Nonclinical testing streamlined with New Approach Methodologies

CDER's primary focus is ensuring the safety and efficacy of human drugs. It has become increasingly clear that new approach methodologies (NAMs)—when combined with knowledge of the pathway, results of pharmacology studies, and other relevant data—may offer ways to achieve this goal more effectively and efficiently than traditional animal testing. Furthermore, NAMs support CDER’s long-standing interest and investment in implementing the 3Rs (Replacement, Reduction, and Refinement) of animal testing in drug development, aligning with both FDA's priorities and those of the public. By embracing NAMs and other methods of replacing and reducing animal testing, CDER aims to enhance its ability to protect public health, improve the drug development process, and respond to evolving scientific advancements in the field of drug evaluation and safety assessment.

Learn more about FDA’s commitment to reducing, replacing, or refining animal testing in drug development.

Drug Development Contexts for Streamlined Nonclinical Programs

The following table contains an inventory of drug development contexts for which CDER is open to a streamlined nonclinical program. This includes but is not limited to reduced sample size, reduced number of species tested, and NAMs use. While the table represents current streamlined approaches, the FDA is committed to further reducing animal testing and sponsors may propose nonclinical tests, including NAMs, as appropriate, and study designs that reduce the number of animals in toxicology studies. Sponsors are encouraged to discuss their nonclinical programs with the FDA review divisions. The Agency will evaluate whether the proposed approaches are acceptable.

This table excludes 505(b)(2), generic, and biosimilar pathways as well as studies related to impurities and excipients. This table provides general information; sponsors should consult guidance documents and appropriate FDA review divisions for more information. The FDA regularly revises existing guidance documents and publishes new guidance documents. This table will be updated as new information becomes available.

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Toxicity Type

Safety Pharmacology General Toxicity Carcinogenicity Developmental and Reproductive Toxicity (DART) Juvenile Animal Toxicology Special Toxicity and Other Types of Toxicity Screens Other Opportunities Applied Regulatory Examples

Reference

Accepted practice Antibody Drug Conjugates: Revisiting the Toxicologic Assessment. Presentation by Melissa Pegues at the Society of Toxicology 2024 Annual Meeting Avila et al. Regul Toxicol Pharmacol. 2023. 139:105345 Chien et al. Regul Toxicol Pharmacol. 2023. 138:105329 Clinical Pharmacology and Biopharmaceutics Review for NDA 215457 (Naloxone Auto-Injector 10 mg) Clinical Pharmacology Review for NDA 21487 (Remdesivir) Draft Guidance for Sponsor-Investigators: Nonclinical Testing of Individualized Antisense Oligonucleotide Drug Products for Severely Debilitating or Life-Threatening Diseases Draft Guidance: Graft-versus-Host Diseases: Developing Drugs, Biological Products, and Certain Devices for Prevention or Treatment E14 and S7B Clinical and Nonclinical Evaluation of QT/QTc Interval Prolongation and Proarrhythmic Potential – Questions and Answers ICH M3(R2): Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals FDA data collection Guidance for Industry: Advanced Prostate Cancer: Developing Gonadotropin-Releasing Hormone Analogues Guidance for Industry: Generally Accepted Scientific Knowledge in Applications for Drug and Biological Products: Nonclinical Information Guidance for Industry: Microdose Radiopharmaceutical Diagnostic Drugs: Nonclinical Study Recommendations Guidance for Industry: Nonclinical Evaluation of Late Radiation Toxicity of Therapeutic Radiopharmaceuticals Guidance for Industry: Nonclinical Evaluation of the Immunotoxic Potential of Pharmaceuticals Guidance for Industry: Nonclinical Safety Assessment of Oligonucleotide-Based Therapeutics Guidance for Industry: Nonclinical Safety Evaluation of Drug or Biologic Combinations Guidance for Industry: Nonclinical Testing of Orally Inhaled Nicotine-Containing Drug Products Guidance for Industry: Oncology Pharmaceuticals: Reproductive Toxicity Testing and Labeling Recommendations Guidance for Industry: Oncology Therapeutic Radiopharmaceuticals: Nonclinical Studies and Labeling Recommendations Guidance for Industry: Population Pharmacokinetics Guidance for Industry: Rare Diseases: Considerations for the Development of Drugs and Biological Products Guidance for Industry: Severely Debilitating or Life-Threatening Hematologic Disorders: Nonclinical Development of Pharmaceuticals ICH M3(R2): Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals ICH S10: Photosafety Evaluation of Pharmaceuticals ICH S11: Nonclinical Safety Testing In Support of Development of Pediatric Pharmaceuticals ICH S1A: The Need for Long-term Rodent Carcinogenicity Studies of Pharmaceuticals ICH S1B(R1): Addendum to S1B Testing for Carcinogenicity of Pharmaceuticals ICH S1B: Testing for Carcinogenicity of Pharmaceuticals ICH S1B(R1): Addendum to S1B Testing for Carcinogenicity of Pharmaceuticals ICH S1C(R2): Dose Selection for Carcinogenicity Studies of Pharmaceuticals ICH S5(R3): Detection of Reproductive and Developmental Toxicity for Human Pharmaceuticals ICH S6(R1): Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals ICH S7A: Safety Pharmacology Studies for Human Pharmaceuticals ICH S8: Immunotoxicity Studies for Human Pharmaceuticals ICH S9: Nonclinical Evaluation for Anticancer Pharmaceuticals ICH S9: Nonclinical Evaluation for Anticancer Pharmaceuticals – Questions and Answers Leighton et al. Regul Toxicol Pharmacol. 2016. 79:142-143 Nelson et al. Science. 2024. 386(6723):724-726 Organisation for Economic Co-operation and Development Test No. 437: Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage Organisation for Economic Co-operation and Development Test No. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method Saber and Leighton. Regul Toxicol Pharmacol. 2015. 71(3):444-452 Saber et al. Regul Toxicol Pharmacol. 2019. 107:104429 Saber et al. Regul Toxicol Pharmacol. 2024. 149:105616 Wange et al. Regul Toxicol Pharmacol. 2021. 123:104953 Simpson et al. Toxicol Sci. 2025. 206(2):219-227 Draft Guidance for Industry: Monoclonal Antibodies: Streamlined Nonclinical Safety Studies Organisation for Economic Co-operation and Development Test No. 467: Defined Approaches for Serious Eye Damage and Eye Irritation Organisation for Economic Co-operation and Development Test No. 497: Defined Approached on Skin Sensitisation Guidance for Industry: Nonclinical Evaluation of the Immunotoxic Potential of Pharmaceuticals Guidance for Industry: Nonclinical Evaluation of the Immunotoxic Potential of Pharmaceuticals Draft Guidance for Industry: Oncology Pharmaceuticals: Streamlined Nonclinical Safety Studies for Biologics and Conjugated Products

