Exploring Greater Flexibility for Chronic Toxicity Study Designs to Support Human Safety Assessment While Balancing 3Rs Considerations

Abstract

Chronic repeated-dose toxicity studies are required to support long-term dosing in late-stage clinical trials, providing data to adequately characterize adverse effects of potential concern for human safety. Different regulatory guidances for the design and duration of chronic toxicity studies are available, with flexibility in approaches often adopted for specific drug modalities. These guidances may provide opportunities to reduce time, cost, compound requirement and animal use within drug development programs if applied more broadly and considered outside their current scopes of use. This article summarizes presentations from a workshop at the 43^rd Annual Meeting of the American College of Toxicology (ACT) in November 2022, discussing different approaches for chronic toxicity studies. A recent industry collaboration between the Netherlands Medicines Evaluation Board (MEB) and UK National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) illustrated current practices and the value of chronic toxicity studies for monoclonal antibodies (mAbs) and evaluated a weight of evidence (WOE) model where a 3-month study rather than a 6-month study might be adequate. Other topics included potential opportunities for single-species chronic toxicity studies for small molecules, peptides and oligonucleotides and whether a 6-month duration non-rodent study can be used more routinely than a 9-month study (similar to ICH S6(R1) for biological products). Also addressed were opportunities to optimize recovery animal use if warranted and whether restriction to one study only (if at all) can be applied more widely within and outside ICH S6(R1).

Introduction

Chronic repeated-dose toxicity studies are required to support long-term, late-stage clinical trials and often vary widely in species selection, study design and duration of dosing, dependent upon the drug modality and relevant regulatory guidances. Small molecules and other drugs such as oligonucleotides and peptides that generally follow ICH M3(R2) guidance ¹ are usually required to provide data from two species (a rodent and a non-rodent) following dosing for 6 months in rodent or 9 months in non-rodent. This requirement differs between regions, with a dosing duration of 6 months accepted for non-rodent data submitted within the EU, although this is not standard practice (as discussed later). A 6-month dosing duration is also generally accepted for biologics following ICH S6(R1) ² and is often performed in a single non-rodent species as the only pharmacologically relevant species. If a molecule is pharmacologically active in both the rodent and non-rodent and similar toxicities were identified in earlier first-in-human (FIH) enabling studies, 6-month data from a single species (preferably the rodent) is generally accepted. ³ For molecules intended for advanced cancer indications following ICH S9 guidance, ⁴ dosing durations of 3 months in a rodent and a non-rodent species are generally considered sufficient.

These long-term dosing studies are often complex, typically integrating assessments of toxicokinetics, clinical chemistry and hematology, and anatomic pathology, plus additional biomarkers and other functional endpoints (such as safety pharmacology) when required, that may assist with clinical trial designs or monitoring. These studies standardly use up to 20 male and 20 female rodents and up to 4 male and 4 female non-rodents per dose group. Alongside these main-test groups, if satellite groups of animals are required to assess recovery (rodents and non-rodents) or toxicokinetics (rodents), these additional animals lead to high numbers required in total. Consequently, various organizations have previously evaluated aspects of chronic toxicity study value and designs, exploring the practices within companies and/or data shared between different companies collaboratively within industry consortia to highlight opportunities for change and/or flexibility in approaches. These broadly cover the following topics: (1) predictivity/translation to adverse effects identified clinically ⁵ ; (2) whether new/further toxicities are identified compared to FIH-enabling data^6-9; and (3) study designs, including appropriate durations of dosing, species use and group sizes.^10,11 In a workshop held in November 2022 as part of the 43^rd Annual Meeting of the American College of Toxicology (ACT), data from a recent industry consortium were shared, along with some new/updated considerations on topics around dosing duration, species use and recovery animals that challenged some common practices and highlighted potential new approaches where reducing or refining the use of animals could be applied to chronic toxicity assessment. The presentations and resulting discussions are summarized herein.

