We are pleased to follow-up with our new subsection in our quarterly newsletter, Preclinical Tales, which highlights exciting approaches and initiatives aligned with the 3R principle. The 3R principle (replace, reduce, refine), introduced by Russell and Burch in 1959, serves as a framework for ethically using animals in research and enhancing scientific quality and integrity in studies involving animals. Get inspired, stay informed, and begin implementation!

A New Era in Preclinical Research: FDA and NIH Take Action to Reduce Animal Testing
Summarized by: Anna E. Rapp, PhD, University of Pennsylvania

Earlier this year, the U.S. Food and Drug Administration (FDA) announced a pivotal shift in how safety testing will be conducted for monoclonal antibody (mAb) therapies and other biologics. The agency plans to move away from traditional animal studies and adopt more human-relevant approaches designed to enhance drug safety, reduce development time and costs, and minimize reliance on animal models. The goal over the next 3 to 5 years is to make animal-based testing the exception rather than the norm. This shift reflects a growing global public trend—especially in Europe and North America—toward greater scrutiny of animal experimentation, demands for transparency, and increased investment in alternatives aligned with the 3Rs.

The FDA’s newly released roadmap outlines a phased strategy to implement scientifically validated “new approach methodologies” (NAMs). These include organ-on-a-chip systems, computational modeling, and advanced in vitro assays using human ex vivo tissues and high-throughput human cell-based screening. The FDA also plans to incorporate existing international data where appropriate and clarify what additional information may be required when non-animal data alone are insufficient. As part of the transition, interim steps will focus on refining current in vivo models and applying them more selectively. The agency acknowledges that successfully moving to NAMs will require careful planning, rigorous science, and targeted training for both researchers and reviewers, and it proposes a set of scientific and technical steps to support FDA adoption of these methods.

Initially, the initiative will focus on monoclonal antibodies but is expected to expand to other biologics and small molecule drugs. This marks a significant departure from traditional preclinical requirements, which have historically relied on repeated-dose toxicity studies, pharmacokinetics, and immune response testing in animals—including non-human primates. A key driver behind this change is the poor translatability of many animal test results to human outcomes: the vast majority of drugs that appear safe and effective in animal studies ultimately fail to gain FDA approval due to safety concerns or lack of efficacy in humans. There have even been cases where adverse effects not detected in animal studies—such as cytokine release syndrome—emerged during human trials. Conversely, some medications considered safe for humans, like aspirin, might not have passed current animal safety tests.

Enforcing this momentum, the National Institutes of Health (NIH) established the Office of Research Innovation, Validation, and Application (ORIVA) in May 2025. This office will coordinate NIH-funded research to support and expand non-animal technologies. In collaboration with the FDA and the Department of Veterans Affairs, this federal effort aims to validate advanced alternatives—such as computational modeling and microphysiological systems—through the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM).

Implications for Musculoskeletal and Orthopaedic Research

While these regulatory changes focus primarily on drug safety evaluation, their implications could extend to musculoskeletal and orthopaedic research. Our field still relies on small and large animal models because the conditions we study involve complex interactions among multiple cell types, as well as the immune, nervous, and endocrine systems – all influenced by biomechanical forces. Nonetheless, researchers have begun exploring promising alternatives, such as organ-on-a-chip platforms that simulate the interfaces between musculoskeletal tissues. However, significant challenges remain, one of them is implementation of the mechanical forces essential for bone, cartilage, and joint health.

The FDA and NIH initiatives signal that investing in the development of NAMs in musculoskeletal and orthopaedic research is both timely and sensible. While NAMs will not fully replace animal research in our field in the near future, they could help reduce the use of animals and incorporate more human-based systems into our everyday research.

For further reading, click on the link below.