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Category Context of Use Current Regulatory Expectations and Opportunities for Reduced Animal Use Reference

Safety Pharmacology General safety pharmacology testing Safety pharmacology endpoints may be incorporated into general toxicity studies in order to reduce animal use. This practice is routine for biologics.

May include non-animal methods.

May be based on a risk assessment that includes information from biodistribution data for radiopharmaceuticals.

• ICH S7A: Safety Pharmacology Studies for Human Pharmaceuticals

• ICH M3(R2): Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals

• ICH S6(R1): Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals

• Guidance for Industry: Oncology Therapeutic Radiopharmaceuticals: Nonclinical Studies and Labeling Recommendations

• ICH S9: Nonclinical Evaluation for Anticancer Pharmaceuticals

• Guidance for Industry: Severely Debilitating or Life-Threatening Hematologic Disorders: Nonclinical Development of Pharmaceuticals

• Draft Guidance: Graft-versus-Host Diseases: Developing Drugs, Biological Products, and Certain Devices for Prevention or Treatment 1

• Guidance for Industry: Rare Diseases: Considerations for the Development of Drugs and Biological Products

Safety Pharmacology Cardiovascular safety pharmacology studies In vitro preparations can be used as test systems (e.g., cell cultures, receptors, ion channels, transporters and enzymes). In vitro systems can be used in supportive studies (e.g., to obtain a profile of the activity of the substance or to investigate the mechanism of effects observed in vivo).

An appropriately qualified proarrhythmia risk prediction model could be used according to its context of use to assess the possibility of torsades de pointes (TdP) in humans.

The analysis of QTc interval together with adequate pharmacokinetic sampling makes it possible to perform dedicated exposure-response modeling similar to concentration-QT analysis for clinical QT studies. Different models, including in silico, in vitro, ex vivo and in vivo models, have the potential to be used as part of an integrated risk assessment strategy to evaluate the proarrhythmic risk of QT-prolonging pharmaceuticals in humans.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a new approach methodology used in regulatory submissions to FDA. FDA encourages submission of these studies that follow best practices, to aid in the review of the clinical relevance of findings in hiPSC-CM assays.

• ICH S7A: Safety Pharmacology Studies for Human Pharmaceuticals

• E14 and S7B Clinical and Nonclinical Evaluation of QT/QTc Interval Prolongation and Proarrhythmic Potential – Questions and Answers

• Simpson et al. Toxicol Sci. 2025. 206(2):219-227

Safety Pharmacology Treatments for severely debilitating or life-threatening diseases (i.e., advanced cancer, several hematologic disorders, and graft-versus-host disease) Endpoints may be integrated into pivotal toxicology studies. In the absence of a specific risk for patients in clinical trials, such studies will not be called for to support clinical trials or for marketing. • ICH S9: Nonclinical Evaluation for Anticancer Pharmaceuticals

• Draft Guidance: Graft-versus-Host Diseases: Developing Drugs, Biological Products, and Certain Devices for Prevention or Treatment 1

• Guidance for Industry: Severely Debilitating or Life-Threatening Hematologic Disorders: Nonclinical Development of Pharmaceuticals

Safety Pharmacology Microdose radiopharmaceutical diagnostic drugs Not warranted • Guidance for Industry: Microdose Radiopharmaceutical Diagnostic Drugs: Nonclinical Study Recommendations

Safety Pharmacology To evaluate seizure risk In vitro or in silico methods could hypothetically be developed and used to predict human-relevant seizure risk and effect on the central nervous system. • Avila et al. Regul Toxicol Pharmacol. 2023. 139:105345

Safety Pharmacology To evaluate drug-induced liver injury In vitro liver models have been developed to predict hepatotoxicity and drug-induced liver injury by assessing changes in liver biomarkers and functional endpoints. • Avila et al. Regul Toxicol Pharmacol. 2023. 139:105345

General Toxicity General toxicity for biologics and conjugated products for the treatment of cancer For biologics, animal toxicology studies should use pharmacologically relevant species. Pharmacology studies should demonstrate binding of the oncology pharmaceuticals to molecular target(s) and elicit the intended pharmacologic effects. In the absence of a pharmacologically relevant species, the safety assessment could be based on a WoE2 risk assessment in lieu of animal toxicology studies.

For biologics that have pharmacological activity similar to humans in both rodent and non-rodent species, general toxicology (1- and 3-month) studies may be conducted in a single rodent species and supplemented with a WoE risk assessment, as appropriate.

For PD-(L)1 blocking monospecific antibodies, an assessment of the products’ chronic effects may be based on a WoE risk assessment in lieu of a 3-month toxicology study. In addition, with justification, the 1-month toxicology study may be non-sacrificial and supplemented with a WoE risk assessment.

For CD3 bispecific T-cell engagers, an assessment of products’ chronic effects may be based on a WoE risk assessment in lieu of a 3-month toxicology study.