Evaluating Optimal Toxicity Study Designs with Monoclonal Antibodies: Results from a MEB/Industry/NC3Rs Survey (Peter van Meer, The Netherlands Medicines Evaluation Board)

To support registration of mAbs for chronic indications, 6-month toxicity studies have historically been conducted as per ICH S6(R1) guidance. Experience with mAb development has shown a relatively benign and well-understood safety profile for this class of products, with most toxicity findings anticipated based on pharmacology. The appropriate/optimal duration of the studies has previously been investigated, with some groups supporting continued use of the 6-month duration, ⁶ with others suggesting that such long-term studies might not be needed.^11,12 Under a European Partnership for Alternative Approaches to Animal Testing (EPAA) supported and funded project, a consortium of 14 pharmaceutical companies, the MEB and the NC3Rs conducted a study to evaluate whether a 6-month toxicity study is still necessary to assess the long-term safety of mAbs. Data on First-in-Human (FIH)-enabling and chronic toxicity studies were shared for 142 mAbs submitted by 11 companies. The full dataset, discussion and recommendations can be found in Chien et al. (2023). ¹³ To compare outcomes of longer-term studies with shorter-term studies, inclusion and exclusion criteria led to a selection of 111 mAbs for the analysis.

For over 85% (96/111) of standard/modified mAbs, studies ≥ 6-months did not reveal any new toxicities of human concern, and for 71% of mAbs, no toxicities or no new toxicities were noted in chronic studies compared to FIH-enabling study findings. New toxicities related to exaggerated pharmacology or antibody-drug-antibody (ADA)-mediated findings were identified in 15.3% of cases; these were defined as not considered to be of human concern. New toxicities of potential concern for human safety or that changed trial design were identified in 13.5% of cases, with 7% being considered critical and 2% leading to program termination. Of interest was the data comparing FIH-enabling studies of ≤4 weeks, between 5 and 11 weeks, and between 12 and 16 weeks, which showed that increasing the duration of the FIH-enabling study decreased the incidence of identifying novel toxicities in the corresponding chronic toxicity study.

Given the high rate of products (both marketed and in development) without clinically relevant safety concerns identified in the longer-term studies; that longer-duration FIH-enabling studies result in fewer novel toxicities of concern in the longer-term toxicity studies; and that toxicities could be generally classified as pharmacology- or immune-mediated, this suggested that risk factors could aid in the identification of those products where toxicity studies of 6 months would be warranted. Therefore, an iterative, WOE model which considers factors that influence the overall risk for a mAb to cause toxicity was developed, with the goal of driving selection of the optimal duration of toxicity study without defaulting to a study of 6-month duration. ¹³ This model enables an evidence-based justification, suggesting when 3-month toxicity studies are likely sufficient to support late-stage clinical development and registration for some mAbs. The final model was tested using a randomized selection of five mAbs from a separate MEB database containing approved mAbs in the EU and a further 16 mAbs selected from the current dataset. Of these 21 case studies, 8 mAbs produced no novel toxicity and 13 mAbs produced findings of human concern identified in their respective longer-term study. For the 8 mAbs without novel toxicity, the model correctly suggested a 3-month study in six cases. For the remaining two cases, an alternative design/longer-term study was suggested as a more conservative approach in absence of more detailed data. The model also correctly anticipated a potential human safety risk in 11 (85%) of the 13 mAbs that produced toxicities. For the remaining two cases, the model suggested a 3-month study, which was based on the fact that new findings were due to exaggerated pharmacology and were therefore at least partially anticipated. When these two cases are included, the model was considered to correctly advise the development strategy for 90% of the mAbs. This data shows that a more science-based WOE approach for mAbs to determine the need for 6-month vs. shorter duration toxicity studies is feasible and should be considered and discussed with regulatory agencies rather than defaulting to a chronic toxicity study of 6-month duration for the majority of mAbs.

Opportunity for Reducing the Duration of the Non-rodent Chronic Toxicity Study (Paul Baldrick, Labcorp)

As previously stated, there is disparity between the recommended duration of non-rodent chronic toxicity studies for small molecules and other modalities following ICH M3(R2), with an option of 6 months in the EU or 9 months in other ICH regions. This generally leads to companies performing the longer duration (i.e., 9-month) study to realize global marketing strategies with a single study (and to avoid a risk of development time delay in having to repeat a shorter duration study to satisfy another region). Given the experience gained using these dual approaches since adoption of ICH M3(R2) more than a decade ago and that non-rodent studies of up to 6 months are recommended for biologics and other molecules following ICH S6(R1), ² this presentation discussed potential opportunities for a wider acceptance of 6-month non-rodent studies, with the potential for refinements in animal use (with 3 months less dosing requiring fewer procedures) and reduced resources (time and costs) for drug development.