FDA Press Release and Roadmap

Refinements in Rodent Analgesia: Our Lab’s Transition to Ethiqa XR
Insights from Research Associate and Veterinarian Kait Link, DVM, Wu Tsai Human Performance Alliance, University of Oregon

The opioid buprenorphine is widely used to prevent pain in rodent orthopedic models. Long-acting analgesics, including those composed of buprenorphine HCL, provide key refinements in preclinical animal research. These long-acting drug formulations reduce handling stress and dosing frequency, while providing consistent plasma levels and effective pain relief [1]. Orthopedic researchers at the University of Oregon recently transitioned from using a compounded sustained-release buprenorphine to the FDA-indexed Ethiqa XR® (NDC 86084-100-30) driven by shifting federal guidance and a commitment to a compliant, refined pain management strategy. Ethiqa XR, developed by Fidelis Animal Health, utilizes their patented Fidelipid LAI technology—a lipid-based suspension that provides up to 72 hours of analgesia following a single subcutaneous injection [2-4].

Why Ethiqa XR?

The key reason behind the switch was to maintain regulatory compliance and accessibility. Ethiqa XR aligns with FDA guidance, which governs the compounding of animal drugs from bulk substances. In April 2023, the FDA declined to include buprenorphine HCl on the list of bulk drug substances approved for compounding [3]. This regulatory shift means that compounded products containing buprenorphine HCl cannot be kept stocked for general office use in research rodents [3,4]. Compounded sustained-release buprenorphine can now only be used legally under a patient-specific prescription with documented justification. When purchasing the previously used product it is now required to confirm that the medication, which is provided by a 503B Outsourcing Facility, will only be administered to or dispensed for patients for whom there is a clinical difference compared with an FDA-approved drug. In contrast, Ethiqa XR is FDA-indexed under Guidance for Industry #256, making it a regulatory-safe alternative which is available through national distributors under a researchers DEA [3,5,6].

Observations in Our Orthopedic Models

Our labs utilize a range of rodent musculoskeletal models—including osteotomies, volumetric muscle loss, and non-invasive knee injury—and we pair our pain relief strategies with a robust postoperative scoring system to monitor animal welfare. Since switching to Ethiqa XR, we’ve observed several notable species-specific responses that differ slightly from those seen with the previous compounded sustained-release formulation.

In rats, we administer Ethiqa XR at the labeled dose of 0.65 mg/kg SC. With delivery at this dose, the mean plasma buprenorphine level reportedly peaks between 6- and 24-hours post-injection and declines linearly thereafter [3]. According to the product label, there is a 4-hour delay before reaching therapeutic levels—a concern when optimal pain management requires immediate intraoperative and early postoperative coverage. This also negates any possible benefit of reducing the minimum alveolar concentration (MAC) of isoflurane achieved by giving buprenorphine preoperatively.  To address this, we co-administer a low dose of standard buprenorphine HCl (FDA approved, non-compounded formulation) at 0.02–0.03 mg/kg SC preoperatively. This bridging strategy has improved recoveries in our hands by ensuring adequate analgesia during this critical early period. In our experience, rats treated with Ethiqa XR exhibit more pronounced signs of sustained piloerection and appear more dysphoric compared to those who have received compounded formulations. We have also observed an increased incidence of pica and suspected gastrointestinal stasis, reflected in reduced fecal output—likely associated with ileus and/or nausea. To address these complications, we are considering the incorporation of maropitant citrate into our treatment protocols to help manage nausea, prevent anorexia, and improve overall recovery; however, this has not yet been implemented. Interestingly, maropitant has been shown to enhance analgesic efficacy when used in combination with morphine, allowing for reduced opioid dosing in rats exposed to noxious thermal stimuli [7]. Additional refinements used at our institution for prevention of pica include the replacement of the standard bedding materials with a cotton-pad cage liner and provision of enrichment items such as Nylabones and paper twists.

We’ve also explored dose titration based on procedure severity. For minor interventions, 0.5 mg/kg has provided adequate analgesia in rats while minimizing side effects. For more invasive procedures, we maintain the full labeled dose. When needed, we supplement with buprenorphine HCl (0.02–0.05 mg/kg SC) at 36–60 hours post initial injection of Ethiqa XR to extend analgesic coverage. Notably, female rats have shown lower plasma buprenorphine levels than males at all time points in a pharmacokinetic study, which may warrant sex-specific dosing considerations [4].

