Review of the use of two species in regulatory toxicology studies: Phase II - molecules following ICH M3(R2)

Phase II of the Two Species project aims to review opportunities to expand the use of a single species in long-term toxicity studies to molecules following the international ICH M3(R2) guidelines [1] for nonclinical safety studies. These guidelines currently require safety and tolerability data from two species – a rodent (e.g. rat or mouse) and a non-rodent (e.g. dog, minipig or non-human primate) – to support human clinical trials, as well as marketing authorisation for pharmaceuticals.

Previously, we have reviewed how and when two species are used within regulatory toxicology studies for molecules following the international ICH S6(R1) guidelines [2] for the development of biotechnology-derived pharmaceuticals (i.e. biologics). For these molecules, a single species can be used for longer-term studies if "similar" toxicities are identified in two species in short-term studies (i.e. supporting the first administration in humans). The evidence collected via survey highlighted opportunities for a single species approach to be used more widely for more molecules following ICH S6(R1), and provided preliminary evidence to support expanding this approach to nonclinical toxicology strategies for other drug modalities (i.e. molecules following ICH M3(R2)) [3]. However, it was recognised that changing the regulatory guidelines would require more robust evidence demonstrating minimal risk/impact for human risk assessment. This is why we decided to start a new phase to investigate these opportunities further.

In 2023, we established an international expert working group consisting of 48 experts from 42 organisations to review opportunities to expand the use of a single species in later-development toxicity studies to molecules following ICH M3(R2). The working group includes representatives from pharmaceutical companies, contract research organisations and regulatory bodies in the UK, elsewhere in Europe, USA and Japan.

The working group developed a survey to collect information on studies carried out to support development of these molecules, to better understand:

• How often similar toxicities are identified in short-term studies across different species.
• How often new or unexpected toxicities manifest in long-term studies, and the differences between rodents and non-rodents.
• The theoretical risk to human safety if using only a single species for long-term studies.

We received information for 75 molecules, including at least two FIH-enabling and two later development studies for each molecule (one in rodent and one in non-rodent species), for a total of 320 studies conducted between 2000 and 2023. Data collected include: details on study design, observed toxicities (severity, monitorability, reversibility), and their impact on decision-making.

Data analysis results suggest there are opportunities to use a single species approach in later development studies for small molecules, without compromising human safety.
 

• Retrospective review indicated that a single species could have been safely used in later development studies for 75% of the molecules analysed.
• Blinded exercises suggested that the decision to use a single species in later development studies could have been made prospectively – based on findings from early and FIH-enabling studies only – for 61% of the molecules analysed, with no impact on human safety.
• Further data analysis is underway to identify the key factors driving these decisions, and to develop a weight‑of‑evidence model to support working group recommendations and inform regulatory guidance.

These results have been shared at various conferences including the EUROTOX Congress in September 2025 [4], the American College of Toxicology (ACT) 46th Annual Meeting in November 2025 and the Society of Toxicology (SOT) 65th Annual Meeting in March 2026 [5].

1. ICH S6(R1) Scientific guideline for preclinical safety evaluation of biotechnology-derived pharmaceuticals.
2. ICH M3(R2) Scientific guideline for non-clinical safety studies for the conduct of human clinical trials for pharmaceuticals.
3. Prior H et al. (2020). Opportunities for use of one species for longer-term toxicology testing during drug development: a cross-industry evaluation. Regulatory Toxicology and Pharmacology 113: e104624. doi: 10.1016/j.yrtph.2020.104624
4. Passini E et al. (2025). Analysis of the use of two species in regulatory toxicology studies for molecules following ICH M3(R2). Toxicology Letters 411:S58-S59. doi: 10.1016/j.toxlet.2025.07.169
5. Passini E et al. (2026). Analysis of the use of two species in regulatory
   toxicology studies for molecules following ICH M3(R2). Poster presented at SOT, March 2026.