The recommendations on the duration of chronic toxicity testing are provided in two ICH guidances, ICH S4 ¹⁴ and ICH M3(R2). ¹ Both support a study of 6-months’ duration in rodents and of 9-months’ duration in non-rodents, although provision for a 6-month non-rodent study is also presented. The 9-month duration was a reduction compared to the original and first revision of ICH M3, which recommended non-rodent studies of 12-month duration, whilst the current guidelines also reduced the need to perform partially duplicative studies for the different regions; the duration of 9 months was proposed as a compromise between the regions following reviews of practices and case-studies with 6- and 12-month data.^15,16 For a number of cases, there were findings observed by 12 months but not by 6 months. It was concluded that these would or could have been detected in a study of 9-month duration, with varying degrees of concern leading to the conclusion that ‘Studies of 12-month duration are usually not necessary and studies of shorter than 9 months duration may be sufficient’. ¹⁴ The FDA also commented in the Federal Register in June 1999 that ‘[the] FDA considers 9-month studies in non-rodents acceptable for most drug development programs’. ¹⁷ However, there was an added recommendation that 12-month testing may be needed on some occasions, e.g., for new pharmacological classes, and despite the newer ICH M3(R2) guidelines recommending ‘9 month non-rodent studies generally support dosing for longer than 6 months in clinical trials’, some companies do still perform a non-rodent study of 12 months to satisfy this FDA requirement (see Table 1).

Table 1.

Reviews of Non-Rodent Chronic Toxicity Study Durations.

Duration of Non-Rodent Chronic Study
	Small Molecule Studies Performed at Labcorp a			Small Molecules or Synthetic Peptides b			Oligonucleotides or Peptides c
	6 Months	9 Months	12 Months	6 Months	9 Months	12 Months	6 Months	9 Months	12 Months
Dog	9 d	97	2 e	2	3	-	-	4 f	-
NHP	9 g	85	1 h	2	6	-	-	13 f	2
Minipig	1	7	-	-	-	-	-	-	-

Note: ‘-’ indicates no studies.

^aNew data, review of small molecule studies performed across Labcorp global sites (2011-2021).

^bData from previous NC3Rs projects reviewing use of two species ¹⁰ .

^cPublicly available data mined from the FDA website ¹⁸ ; review of oligonucleotides and peptides approved by the FDA between 2019 and 2021 ¹⁸ .

^dIncluded local/tolerance ocular evaluation and subcutaneous implant work.

^eBoth inhalation testing.

^fOne study was 10-month duration.

^gIncluded local/tolerance ocular evaluation, advanced cancer drugs and antiviral drugs.

^hFor Asian market.

With available guidance in mind, the current testing paradigm was examined retrospectively from a number of data sources (Table 1), including Labcorp internal data (2011-2021, across global sites) for non-rodent toxicity studies with small molecules. Data was also available within a database of studies collected in a previous NC3Rs collaboration, ¹⁰ where nine small molecules and four synthetic peptides following ICH M3(R2) had performed non-rodent chronic toxicity studies. Another set of data was generated within NC3Rs by reviewing submissions of 10 oligonucleotides and nine peptides approved by the FDA between 2019 and 2021. ¹⁸

The above evaluations showed that a non-rodent chronic toxicity study duration of 9 months is currently the usual default for small molecules following ICH S4 and ICH M3(R2) guidelines and is also the case for most oligonucleotides and peptides. Exceptions using, for example, 6-month testing only rarely occurred and appeared to be related to a specific need, such as a local/tolerance ocular evaluation or for synthetic peptides following ICH S6(R1) recommendations.