Using Ethiqa XR in Mice at the labeled 3.25 mg/kg SC has proven very successful in providing pain management for our mouse models without additional adverse effects. We’ve noted improved lameness grades, grooming and nest building behaviors—an overall general improvement in the score received using our monitoring system. Another important point about Ethiqa XR in mice is that therapeutic levels are reached within 30 minutes, eliminating the delayed onset concerns observed in rats. One challenge of this product in the mouse is the small administration volumes, note that Ethiqa XR cannot be diluted. So far, we’re very satisfied with the product in mice.

Although multimodal analgesia is often considered the gold standard, we avoid the use of NSAIDs in most of our models due to their known effects on inflammatory pathways involved in musculoskeletal repair, which could confound our scientific outcomes. In situations where NSAIDs are not contraindicated, it is important to note that concurrent administration of NSAIDs with Ethiqa XR has been associated with sudden deaths in mice. Although causality has not been definitively established, we experienced a similar incident in a rat that received meloxicam concurrently with Ethiqa XR prior to surgery under general anesthesia. To minimize risk, the manufacturer recommends spacing NSAIDs and Ethiqa XR by at least 30 minutes perioperatively, ideally with NSAIDs administered pre-operatively and Ethiqa XR post-operatively [3]. The use of sub-anesthetic doses of ketamine, maropitant (as mentioned above) or local anesthetic agents such as lidocaine may offer alternative strategies for achieving multimodal analgesia. These approaches can help reduce the required buprenorphine dose to reach therapeutic levels while minimizing unwanted side effects and should be considered for incorporation into your analgesic protocols.

Other notes on administration; Ethiqa XR forms a subcutaneous depot, and we’ve observed granulomatous nodules at injection sites. Also, once broached, the vial must be discarded after 90 days.

As regulatory oversight tightens, the use of FDA-indexed products like Ethiqa XR will become increasingly standard in preclinical pain management. Ethiqa XR isn’t a perfect one-size-fits-all solution, but it’s a compliant, effective option for extended analgesia in rodents—especially for studies that preclude NSAIDs (like ours in osteoimmunology). Like any medication, it requires careful dosing strategy and awareness of species-specific effects. The key is careful monitoring of research subjects and pain management protocol customization to optimize animal welfare and research outcomes.

Have you made the switch? We’d love to hear your experience.

References:

  1. Huss MK, Pacharinsak C. A review of long-acting parenteral analgesics for mice and rats. J Am Assoc Lab Anim Sci. 2022;61(6):597–604.
  2. Fidelis Animal Health. Ethiqa XR® (Buprenorphine Extended-Release Injectable Suspension) [Product Information]. 2022.
  3. Fidelis Animal Health. Ethiqa XR® Frequently Asked Questions. https://ethiqaxr.com/faqs/. Accessed June 3, 2025.
  4. Levinson BL, Leary SL, Bassett BJ, Cook CJ, Gorman GS, Coward LU. Pharmacokinetic and histopathologic study of an extended-release, injectable formulation of buprenorphine in Sprague-Dawley rats. J Am Assoc Lab Anim Sci. 2021;60(4):462–469. doi:10.30802/AALAS-JAALAS-20-000149.
  5. U.S. Food and Drug Administration. List of bulk drug substances for compounding office stock drugs for use in nonfood-producing animals. https://www.fda.gov/animal-veterinary/animal-drug-compounding/list-bulk-drug-substances-compounding-office-stock-drugs-use-nonfood-producing-animals. Accessed June 4, 2025.
  6. U.S. Food and Drug Administration. Guidance for Industry #256: Compounding Animal Drugs from Bulk Drug Substances. August 2022. https://www.fda.gov/media/132567/download. Accessed June 4, 2025.
  7. Karna SR, Kongara K, Singh PM, Chambers P, Lopez-Villalobos N. Evaluation of analgesic interaction between morphine, dexmedetomidine and maropitant using hot-plate and tail-flick tests in rats. Vet Anaesth Analg. 2019;46(4):476–482. doi:10.1016/j.vaa.2018.12.009.