Accelerating NDA filing through faster carcinogenicity assessment

Dr Megan MacBride and Dr Caroline Horizny Mitchell, Taconic, examine recent changes to animal testing in drug design.

Drug discovery and development is an arduous process that can cost upwards of $2.6 billion and take over 10 years. While the discovery phase – which includes target discovery, target validation, lead compound identification, and lead compound optimisation – averages several years and $200 million, the majority of the research, time, and financial spend takes place downstream in drug development. This stage is more highly regulated and consists of both preclinical testing and clinical trials. Due to the high costs associated with new drug development, pharmaceutical companies are under pressure to get effective drugs to market as fast as possible while ensuring they are safe for the intended patient population and dosing timeline, which can be lifelong for some drugs. The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) issues guidelines and develops testing strategies for evaluating carcinogenicity along with other risks such as genotoxicity, immunotoxicity, and reproductive toxicity. Until recently, drug carcinogenicity assessment required two different in vivo studies, one of which was typically a two-year lifetime study in rats. While historically used to ensure that new small molecule drugs going through development are non-carcinogenic in humans, two-year rodent studies present both financial and timeline burdens and may not always be more informative on human safety compared to alternative approaches. In August 2022, the ICH issued an addendum to its S1B guidelines that allows certain drugs to forego two-year rat studies1. This change in guidelines enables pharmaceutical companies to use only one species (mice) for their carcinogenicity testing—and by using transgenic rasH2 mice, they can condense the in-life portion of testing to six months, significantly decreasing time to New Drug Application (NDA) filing.

Drug discovery and development

New pharmaceutical small molecule drugs require extensive safety testing before the manufacturer can obtain regulatory approval for use in humans. Specific guidelines set forth to regulate these testing processes are issued by the ICH, a non-profit organisation with representation from global regulatory agencies and pharmaceutical companies. These guidelines are in place to evaluate a pharmaceutical’s potential health risks, including genotoxicity, immunotoxicity, and reproductive toxicity. In particular, carcinogenicity studies have long been a high priority in determining the overall risk to human health, given that some classes of pharmaceuticals exhibit known carcinogenic properties (the potential for a compound to induce tumours, decrease the timeline to tumour formation, or increase the malignancy of tumours). Regulatory agencies such as the US Food & Drug Administration (FDA), European Medicines Agency (EMA), and the China National Medical Products Administration (NMPA) adhere to these guidelines and require that novel small molecule compounds intended for continuous or intermittent use undergo specific types of carcinogenicity testing studies.

Figure 1: Comparing Carcinogenicity Testing Timelines in Transgenic vs. Standard Mice.

Carcinogenicity testing

For decades, the two-year rodent bioassay has been the regulatory standard used to determine the carcinogenic potential of new drugs. This process historically involved lifetime carcinogenicity bioassays in two species. In practice, this was typically conducted in rats and mice. However, in the late 1990s, regulatory agencies including the FDA began to accept the use of shorter medium-term bioassays in transgenic mice as a substitute for one of the two required lifetime studies.

Lifetime (two-year) carcinogenicity bioassays

Typical carcinogenicity study design involves the exposure of both sexes of the test species to at least three different doses of a drug. A control group is usually included which receives vehicle treatment. Upon reaching a defined endpoint, a comprehensive whole-body analysis of each animal is carried out, including a histopathological examination of tissues and organs. Although the two-year rodent bioassay has proven useful in identifying several human carcinogens, the assay is not without limitations:

Long Study Period – Given the long duration of the two-year rodent carcinogenicity bioassay, the time from study start to finish can take up to three years. This timeline is far from ideal, particularly for novel pharmaceuticals that, if authorised for market release, often have significant potential to benefit human health. There is also the investment to take into account: at least 50 animals per sex per dose/ group are recommended to obtain meaningful biological and statistical results. When including the costs of animal housing, quantity of drug needed, and labour, at current estimates, a single study can cost in excess of $2.5 million USD.

Spontaneous Tumours – A well-documented limitation of the lifetime assay is the high background incidence of tumours in both mice and rats as they age. The presence of spontaneous tumours that arise independent of chemical exposure can lead to false positive results, which can confound data interpretation.

Short or medium-term carcinogenicity bioassays

In the early 1990s, initial evaluations at multiple research centres worldwide—including the US National Toxicology Program and the Central Institute for Experimental Animals (CIEA) in Japan – brought transgenic mouse models to the forefront of carcinogenicity discussions. Although initial testing was carried out in five different transgenic mouse models, two models – the heterozygous p53 knockout mouse and the rasH2 mouse (also referred to as Tg.rasH2) – emerged as the most suitable models. The loss of a key tumour suppressor gene (p53), in the case of the p53 knockout mouse, or the overexpression of a human proto-oncogene, in the case of the rasH2 mouse, both result in increased susceptibility to tumour formation following carcinogen exposure. While the p53 knockout mouse model was appropriate only for the detection of genotoxic compounds, the rasH2 model developed by CIEA could detect both genotoxic and non-genotoxic carcinogens. The mutations in these transgenic mice resulted in a faster tumorigenic response to carcinogens, thereby allowing assessment of carcinogenic potential in just six months.

The original ICH S1B guideline allowed for the use of alternative bioassays, such as shorter studies using transgenic animals as an alternative to the two-year lifetime mouse study, and the use of these became accepted by regulatory authorities and widespread by pharmaceutical companies over the following decades2.