Overall, other modalities have reduced duration testing, with 6 months acceptable for non-rodents with biopharmaceuticals ² and, although only for life-threatening cancer, a duration of 3 months is recommended for oncology drugs. ⁴ Clear opportunities lay with the drug modalities that ‘span’ the small molecule and biologics space, such as oligonucleotides and peptides, which follow ICH M3(R2) but also adopt some aspects of ICH S6(R1) within their drug development programs. These molecules, using pharmacologically relevant species for testing, tend to exhibit a high degree of selectivity and thus could also adopt the shorter chronic study duration common with other biologics. For change to occur for small molecules, guidance documents will need to go through an ICH update process, and a re-evaluation of the early 1990s findings would likely be required to investigate the original concerns around why 6-month testing in non-rodents was not deemed sufficient. However, risk assessment evaluation has moved on, and although there are probably limited examples, examination of any more recent data comparing 6-month to 9-month or longer data available in drug development company databases would also be beneficial. Results would help put to bed the question of whether the additional 3-month dosing of non-rodents identifies any new toxicities which may be of concern for human safety.

In summary, a reduction in study duration in non-rodents from 9 to 6 months is an important consideration to discuss across the industry, with opportunities to refine study designs allowing a shorter period on study for the animals involved. Secondary wins would be reduced use of test compounds, cost savings and time savings.

Inclusion of Recovery Animals: Opportunity for Further Optimization Remains (Helen Prior, NC3Rs)

If findings with potential adverse clinical impact are identified in nonclinical toxicity studies, an assessment of reversibility is required, but there is flexibility around how and when this is performed. Although this assessment can sometimes be made by scientific judgement of existing data and well-established knowledge of specific pathophysiologies, it is common to include additional groups of ‘recovery’ animals within a study to assess whether effects observed during the dosing period persist, or fully or partially reverse once dosing ends. The main ICH guidelines covering pharmaceutical development, ICH M3(R2), ICH S9 and ICH S6(R1), re-iterate/infer that recovery groups should not automatically be included in all general toxicology studies but that, if warranted, an option for inclusion of recovery animals in a single study within a package can be sufficient. ¹⁹ A specific note is also included in ICH M3(Q&A) ²⁰ which provides information on when use of recovery animals is warranted (or not). Yet in practice, recovery animals are widely included in more than one of the toxicity studies within the regulatory package,^21,22 increasing animal use, study costs and timelines. Data from recent industry collaborations were reviewed to evaluate current practices for small molecules and mAbs to identify opportunities to reduce the use of recovery animals within individual studies and/or development programs.

Data for mAb programs (the FIH-enabling and later-development studies) were collected as part of the MEB/Industry/NC3Rs survey described previously in this manuscript, ¹³ with detailed study information pertaining to recovery animal use reviewed in this analysis. To examine practices within a reasonably recent timeframe, and to establish any changes in practice since previous work ²¹ which recommended inclusion in later development (if deemed necessary) rather than in studies to support FIH and guideline updates,^2,23 only mAbs with at least one study starting in/after 2015 were included (51 mAbs, with study dates ranging from 2015 to 2019). There were examples of programs with no recovery animal use (four mAbs) with no detrimental impact on clinical/regulatory decisions, but recovery animals were routinely included in a high number of toxicity studies – 68% of FIH-enabling and 69% of later-development studies – and often in multiple phases and species in the same program (28 mAbs). When recovery animals were included in only one phase, this was the FIH-enabling studies for 12 mAbs and the later-development studies for 7 mAbs. In the studies where recovery animals were included (55 non-rodent and 9 rodent studies), appropriate justification for inclusion was made for 28% of studies (to assess reversibility of effects previously identified or to monitor reversal of primary pharmacologies). However, the most common reasons for inclusion may not be appropriate scientific justification: in case new effects were identified (77% of studies) or as a default approach for the specific study duration (33%). ICH S6(R1) discourages inclusion of recovery animals to assess delayed toxicities or the potential for immunogenicity; however, these reasons were cited for 31% and 19% of studies, respectively. The number of recovery groups was often minimized to control + one test article-dosed group (usually the high dose) for non-rodent studies (51% of studies), but most rodent studies (78%) and many non-rodent studies (40%) included recovery groups on all test article-dosed groups. The non-rodent recovery group size was typically two males and two females (2M+2F; 82% of studies) whilst the rodent recovery group size was more variable, commonly ranging from 4 to 6 M + F for wild-type rats or mice (78%), whilst two studies with transgenic mice used higher recovery group sizes of 10 or 12 M + F. The full dataset, discussion and recommendations can be found in Prior et al. (2023). ²⁴