For antibody-drug conjugates (ADCs) with cytotoxic payloads, when the safety of the payload is well-characterized (e.g., same payload as in approved ADCs) and the payload is the main driver of toxicities, the 3-month toxicology study may be conducted in rodents only, irrespective of ADC binding to the target antigen. The rodent study may use the ADC or the payload, as appropriate. When the target of the antibody is novel and binding to the target does not occur in the rodent species, a WoE risk assessment should also be submitted.

• Draft Guidance for Industry: Oncology Pharmaceuticals: Streamlined Nonclinical Safety Studies for Biologics and Conjugated Products1

General Toxicity Acute toxicity studies for small molecules When appropriately conducted dose-escalation studies or short-duration dose-ranging studies are available to inform on the acute toxicity risk for small molecules, stand-alone, single-dose acute toxicity studies are not warranted. • ICH M3(R2): Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals

General Toxicity Chronic toxicology studies for small molecule drugs (6-9 months; rodent and non-rodent, respectively) Toxicokinetic and pharmacokinetic analyses may be conducted during general toxicity studies. • ICH M3(R2): Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals

General Toxicity Chronic toxicology studies for biologics If both rodents and non-rodents (nonhuman primates or NHPs) are pharmacologically relevant, use a single species if no differences are noted in short-term studies.

Use the phylogenetically lower species.

Chronic toxicology studies can be six months long.

Information on recovery can be obtained by an understanding that the particular effect observed is generally reversible/nonreversible; thus, inclusion of recovery animals is not necessarily warranted. When warranted, they only need to be included in one toxicology study.

• ICH S6(R1): Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals

• Wange et al. Regul Toxicol Pharmacol. 2021. 123:104953

General Toxicity Monoclonal antibodies that recognize a single molecular target Chronic (long-term) toxicology: In general, studies longer than 3 months in nonrodent species (e.g., NHPs, dogs, and mini-pigs) are not warranted to evaluate toxicities from chronic administration of monospecific antibodies when data from 3-month studies are supplemented with a weight-of-evidence (WoE) risk assessment.

When an assessment of long-term safety is warranted, sponsors should decide whether a 3-month (or longer duration) toxicology study in animals will generate relevant information.

Short- and long-term toxicology: For antibodies that have pharmacological activity similar to humans in both rodent and nonrodent species, general toxicology studies (short- and long-term) conducted in a single rodent species may provide sufficient and appropriate nonclinical data.

• Draft Guidance for Industry: Monoclonal Antibodies: Streamlined Nonclinical Safety Studies 1

General Toxicity Microdose radiopharmaceutical diagnostic drugs Repeat-dose toxicity is not warranted. • Guidance for Industry: Microdose Radiopharmaceutical Diagnostic Drugs: Nonclinical Study Recommendations

General Toxicity Treatments of severely debilitating or life-threatening diseases (i.e.,several hematologic disorders, advanced cancer, graft-versus-host disease) Small molecule drugs and biologics: chronic toxicity is of three-month duration. When the investigational pharmaceutical extends survival or lessens the severity or the frequency of a debilitating event, toxicology studies of six to nine months duration are generally not warranted.

For an individualized antisense oligonucleotide, a single three-month toxicity study is considered adequate to assess safety for initiating human dosing, dose escalation, and chronic treatment.

• ICH S9: Nonclinical Evaluation for Anticancer Pharmaceuticals

• Draft Guidance: Graft-versus-Host Diseases: Developing Drugs, Biological Products, and Certain Devices for Prevention or Treatment 1

• Guidance for Industry: Severely Debilitating or Life-Threatening Hematologic Disorders: Nonclinical Development of Pharmaceuticals

• Draft Guidance for Sponsor-Investigators: Nonclinical Testing of Individualized Antisense Oligonucleotide Drug Products for Severely Debilitating or Life-Threatening Diseases 1

General Toxicity Orally inhaled nicotine products (for use as medical product, [e.g., smoking cessation]) If the exposure is within range of exposure from lawfully marketed tobacco products or an approved product, no new nonclinical toxicity data is required. • Guidance for Industry: Nonclinical Testing of Orally Inhaled Nicotine-Containing Drug Products

General Toxicity General toxicity for a combination of two or more previously marketed drugs or biologics A bridging study may be appropriate, provided the duration is sufficient to elicit the toxicity of concern. For example, a three-month general toxicity bridging study could be considered for a chronic indication. If the two products have sufficient nonclinical and clinical characterization, combination animal studies may not be warranted. • Guidance for Industry: Nonclinical Safety Evaluation of Drug or Biologic Combinations

General Toxicity General toxicity for a combination of two or more previously marketed drugs or biologics In general, toxicology studies investigating the safety of combinations of pharmaceuticals intended to treat patients with advanced cancer are not warranted.

If sufficient clinical data (e.g., a completed phase 1 or a monotherapy phase within phase 1) are available with the individual pharmaceuticals, additional nonclinical toxicology data may not be warranted. A rationale to support the combination should be provided, which can include in vitro or in vivo pharmacology data or a literature assessment.

If there is no or very limited human safety data for one of the combination components, a nonclinical pharmacology study of the combination should be considered, in addition to the toxicology studies with the single agents.

For pharmaceuticals that are pharmacologically inactive in animal species, assessment of combination can be based on relevant in vitro tests and/or a mechanistic understanding of target biology.

• ICH S9: Nonclinical Evaluation for Anticancer Pharmaceuticals

• ICH S9: Nonclinical Evaluation for Anticancer Pharmaceuticals – Questions and Answers

Carcinogenicity Carcinogenicity studies are not always needed Carcinogenicity studies are not generally needed for endogenous substances given as replacement therapy (i.e., physiological levels). When a drug is unequivocally genotoxic, a carcinogenicity study is not warranted as the drug is presumed to be trans-species carcinogen.

