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Microsampling improves animal welfare (refinement and reduction) and enables to evaluate the relationship between safety profile and drug exposure in the same animal. Many facilities have reported the relationship between microsampling and anemia, but that between microsampling and hematopoiesis caused by anemia has little been evaluated. We examined anemia- and hematopoiesis-related changes at endpoints of 1, 3, and 7 days after blood sampling by clinical pathology and histopathology. Seven-day oral dosing with microsampling was also conducted to mimic a 1-week screening toxicity study with toxicokinetics. Methods Six-week-old female Crl:CD(SD) rats (5 animals/group) were used, and microsampling was conducted via the jugular vein without anesthesia. Experiment 1: Blood was collected by serial sampling (6 × 0.05, 0.10, and 0.15 mL of blood; approximately 3, 6, and 9% of the circulating blood volume, respectively. Sampling points were 0, 1, 2, 4, 8, 24 h) on Day 1, and the animals were necropsied on Day 2 or Day 4. Untreated control and sham groups (restraint and needle puncture) were also set. Experiment 2: Animals received 10 mL/kg of water orally, once daily for 7 days. Animals underwent serial microsampling (6 × 0.05 mL at 0.5, 1, 2, 4, 8, 24 h on Day 1 and 7 × 0.05 mL on Day 7). Untreated control and sham groups were also set. Results Experiment 1: The 0.05 mL sampling at 6 points did not affect results in any examination. Anemia and hematopoiesis-related changes were noted in 0.10 mL and 0.15 mL samplings. The 0.10 mL sampling caused slightly low Ht on Day 2 and high RET and extramedullary hematopoiesis in the spleen and liver on Day 4. The 0.15 mL sampling caused low RBC, Hb, and Ht, increased hematopoiesis of erythroid cells in the femoral bone marrow, and extramedullary hematopoiesis in the liver on Day 2, and low RBC, Hb, and Ht, high RET, slightly high spleen weight, and increased extramedullary hematopoiesis in the spleen and liver on Day 4. Experiment 2: Microsampling did not affect results in any examination during 7-day oral administration. In both Experiments 1 and 2, changes related to handle-restraint and needle puncture were noted, including high neutrophils and AST, and thymic hemorrhage. Conclusion Microsampling with 6% and 9% of the circulating blood volume caused anemia and/or hematopoiesis, but 3% did not. Microsampling within 3% of circulating blood volume enabled toxicity to be appropriately evaluated.

Kubota, S. Takenoue, T. Nagaoka, K. Sakamoto,

H. Minomo, and H. Tsusaki

Shin Nippon Biomedical Laboratories, Ltd., Kagoshima, Japan.