Data for small molecules were collected as part of the NC3Rs review of two species use, ¹⁰ limited to those with at least one study starting in/after 2014 (62 small molecules, with study dates ranging from 2014 to 2017) to establish any changes in practice since previous work published in 2014. ²¹ In the more recent dataset, there were more examples of small molecules that did not include recovery animals in any of the studies to support FIH packages (34% vs. 23% in the previous dataset ²¹ ) and more small molecules that included recovery animals in some studies (27% vs. 12%), often in only one species, rather than all studies in both species to support FIH packages (39% vs. 65%). The number of recovery groups was similar to the previous dataset, with a high number of studies including recovery in control + one test article-dosed group only (70% vs. 75% in the previous dataset). However, the more recent dataset included more examples of specific study designs to minimize recovery animals, e.g., high dose only (no controls) and males only.

The recent data indicates that some companies are moving towards a more case-by-case approach for recovery animal use for both mAb and small molecule development and that packages without recovery animals are feasible. However, despite recommendations in 2014 ²¹ to move away from default inclusion of recovery animals and, in particular, consider inclusion later in clinical development once more information on the toxicity profile is known, for many companies the default still seems to be inclusion of recovery animals in both species, often on multiple studies across the FIH-enabling and chronic toxicity package. However, some optimization of study designs is apparent, and recovery animals are now being included in fewer dose groups. Limiting inclusion of recovery to a single nonclinical toxicology study and species, study design optimization and use of existing knowledge such that additional recovery groups are not required at all, provide opportunities to further reduce and refine animal use within drug development programs.

Species use for chronic toxicity studies: further opportunities for use of a single species (David Clarke, Eli Lilly and Company)

As noted, short- and long-term repeat-dose toxicology assessments of experimental therapeutic molecules are generally conducted in both a rodent and a non-rodent species, consistent with existing regulatory guidance and an expectation of health authorities. Exceptions exist, particularly for some biotherapeutic molecules. Many mAbs and antibody-like molecules cross-react with the orthologous pharmacological target only in a NHP species, with repeat-dose toxicology studies therefore conducted in that species alone. For biotherapeutic molecules that are pharmacologically active in both a rodent and a non-rodent species, ICH S6(R1) provides an opportunity to reduce to a single species (preferably the rodent) for long-term toxicology studies, where scientifically justified, largely based on similarity of toxicity findings identified in each of the two species from early, typically FIH-enabling, toxicology studies. Considerations for reducing to one species or maintaining two species for long-term studies were a topic of focus at a previous ACT meeting. ³ Experiences presented by industry, US FDA and UK MHRA, collectively indicated that a similar toxicity profile (or absence of toxicity) in two species is not the only factor to be considered; others being the profile and nature of both adverse and non-adverse findings, exposure/immunogenicity, the extent of target(s) engagement, signalling and pharmacological response, concerns based on mechanism of action, and consideration for the clinical population and whether the targeted disease is severely debilitating or life-threatening (SDLT). Further, in response to constrained supply of NHP species for safety testing due to the COVID-19 pandemic, the US FDA issued guidance early in 2022 that discouraged NHP use where possible for any modality and, on a case-by-case basis, permitted toxicity testing using only the rodent for a biotherapeutic that is active in the rodent and acts on a well-characterized target. ²⁵ While the guidance was withdrawn with the end of the COVID-19 Public Health Emergency in the US, its principles/approaches that minimize NHP use continue to be recognized by the US FDA. ²⁶