Pharmaceuticals administered infrequently or for short duration of exposure (e.g., anesthetics and radiolabeled imaging agents) do not need carcinogenicity studies unless there is cause for concern.

• ICH S1A: The Need for Long-term Rodent Carcinogenicity Studies of Pharmaceuticals

Carcinogenicity General replacement of two long-term carcinogenicity studies for pharmaceuticals The two-year study in rats may be substituted with a WoE risk assessment. A WoE assessment may inform whether a two-year rat study is needed.

An alternative to a two-year mouse study may be a six-month study in transgenic strains of mice, which typically employ fewer animals.

• ICH S1B: Testing for Carcinogenicity of Pharmaceuticals

• ICH S1B(R1): Addendum to S1B Testing for Carcinogenicity of Pharmaceuticals

• Wange et al. Regul Toxicol Pharmacol. 2021. 123:104953

Carcinogenicity Biologics Standard carcinogenicity bioassays are generally inappropriate. Product-specific assessment for carcinogenic potential is recommended. Use all available information (e.g., knock-out or animal disease models, human genetic diseases, class effects, and target biology). • ICH S6(R1): Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals

Carcinogenicity Treatments of severely debilitating or life-threatening diseases (i.e., advanced cancer, certain hematologic disorders; graft-versus-host disease) Animal carcinogenicity studies are generally not warranted when any of these applies: Carcinogenicity signals have been identified in general toxicology studies or in humans; the drug is a genotoxic cytotoxic agent; a WoE risk assessment was conducted that can determine the outcome; product-specific agreement (see the guidance for GnRH analogues in prostate cancer).

When an animal study is warranted, it could be conducted postapproval, as appropriate (e.g., when clinical development is short and carcinogenicity studies would delay product approval).

• ICH S9: Nonclinical Evaluation for Anticancer Pharmaceuticals

• Draft Guidance: Graft-versus-Host Diseases: Developing Drugs, Biological Products, and Certain Devices for Prevention or Treatment 1

• Guidance for Industry: Severely Debilitating or Life-Threatening Hematologic Disorders: Nonclinical Development of Pharmaceuticals

• ICH S1B(R1): Addendum to S1B Testing for Carcinogenicity of Pharmaceuticals

• Guidance for Industry: Advanced Prostate Cancer: Developing Gonadotropin-Releasing Hormone Analogues

Carcinogenicity Rare diseases Carcinogenicity study results may be submitted with the marketing application. In certain circumstances, submission of these data may be deferred to after approval. • Guidance for Industry: Rare Diseases: Considerations for the Development of Drugs and Biological Products

Developmental and Reproductive Toxicity (DART) General reduction of fertility, embryofetal development, and/or pre- and postnatal development studies Studies may be submitted at the time of marketing application in certain circumstances.

A number of alternative in vitro, ex vivo, and nonmammalian in vivo assays (alternative assays) have been developed to detect potential hazards to embryo-fetal development. The use of alternative assays for these purposes is encouraged.

If alternative approaches are used for embryofetal development studies, multiple alternative assays used within a tiered or battery approach should provide a level of confidence for human safety assurance at least equivalent to that provided by the current testing paradigms.

If in vivo studies are used, use of the same species and strain as that in already completed toxicity studies can eliminate the need to use additional animals or conduct additional studies.

Combination studies can be employed to assess all relevant stages of the reproductive process using fewer animals.

• ICH M3(R2): Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals

• ICH S5(R3): Detection of Reproductive and Developmental Toxicity for Human Pharmaceuticals

• Guidance for Industry: Rare Diseases: Considerations for the Development of Drugs and Biological Products

Developmental and Reproductive Toxicity (DART) Treatments of severely debilitating or life-threatening diseases (i.e., advanced cancers, certain hematologic disorders, graft-versus-host disease) Embryofetal developmental studies are not considered essential for pharmaceuticals that are genotoxic and target rapidly dividing cells or belong to a class that has been well characterized as causing developmental toxicity.

In cases where an embryofetal developmental toxicity study (including pilot non-GLP study) is positive for embryofetal lethality or teratogenicity, a confirmatory study in a second species is usually not warranted.

A WoE approach showing potential for reproductive toxicity may eliminate the need to conduct a dedicated study.

A WoE risk assessment can be used for small molecule drugs and biopharmaceuticals. The WoE can include a literature assessment of target biology, use of alternative assays such as fit-for-purpose in vitro or ex vivo, or nonmammalian in vivo assays.

• Guidance for Industry: Severely Debilitating or Life-Threatening Hematologic Disorders: Nonclinical Development of Pharmaceuticals

• Draft Guidance: Graft-versus-Host Diseases: Developing Drugs, Biological Products, and Certain Devices for Prevention or Treatment 1

• ICH S9: Nonclinical Evaluation for Anticancer Pharmaceuticals – Questions and Answers

• ICH S9: Nonclinical Evaluation for Anticancer Pharmaceuticals

• Guidance for Industry: Oncology Pharmaceuticals: Reproductive Toxicity Testing and Labeling Recommendations

• Guidance for Industry: Advanced Prostate Cancer: Developing Gonadotropin-Releasing Hormone Analogues

Developmental and Reproductive Toxicity (DART) Biologics When the product is active in rodents and rabbits, sponsors should use these species (instead of NHPs) for EFD assessment. When a study is positive, a study in the second species is not warranted.

DART studies may not be warranted if the WoE risk assessment suggests an adverse effect on fertility or pregnancy.

If a biologic is only active in NHPs, conduct a single enhanced pre-/postnatal development study. Additionally, fertility can be assessed by evaluation of the reproductive organs in general toxicology studies rather than a standalone study.

If clinical studies include sufficient precautions to prevent pregnancy, studies may be conducted during phase 3. An alternative model can be used in place of NHPs if appropriate scientific justification is provided.