Carcinogenicity bioassays using the rasH2 mouse model require significantly fewer animals (25 per sex, per group) dosed for a short six-month in-life portion, and the resulting study read-out is simplified as the smaller cohort numbers mean fewer tissues to analyse and much lower incidence of background spontaneous tumours3. Thus, the use of rasH2 mice in carcinogenicity studies represents a major advance toward the principle of the 3Rs (Replacement, Reduction, and Refinement) for the ethical use of animals in research. A short-term transgenic study in the rasH2 mouse can save ~$1.5 million USD over a standard two-year mouse bioassay, and in parallel, accelerates the evaluation of the carcinogenic risk of compound exposure in humans.

Updated ICH S1B guidelines

As previously mentioned, the original ICH guideline required a two-year lifetime carcinogenicity study, generally in rats, as well as a bioassay in a second species. This second assay could be completed as a two-year lifetime mouse study alongside the rat study, but pharmaceutical companies have increasingly opted for a six-month transgenic mouse study in the rasH2 mouse. The two-year rat study is expensive, labour-intensive, and has long reporting timelines, and it comes with spontaneous tumour risk. Researchers who opted for the six-month rasH2 mouse study as the second bioassay saw cost savings, but the overall timeline for carcinogenicity assessment was limited by the two-year rat study timeline, which slowed time-to-market and increased overall animal use and study costs. In August 2022, after over 14 years of work by stakeholders across drug regulatory agencies and industry, the ICH revised the S1B guidelines (addendum (R1)) to introduce a Weight of Evidence (WoE) approach for the assessment of human carcinogenic risk which could eliminate the need for a two- year rat study for drugs in which carcinogenic potential in humans is unlikely. In those cases, carcinogenicity testing would generally take place only in mice4.

Figure 2: Process for Evaluating Key Factors to Inform a Carcinogenicity Assessment Strategy. Credit: Frank D Sistare and Taconic Biosciences.

Weight of evidence-based approach

This change in guidelines is backed by a WoE criteria-based approach that evaluates drugs on a case-by-case basis and determines whether carcinogenic potential in humans is likely, unlikely, or uncertain. It is estimated that two-thirds of the drugs that will be evaluated in this manner will not require the two- year rat study. These guidelines “leverage computational, in vitro, and emerging methodologies as well as data and tissue samples from in vivo studies that are already complete. It is anticipated that rat studies will decrease by 40% initially, and over time we could see a further reduction.” When a rat study is not required, choosing a short-term transgenic bioassay over a standard two- year mouse assay shortens the overall timeline for carcinogenicity assessment significantly.

Pharmaceutical companies can still choose to hew to the old historical standards and perform a two-year rat study. But toxicologists now have the freedom to ask: Will a two-year rat study truly add value and improve human safety? Or, will it simply take time and resources away from moving a potential new game-changing therapeutic to market faster?

Dosing specifications for transgenic mouse studies

This change in guidelines also provides clarity on high-dose selection endpoints for the bioassays using the rasH2 mouse. Previously, high dose selection in transgenic models was not standardised, with resulting concern around potentially causing extremely high doses which yielded results that weren’t physiologically relevant. This ambiguity on dose selection created hesitation for pharmaceutical companies to use the transgenic rasH2 mouse, even if it meant shorter studies and lower costs. With the updated guidelines specifying an acceptable high dose based on 50-fold plasma exposure ratio (rodent:human) is likely to encourage greater use of the rasH2 mouse and more reliable results5.

Moving forward

The latest revisions to ICH S1B represent a significant modernisation of carcinogenicity assessment of small molecule drugs. They encourage an integrated analysis of risk considering many factors, including drug target biology and evidence for hormonal or immunological perturbation. While animal studies are still critical to assess human safety of new drugs, the updated guidance encourages thoughtful planning and assessment to get more information out of required studies and not perform unnecessary studies which do not add value to evaluation of risk in humans. This is not only a massive step forward in reducing the use of animals in toxicology, but it can potentially move up NDA filing and subsequent drug approvals by two-three years, which is a win for both humans and research animals.

DDW Volume 24 – Issue 2, Spring 2023


  1. Testing for Carcinogenicity of Pharmaceuticals. Guideline S1B(R1).
  2. Storer RD, Sistare FD, Reddy MV, DeGeorge JJ. An industry perspective on the utility of short- term carcinogenicity testing in transgenic mice in pharmaceutical development. Toxicologic Pathology. 2010;38(1):51-61. doi:10.1177/01926 2330935171809351718
  3. Hisada S, Tsubota K, Inoue K, Yamada H, Ikeda T, Sistare FD. Survey of tumorigenic sensitivity in 6-month rasH2-Tg mice studies compared with 2-year rodent assays. Journal of Toxicologic Pathology. 2022;35(1):53-73. doi:10.1293/ tox.2021-0031
  4. The rasH2TM
  5. Sistare FD, Morton D, Alden C, et al. An Analysis of Pharmaceutical Experience with Decades of Rat Carcinogenicity Testing: Support for a Proposal to Modify Current Regulatory Guidelines. Toxicologic Pathology. 2011;39(4):716-744. doi:10.1177/0192623311406935

About the authors:

Dr Megan MacBrideDr Megan MacBride, Director, Product Marketing at Taconic has 16 years of experience in product management and commercialisation of animal models, including genetically engineered and humanised immune system mice. She has an AB in chemistry from Princeton University and PhD in chemistry from the Pennsylvania State University.


Dr Caroline Horizny Mitchell

Dr Caroline Horizny Mitchell, Senior Manager, Content Strategy at Taconic has experience in scientific writing, content creation, and RNA biology and nanotechnology. She obtained her BS and MS in biology from Rensselaer Polytechnic Institute and PhD in Nanoscale Science from the University at Albany.

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