The question posed in the 2022 presentation was whether opportunities exist for modalities other than biotherapeutics, to reduce to a single species for long-term/chronic toxicology assessments; that is, can a similar case be made under certain circumstances for small molecules, peptides and RNA- and nucleotide-based modalities? Presently, US FDA guidance provides this for individualized antisense oligonucleotide drug products being developed to treat SDLT diseases, permitting a single toxicology study to be conducted in a single species, ideally one that is pharmacologically relevant. ²⁷ Additionally, findings from a survey conducted to understand nonclinical practices and regulatory expectations for safety assessment of oligonucleotide therapeutics indicate that single-species programs, given sufficient justification, have been accepted by health authorities. ²⁸ Whilst about three-quarters of the 22 companies who responded to the survey always used two species (rodent and non-rodent), others had justified a single species, typically the non-rodent, based on limitations using the rodent (lack of pharmacological relevance, impracticality of administration via the intended clinical route) and low risk of systemic class-related or off-target toxicity based on route of administration.

Insights as to whether two species are warranted for long-term toxicology studies for small molecules and synthetic peptides (as well as recombinant proteins, mAbs and ADCs) were provided by a NC3Rs survey and retrospective analysis conducted previously. ¹⁰ Analysis of anonymized data for 172 drug candidates from 18 organizations identified that two-species initial toxicology programs reduced to a single species for long-term (post-FIH-enabling) toxicology studies for about 36% to 40% of mAbs and ADCs, in contrast to 1% (1/25) of small molecules and 0% (0/5) of synthetic peptides. Yet, remarkably, the results of both the initial and long-term toxicology studies combined for individual molecules, identified either no toxicity, the same toxicity profile or a similar toxicity profile in the two species used for 32% (24/75) of small molecules and 42% (5/12) of synthetic peptides; these figures were compared with 36% (4/11) of recombinant proteins, 25% (1/4) of ADCs and 85% (11/13) of mAbs. Moreover, when asked if, with the benefit of hindsight, a single species would have been sufficient to generate the longer-term toxicology data and make development decisions without compromising human safety, survey respondents stated this to be the case for 66% small molecules and 62% synthetic peptides (also the case for 58% of recombinant proteins, 67% of ADCs and 89% of mAbs). The author group conducted a further exercise in which they evaluated the FIH-enabling toxicology data for the potential to reduce from two species to a single species for longer-term toxicology assessment, whilst being blinded to the actual longer-term toxicology study data. For 23 small molecules evaluated in this exercise, the group identified 14 for which a single species might have been sufficient to assess longer-term toxicity; this was later scored a ‘correct’ decision for 9 molecules (64% concordance) given no, same or similar toxicity in the two species actual longer-term toxicity data, but was ‘incorrect’ for five molecules given there were species differences in the longer-term toxicology data. For the other nine small molecules, the group determined that two species should be retained to assess longer-term toxicity, which was a ‘correct’ decision for seven molecules (78% concordance) when unblinded to the actual longer-term toxicology data; a single species could, in hindsight, have been used for the other two molecules. The group considered that their decision making would likely have been improved if comprehensive toxicology, pharmaco-/toxicokinetic, drug disposition and pharmacology data had been available for individual molecules rather than the top-level toxicology results they needed to rely on.

If the pharmaceutical industry and regulators are willing to consider a pathway for single-species longer-term toxicology assessment for modalities other than biologics, how might we be able to achieve this? As described by Prior et al. (2022) ³ with regard to biotherapeutic molecules, in addition to ‘similar’ initial toxicity profiles in the rodent and non-rodent species to justify reducing to one species, there are other considerations that can weigh into the decision. These include the target biology of each species as well as the pharmacological action of the therapeutic in each species and how these represent the biology and pharmacology in humans, the novelty of the target, the benefit-risk for the clinical population(s) investigated, and any early clinical data that might be available. The temporal aspect of generating longer-term toxicology data to support the overall development plan and the ability to seek timely regulatory feedback is also a consideration, though this ought not to be a hurdle for most programs with adequate forward planning. In general, these same considerations would apply to non-biologic modalities; species metabolic relevance to humans would be an additional key consideration for small molecules. An established set of well-thought-out considerations and criteria could provide a practical way to consider a single species program, or to potentially reduce from two to one species for longer-term toxicity assessments, for most modalities. When used in concert with an appropriate decision algorithm, the overall framework would provide for a WOE-based approach to selecting one or two species for toxicology assessments, either for FIH-enabling or longer-term toxicology studies. Various criteria- or questionnaire-guided WOE-based frameworks to decide on appropriate nonclinical safety assessment strategies are already being used, e.g., regarding the need for juvenile toxicology studies, ²⁹ for a one vs. two species assessment of embryo-fetal development, ³⁰ and on the need for a 2-year rat carcinogenicity study, ³¹ or have been recommended, e.g., for the selection of FIH starting dose, ³² and (as described above) regarding the need for chronic toxicology studies for mAbs. ¹³