• ICH S6(R1): Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals

Developmental and Reproductive Toxicity (DART) Monoclonal antibodies that recognize a single molecular target Assessment of reproductive and developmental toxicities should begin with a WoE risk assessment. A study in a relevant animal species may be warranted when a WoE risk assessment or other approaches (e.g., alternative assays) cannot adequately address safety. For products intended to directly or indirectly target gametes or have an indication for pregnancy-specific conditions, sponsors should consult the appropriate FDA review division regarding the appropriate approach for assessing risk. • Draft Guidance for Industry: Monoclonal Antibodies: Streamlined Nonclinical Safety Studies 1

Developmental and Reproductive Toxicity (DART) Oligonucleotides There can be cases where the WoE from existing data may be considered sufficient to communicate the risk to reproduction and embryofetal development, and no additional nonclinical studies are warranted. • Guidance for Industry: Nonclinical Safety Assessment of Oligonucleotide-Based Therapeutics

Developmental and Reproductive Toxicity (DART) Microdose radiopharmaceutical diagnostic drugs Not warranted • Guidance for Industry: Microdose Radiopharmaceutical Diagnostic Drugs: Nonclinical Study Recommendations

Developmental and Reproductive Toxicity (DART) Therapeutic radiopharmaceuticals Not warranted • Guidance for Industry: Oncology Therapeutic Radiopharmaceuticals: Nonclinical Studies and Labeling Recommendations

Juvenile Animal Toxicology General reduction of juvenile animal studies (JAS) WoE-based decisions should be made. Juvenile animal studies (JAS) are not considered important to support short-term pharmacokinetic studies in pediatric populations, and a WoE-based decision should be made. Changing the design of a traditional nonclinical program can address pediatric concerns (e.g., dosing at a younger age in repeat-dose toxicity). If JAS are conducted, combine assessment of endpoints in the same subset of animals. • ICH S11: Nonclinical Safety Testing In Support of Development of Pediatric Pharmaceuticals

• Guidance for Industry: Rare Diseases: Considerations for the Development of Drugs and Biological Products

• Draft Guidance for Industry: Monoclonal Antibodies: Streamlined Nonclinical Safety Studies 1

Juvenile Animal Toxicology Treatments of severely debilitating or life-threatening diseases (i.e., advanced cancers, certain hematologic disorders, graft-versus-host disease) Studies in juvenile animals are not usually conducted to support the inclusion of pediatric populations for the treatment of cancer.

JAS are not needed to initiate clinical trials in pediatric populations when clinical data in adults are available.

• ICH S9: Nonclinical Evaluation for Anticancer Pharmaceuticals

• Leighton et al. Regul Toxicol Pharmacol. 2016. 79:142-143.

• Guidance for Industry: Severely Debilitating or Life-Threatening Hematologic Disorders: Nonclinical Development of Pharmaceuticals

• Draft Guidance: Graft-versus-Host Diseases: Developing Drugs, Biological Products, and Certain Devices for Prevention or Treatment 1

Special Toxicity and Other Types of Toxicity Screens Immunotoxicity studies Immunotoxicity could be addressed through in vitro assays, a WoE assessment, or by adding endpoints into the design of general toxicity or proof-of-concept studies. Stand-alone animal immunotoxicity studies should only be conducted if the WoE from the general animal toxicology studies, knowledge of the target biology and/or pharmacokinetic/pharmacodynamic data suggest a risk that should be better characterized.

For most anticancer pharmaceuticals, the design components of the general toxicology studies are considered sufficient to evaluate the immunotoxic potential and support marketing.

For pharmaceuticals activating the immune system, the sponsor can consider additional endpoints (such as immunophenotyping by flow cytometry) in the toxicology or proof-of-concept study design.

To assess immune activation, an appropriate assay to be considered. Examples include cytokine release assay, antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity assays.

• ICH M3(R2): Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals

• ICH S8: Immunotoxicity Studies for Human Pharmaceuticals

• ICH S9: Nonclinical Evaluation for Anticancer Pharmaceuticals

• Guidance for Industry: Severely Debilitating or Life-Threatening Hematologic Disorders: Nonclinical Development of Pharmaceuticals

• Guidance for Industry: Nonclinical Evaluation of the Immunotoxic Potential of Pharmaceuticals

Special Toxicity and Other Types of Toxicity Screens Phototoxicity studies for small molecules Non-animal methods or clinical data are typically used for photosafety assessment.

A risk assessment should be considered, such as photochemical properties and findings from nonclinical and clinical studies.

• ICH M3(R2): Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals

• ICH S10: Photosafety Evaluation of Pharmaceuticals

• ICH S9: Nonclinical Evaluation for Anticancer Pharmaceuticals

• Guidance for Industry: Severely Debilitating or Life-Threatening Hematologic Disorders: Nonclinical Development of Pharmaceuticals

• Draft Guidance: Graft-versus-Host Diseases: Developing Drugs, Biological Products, and Certain Devices for Prevention or Treatment 1

Special Toxicity and Other Types of Toxicity Screens Special toxicity in microdose radiopharmaceutical diagnostic drugs or when the drug is a GnRH analogue in advanced prostate cancer Not warranted • Guidance for Industry: Advanced Prostate Cancer: Developing Gonadotropin-Releasing Hormone Analogues

• Guidance for Industry: Microdose Radiopharmaceutical Diagnostic Drugs: Nonclinical Study Recommendations

Special Toxicity and Other Types of Toxicity Screens Secondary pharmacology for biological products In cases where there are no pharmacologically relevant species, human tissue cross reactivity or alternative methods should be considered for the first-in-human (FIH) study. • ICH S9: Nonclinical Evaluation for Anticancer Pharmaceuticals – Questions and Answers

• Draft Guidance: Graft-versus-Host Diseases: Developing Drugs, Biological Products, and Certain Devices for Prevention or Treatment 1

