Balancing Blood Sample Volume with 3Rs: Implementation and Best Practices for Small Molecule Toxicokinetic Assessments in Rats

Abstract

Improved small molecule bioanalytical sensitivity and concomitant decreased sample volume requirements provide an opportunity to reconsider how toxicokinetic (TK) data are collected in rat toxicity studies. Often, satellite groups of rats are designated to separate procedural effects of TK blood collection from the primary toxicity evaluation. Blood microsampling (i.e., ≤50 μL) decreases the blood volume collected such that TK samples can be collected from toxicity groups without impacting toxicity assessment. Small plasma sampling uses slightly higher blood volumes (i.e., 200 μL) with comparable technical feasibility and, importantly, allows multiple analyses with no negative impact on study interpretation. Our "base case" study designs utilize sparse TK sampling from sample toxicity group rats (1-2 samples/rat). Alternate designs with satellite animals may still be warranted based on study objectives (e.g., biomarkers), intolerability, or smaller rat strains; however, we propose these as exceptions rather than standard practice and with a focus to use the fewest animals possible. We review the state of knowledge in bioanalytical and blood sampling techniques and support the paradigm whereby TK sampling of main study animals significantly decreases the overall number of rats required for toxicity assessments and refines study interpretation with additional data options. These efforts maintain a commitment to the 3Rs (replacement, reduction, and refinement) while maintaining high-quality TK evaluations on toxicity studies.

Multiwell three-dimensional systems enable in vivo screening of immune reactions to biomaterials: a new strategy toward translational biomaterial research

“…This represents a large saving in the number of required animals. These approaches represent a responsible means of dealing with animal experiments according to the principles of the 3Rs (Replacement, Reduction and Refinement) to enhance the effectiveness of animal experiments and also combines a more economical and ethically acceptable path for future research [21][22][23].…”

Reducing blood volume requirements for clinical pathology testing in toxicologic studies—points to consider

“…47,48,50 Other effects that have been reported include minor decreases in urea and increases in sodium. 50 Collecting TK blood from toxicity animals may induce a regenerative hematopoietic response that could mask subtle test article-related hematotoxicity, normally detected as a decrease in reticulocyte counts. Based on the variability of drug effects, hematopoietic responses, and clinical/tolerability impacts, TK collections in toxicity animals are not recommended when hematotoxicity or hemodynamic effects are anticipated or when pushing high dose tolerability.…”

“…Collection of blood volumes up to 1.2 mL (6 time points of 200 μL each) over 24 hours for up to one month in rats did not interfere with toxicity assessment in regulatory toxicity studies. Generally speaking, expected procedure‐related effects may include decreased RBC mass, and increased reticulocyte counts with associated increased splenic weights and splenic extramedullary hematopoiesis . Other effects that have been reported include minor decreases in urea and increases in sodium .…”

“…Generally speaking, expected procedure‐related effects may include decreased RBC mass, and increased reticulocyte counts with associated increased splenic weights and splenic extramedullary hematopoiesis . Other effects that have been reported include minor decreases in urea and increases in sodium . Collecting TK blood from toxicity animals may induce a regenerative hematopoietic response that could mask subtle test article‐related hematotoxicity, normally detected as a decrease in reticulocyte counts.…”

Opportunities to Apply the 3Rs in Safety Assessment Programs

“…The biggest opportunity to reduce animal numbers, particularly rodents, in regulatory toxicology studies is in the collection of blood samples for TK evaluation ( Harstad et al 2016 ; Sparrow et al 2011 ), as separate groups of animals, termed satellite animals, are often included for this purpose. TK analysis is carried out to assess systemic exposure and is a required component of repeat-dose toxicity studies ( ICH S3A ).…”