Concluding Remarks

Chronic toxicity studies range in duration between 3 and 9 months (and occasionally 12 months) according to the drug modality and therapeutic indication. The purpose of the ACT session was to discuss the value of current chronic toxicity study expectations and designs whilst considering the potential for harmonization and adoption of methods that incorporate 3Rs approaches to reduce animal use and/or refine procedures (by adoption of shorter dosing durations) without compromising human safety.

Chronic toxicity studies are currently required to provide data in support of long-term dosing in Phase II and III trials in patient populations. The data presented from the MEB/Industry/NC3Rs consortia show that whilst new toxicities are identified with longer dosing durations than the FIH-enabling studies, these rarely translate to humans or modify clinical development. However, the provision of this data supports clinical development by confirming absence or presence of earlier findings and supports progression with appropriate monitoring, which could still be achieved using shorter studies. The data also provides useful information for dose setting for rat carcinogenicity studies (when required) or for WOE considerations (when appropriate) that mitigate the need for these studies. ³¹

Opportunities to reduce animal use for chronic toxicity studies were highlighted for mAbs; many companies perform 4-, 13- and 26-week duration studies to support FIH and longer duration dosing trials, but for many mAbs, a 13-week (3-month) study may be sufficient for supporting long-term dosing in humans. This potentially eliminates the 26-week (6-month) study, providing savings in animals, test articles and timelines. Other opportunities for reducing animal use for mAbs include restricting the inclusion of recovery animals to a single study within the package (if required at all) and restricting chronic toxicity studies to a single species (ideally rodent) for mAbs with multiple pharmacologically relevant species. Each of these latter opportunities is already outlined within regulatory guidance, ² and therefore could be more widely adopted with low risk of rejection from health authorities.

Certain principles described in ICH S6(R1) for biotherapeutic molecules, such as use of a single species for chronic toxicity studies and a 6-month duration non-rodent study, were discussed as opportunities for potential adoption with other drug modalities that currently fall outside the remit of this guidance. Some drug modalities, such as oligonucleotides and other peptides, span the small molecule and biologics space but often follow ICH M3(R2) guidance, such that chronic studies are performed in two species, including a 9-month duration study for the non-rodent. As some oligonucleotides have successfully justified single-species programs, ²⁸ more flexibility towards ICH S6(R1) rather than ICH M3(R2) approaches for these molecule types may be justified. Harmonization of non-rodent chronic toxicity studies to 6-month durations for small molecules and other drug modalities following ICH M3(R2) guidance would be a refinement via fewer procedures within a shorter dosing period but also reduce test article requirements and shorten timelines. There could even be scope for some small molecules to use a single species for chronic toxicity studies when similar toxicities are identified in earlier FIH-enabling studies, ¹⁰ and this will be evaluated further in a new phase of the NC3Rs project in 2024. Furthermore, for any modality, the inclusion of recovery animals in chronic toxicity studies for any drug modality only when necessary and scientifically justified, rather than in multiple studies/species across a package, is another means to potentially reduce animal use whilst also shortening timelines. Notably, the US FDA’s ‘COVID-19’ pandemic-era guidance to minimize NHP use provided some such opportunities where scientifically justified and, encouragingly, the FDA appears to have continued interest in such approaches.^25,26 The door has been opened for regulatory agencies worldwide, and the industry as a whole, to consider broader flexibility and new approaches in the assessment of chronic toxicity potential/characterization for a variety of drug modalities.

Abbreviations

ADA

Anti-drug antibody

ICH

International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use

mAb

Monoclonal antibody

NHP

Non-human primate

ORCID iD

Fiona Sewell https://orcid.org/0000-0001-5589-9453