Special Toxicity and Other Types of Toxicity Screens Eye irritation studies Non-animal methods, such as reconstructed human cornea-like epithelium models, may be used to assess eye irritation potential of topical and ocular products. • Organisation for Economic Co-operation and Development Test No. 437: Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage

• Nelson et al. Science. 2024. 386(6723):724-726

• Organisation for Economic Co-operation and Development Test No. 467: Defined Approaches for Serious Eye Damage and Eye Irritation

Special Toxicity and Other Types of Toxicity Screens Dermal irritation studies Non-animal methods may be used to assess dermal irritation. A 3D reconstructed human epidermis model is accepted for human pharmaceuticals. • Organisation for Economic Co-operation and Development Test No. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method

Special Toxicity and Other Types of Toxicity Screens Skin sensitization Several Defined Approaches for skin sensitization are described in the Organisation for Economic Co-operation and Development Test Guideline No. 497 and may be used to assess skin sensitization where applicable • Organisation for Economic Co-operation and Development Test No. 497: Defined Approached on Skin Sensitisation

• Guidance for Industry: Nonclinical Evaluation of the Immunotoxic Potential of Pharmaceuticals

Other Opportunities Many guidances include a general statement that alternative methods can be considered in place of standard models Discussion with FDA about the acceptability of an alternative method. Examples (not all inclusive):

• ICH M3(R2): Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals

• ICH S6(R1): Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals

• Guidance for Industry: Oncology Pharmaceuticals: Reproductive Toxicity Testing and Labeling Recommendations

• Guidance for Industry: Nonclinical Evaluation of Late Radiation Toxicity of Therapeutic Radiopharmaceuticals

• ICH S5(R3): Detection of Reproductive and Developmental Toxicity for Human Pharmaceuticals

Other Opportunities In vivo metabolism data In the absence of appropriate in vivo metabolism data, in vitro metabolism data (e.g., from liver slices or uninduced microsomal preparations) can provide appropriate support for the similarity of metabolism across species. • ICH S1C(R2): Dose Selection for Carcinogenicity Studies of Pharmaceuticals

Other Opportunities In vivo protein binding data While in vivo determinations of unbound drug might be the best approach, in vitro determinations of protein binding using parent and/or metabolites as appropriate might be used in the estimation of AUC unbound. • ICH S1C(R2): Dose Selection for Carcinogenicity Studies of Pharmaceuticals

Other Opportunities Organ radioactivity For oncology therapeutic radiopharmaceuticals when there is experience with the radionuclide or the ligand components of the radiopharmaceutical being developed, the nonclinical program can be abbreviated as needed, and the FIH dose can be based on clinical data. • Guidance for Industry: Oncology Therapeutic Radiopharmaceuticals: Nonclinical Studies and Labeling Recommendations

Other Opportunities Anti-tumor activity of anticancer pharmaceuticals If in vitro systems that are used for pharmacology studies of anti-tumor activity are demonstrated to generate relevant data, then they should be considered sufficient. • ICH S9: Nonclinical Evaluation for Anticancer Pharmaceuticals – Questions and Answers

Other Opportunities Exaggerated pharmacology in oligonucleotide-based therapeutics If the effects of exaggerated pharmacology cannot be evaluated in toxicology studies, a literature-based WoE risk assessment of the potential adverse effects should be provided. • Guidance for Industry: Nonclinical Safety Assessment of Oligonucleotide-Based Therapeutics

Other Opportunities First-in-human (FIH) dosing Population pharmacokinetics analysis is a well-established, quantitative method that can explain variability in drug concentrations among individuals. It has the potential to replace some animal pharmacokinetic studies to determine FIH dosing. For certain anticancer biological products, doses given to patients with related/relevant biological products may inform on FIH dosing. • Guidance for Industry: Population Pharmacokinetics

• Saber et al. Regul Toxicol Pharmacol. 2024. 149:105616

Other Opportunities Active ingredients that are endogenous substances Where the drug is an unmodified endogenous substance and a patient’s exposure to the drug reflects the same level of exposure and distribution of the endogenous substance seen in a healthy individual, generally accepted scientific knowledge regarding the safety of such a drug may be used to obviate the need for certain nonclinical data. • Guidance for Industry: Generally Accepted Scientific Knowledge in Applications for Drug and Biological Products: Nonclinical Information

Other Opportunities Drugs with well-known biological pathways It may be appropriate to rely on generally accepted scientific knowledge regarding the impact of the pathway rather than to conduct specific pharmacology and/or toxicology studies intended to measure the impact of the pathway. • Guidance for Industry: Generally Accepted Scientific Knowledge in Applications for Drug and Biological Products: Nonclinical Information

Applied Regulatory Examples Drug efficacy for genetic variants of cystic fibrosis and Fabry disease An in vitro approach was used to assess the functional and biochemical response of mutated or dysfunctional protein(s) in the presence of drug to make inferences about the potential for response in vivo. The findings from these data supported expanding the indications for the drugs to mutations not tested clinically. • Nelson et al. Science. 2024. 386(6723):724-726

Applied Regulatory Examples Drug safety of remdesivir In silico secondary pharmacology approaches were used to predict the potential renal safety risk of remdesivir based on its structure, physiochemical properties, and affinity for targets. • Clinical Pharmacology Review for NDA 21487 (Remdesivir)

Applied Regulatory Examples Drug efficacy for a new naloxone indication The FDA’s independent modeling and simulation supports the sponsor’s claim that administration of the naloxone auto-injector (NAI) 10 mg resulted in a higher percentage of subjects recovering from respiratory depression for middle and high opioid doses compared to NAI 2 mg. In addition, the FDA’s independent modeling and simulation supported the sponsor’s second claim that administration of NAI 10 mg before fentanyl or carfentanil exposure can prevent rapid and profound opioid-induced respiratory depression. • Clinical Pharmacology and Biopharmaceutics Review for NDA 215457 (Naloxone Auto-Injector 10 mg)

Applied Regulatory Examples Antibody-drug conjugates (ADCs) Data collected for ADCs with cytotoxic payloads indicate that toxicities of these products are mainly from the payload. Toxicity profiles of ADCs that contain the same payload were comparable in animals, independent of the monoclonal antibody moiety. Thus, streamlined approaches reducing use of NHPs may be applicable for certain ADCs.

