2

Figure 1 Typical vaginal smear characteristics from adult female rats at each stage of estrous cycleNote：A， proestrus； B， estrus； C， metestrus； D， diestrus. The black arrow indicates nucleated epithelial cells； the red arrow indicates anucleated epithelial cells； the green arrows indicate leukocytes. The staining method of the smear was hematoxylin and eosin （HE） staining. The magnification of the images is 40 and the scale bars are 100 μm.

Figure 2 Ultrasound cross-sectional view of gestational sac in a female monkey during early pregnancyNote：The area indicated by the arrow in the figure shows a well-formed gestational sac structure.

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