Studies of the linker or the monoclonal antibody alone are not warranted.

• Saber and Leighton. Regul Toxicol Pharmacol. 2015. 71(3):444-452

• Saber et al. Regul Toxicol Pharmacol. 2019. 107:104429

• Antibody Drug Conjugates: Revisiting the Toxicologic Assessment. Presentation by Melissa Pegues at the Society of Toxicology 2024 Annual Meeting

• ICH S9: Nonclinical Evaluation for Anticancer Pharmaceuticals – Questions and Answers

• Draft Guidance for Industry: Oncology Pharmaceuticals: Streamlined Nonclinical Safety Studies for Biologics and Conjugated Products 1

Applied Regulatory Examples Monoclonal antibodies that recognize a single molecular target Examples of when 3-month studies (or studies of longer duration) are not warranted. The concept may be applied to other safety studies, as appropriate:

• When results of additional studies are likely to be confounded, for example, due to formation of neutralizing or clearing anti-drug antibodies observed in a completed short-term toxicology study conducted with the investigational monospecific antibody.

• When a 3-month toxicology study in animals is not feasible, for example, because severe immune suppression or anemia in a short-term toxicology study suggests that a longer duration study is likely to lead to mortality.

• When pharmacology data indicate that the monospecific antibody does not bind to the target in any nonclinical species or binding does not elicit pharmacological activity. In these cases, no animal toxicology studies are warranted.

• When substantial animal data with other monospecific antibodies against the same molecular target indicate that the animal data have not been predictive of human toxicities. In these cases, no animal toxicology studies are warranted

• Draft Guidance for Industry: Monoclonal Antibodies: Streamlined Nonclinical Safety Studies 1

Applied Regulatory Examples Other Flexibility and adherence to 3Rs discussed in several FDA publications (e.g., reprotoxicity, safety pharmacology, acute toxicity, local tolerance, juvenile animal toxicity, immunotoxicity, phototoxicity, and liver toxicity), and data-driven external publications.

For oncology indications, sponsors can choose to conduct the three-month toxicology study before initiating a phase 1 human study, in which case a one-month toxicology study (generally warranted to support a phase 1 study) won’t be needed.

• Wange et al. Regul Toxicol Pharmacol. 2021. 123:104953

• Avila et al. Regul Toxicol Pharmacol. 2023. 139:105345

• Chien et al. Regul Toxicol Pharmacol. 2023. 138:105329

• Accepted practice

References

Guidance from the U.S. Food and Drug Administration

• Guidance for Industry: Advanced Prostate Cancer: Developing Gonadotropin-Releasing Hormone Analogues (2022)

• Guidance for Industry: Generally Accepted Scientific Knowledge in Applications for Drug and Biological Products: Nonclinical Information (2023)

• Guidance for Industry: Microdose Radiopharmaceutical Diagnostic Drugs: Nonclinical Study Recommendations (2018)

• Guidance for Industry: Nonclinical Evaluation of Late Radiation Toxicity of Therapeutic Radiopharmaceuticals (2011)

• Guidance for Industry: Nonclinical Evaluation of the Immunotoxic Potential of Pharmaceuticals (2023)

• Guidance for Industry: Nonclinical Safety Assessment of Oligonucleotide-Based Therapeutics (2024)

• Guidance for Industry: Nonclinical Safety Evaluation of Drug or Biologic Combinations (2006)

• Guidance for Industry: Nonclinical Studies for the Safety Evaluation of Pharmaceutical Excipients (2005)

• Guidance for Industry: Nonclinical Testing of Orally Inhaled Nicotine-Containing Drug Products (2020)

• Guidance for Industry: Oncology Pharmaceuticals: Reproductive Toxicity Testing and Labeling Recommendations (2019)

• Guidance for Industry: Oncology Therapeutic Radiopharmaceuticals: Nonclinical Studies and Labeling Recommendations (2019)

• Guidance for Industry: Population Pharmacokinetics (2022)

• Guidance for Industry: Rare Diseases: Considerations for the Development of Drugs and Biological Products (2023)

• Guidance for Industry: Severely Debilitating or Life-Threatening Hematologic Disorders: Nonclinical Development of Pharmaceuticals (2019)

• Draft Guidance: Graft-versus-Host Diseases: Developing Drugs, Biological Products, and Certain Devices for Prevention or Treatment (2023)

• Draft Guidance for Sponsor-Investigators: Nonclinical Testing of Individualized Antisense Oligonucleotide Drug Products for Severely Debilitating or Life-Threatening Diseases (2021)

• Draft Guidance for Industry: Monoclonal Antibodies: Streamlined Nonclinical Safety Studies (2025)

• Draft Guidance for Industry: Oncology Pharmaceuticals: Streamlined Nonclinical Safety Studies for Biologics and Conjugated Products (2026)

Guidance from the International Council for Harmonisation (ICH)

• E14 and S7B Clinical and Nonclinical Evaluation of QT/QTc Interval Prolongation and Proarrhythmic Potential – Questions and Answers (2022)

• ICH M3(R2): Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals (2010)

• ICH M7(R2): Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals To Limit Potential Carcinogenic Risk (2023)

• ICH S1A: The Need for Long-term Rodent Carcinogenicity Studies of Pharmaceuticals (1996)

• ICH S1B: Testing for Carcinogenicity of Pharmaceuticals (1998)

• ICH S1B(R1): Addendum to S1B Testing for Carcinogenicity of Pharmaceuticals (2022)

• ICH S1C(R2): Dose Selection for Carcinogenicity Studies of Pharmaceuticals (2008)

• ICH S5(R3): Detection of Reproductive and Developmental Toxicity for Human Pharmaceuticals (2021)

• ICH S6(R1): Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals (2012)

• ICH S7A: Safety Pharmacology Studies for Human Pharmaceuticals (2001)

• ICH S8: Immunotoxicity Studies for Human Pharmaceuticals (2006)

• ICH S9: Nonclinical Evaluation for Anticancer Pharmaceuticals (2010)

• ICH S9: Nonclinical Evaluation for Anticancer Pharmaceuticals – Questions and Answers (2010)

• ICH S10: Photosafety Evaluation of Pharmaceuticals (2015)

• ICH S11: Nonclinical Safety Testing In Support of Development of Pediatric Pharmaceuticals (2021)

Publications and Presentations

• Avila AM, Bebenek I, Mendrick DL, Peretz J, Yao J, Brown PC. Gaps and challenges in nonclinical assessments of pharmaceuticals: An FDA/CDER perspective on considerations for development of new approach methodologies. Regul Toxicol Pharmacol. 2023 Mar;139:105345. doi: 10.1016/j.yrtph.2023.105345. PMID: 36746323.

• Chien HT, Prior H, Andrews L, van Aerts L, Cauvin A, Clarke DO, Datta K, Dempster M, Dybdal N, Freebern W, de Haan L, Herzyk D, Hey A, Kissner T, Kronenberg S, Leach MW, Lee D, Schutte K, Sewell F, Trouba K, Ulrich P, Weir L, van Meer P. Re-evaluating the need for chronic toxicity studies with therapeutic monoclonal antibodies, using a weight of evidence approach. Regul Toxicol Pharmacol. 2023 Feb;138:105329. doi: 10.1016/j.yrtph.2022.105329. PMID: 36592682.

• Leighton JK, Saber H, Reaman G, Pazdur R. An FDA oncology view of juvenile animal studies in support of initial pediatric trials for anticancer drugs. Regul Toxicol Pharmacol. 2016 Aug;79:142-143. doi: 10.1016/j.yrtph.2016.03.001. PMID: 26952647.

• Nelson CP, Brown P, Fitzpatrick S, Ford KA, Howard PC, MacGill T, Margerrison EEC, O'Shaughnessy J, Patterson TA, Raghuwanshi R, Rouse R, Stromgren S, Sung KE, Valerio LG Jr, Ward JL, Bumpus NN. Advancing alternative methods to reduce animal testing. Science. 2024 Nov 15;386(6723):724-726. doi: 10.1126/science.adg6228. PMID: 39541448.

• Saber H, Leighton JK. An FDA oncology analysis of antibody-drug conjugates. Regul Toxicol Pharmacol. 2015 Apr;71(3):444-52. doi: 10.1016/j.yrtph.2015.01.014. PMID: 25661711.

• Saber H, Simpson N, Ricks TK, Leighton JK. An FDA oncology analysis of toxicities associated with PBD-containing antibody-drug conjugates. Regul Toxicol Pharmacol. 2019 Oct;107:104429. doi: 10.1016/j.yrtph.2019.104429. PMID: 31325532.

• Saber H, Thompson MD, Leighton JK. Pharmacokinetic models for first-in-human dose selection of immune-activating products in oncology. Regul Toxicol Pharmacol. 2024 May;149:105616. doi: 10.1016/j.yrtph.2024.105616. PMID: 38561147.

• Simpson NE, Bourcier T, Sadrieh N. A Food and Drug Administration/Center for Drug Evaluation and Research nonclinical perspective on the use of human-induced pluripotent stem cell-derived cardiomyocyte data for cardiovascular safety assessment and regulatory decisions. Toxicol Sci. 2025 Aug 1;206(2):219-227. doi: 10.1093/toxsci/kfaf064. PMID: 40341948.

• Wange RL, Brown PC, Davis-Bruno KL. Implementation of the principles of the 3Rs of animal testing at CDER: Past, present and future. Regul Toxicol Pharmacol. 2021 Jul;123:104953. doi: 10.1016/j.yrtph.2021.104953. PMID: 33984412.

• Antibody Drug Conjugates: Revisiting the Toxicologic Assessment. Presentation by Melissa Pegues at the Society of Toxicology 2024 Annual Meeting.

Regulatory Review Documents

• Clinical Pharmacology Review for NDA 21487 (Remdesivir)

• Clinical Pharmacology and Biopharmaceutics Review for NDA 215457 (Naloxone Auto-Injector 10 mg)

Test Methods

• Organisation for Economic Co-operation and Development Test No. 437: Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage (2025)

• Organisation for Economic Co-operation and Development Test No. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method

• Organisation for Economic Co-operation and Development Test No. 467: Defined Approaches for Serious Eye Damage and Eye Irritation

• Organisation for Economic Co-operation and Development Test No. 497: Defined Approached on Skin Sensitisation

1 This draft guidance, when finalized, will represent the current thinking of the Food and Drug Administration (FDA or Agency) on this topic.

2 As defined in Draft Guidance for Industry: Oncology Pharmaceuticals: Streamlined Nonclinical Safety Studies for Biologics and Conjugated Products, a WoE risk assessment may include the following information:

• Nonclinical and clinical data generated with the investigational product, such as pharmacology, safety, and pharmacokinetics

• A literature-based assessment of potential toxicities associated with the molecular target (e.g., based on the expression profile or roles of the molecular target in physiological processes, in healthy and disease states)

• Toxicity findings in animals and humans, such as when the investigational product is in a class of pharmaceuticals with extensive information published on toxic effects

• Other data, as appropriate, e.g., fit-for-purpose NAMs

CDER oncology review divisions will determine whether the information included in the WoE risk assessment is sufficient to address the safety risks based on the totality of evidence.