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.

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.

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. Safety Pharmacology Microdose radiopharmaceutical diagnostic drugs Not warranted 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. 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. 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. 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. 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.

General Toxicity Microdose radiopharmaceutical diagnostic drugs Repeat-dose toxicity is not warranted. 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.  

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. 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. 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.  

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.

Carcinogenicity General replacement of two long-term carcinogenicity studies for pharmaceuticals 

The two-year study in rats may be substituted with a weight of evidence (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.

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).  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). 

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. 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.

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.

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.

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. Developmental and Reproductive Toxicity (DART) Microdose radiopharmaceutical diagnostic drugs Not warranted Developmental and Reproductive Toxicity (DART) Therapeutic radiopharmaceuticals Not warranted Juvenile Animal Toxicology General reduction of juvenile animal studies (JAS) 

Juvenile animal studies (JAS) should only be conducted when human adult data and data from general animal toxicology studies are not adequate for assessing risk to the intended pediatric population. 

WoE-based decisions should be made. 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.

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.

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.

Special Toxicity and Other Types of Toxicity Screens Phototoxicity studies for small molecules

Typically non-animal evaluations. 

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

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 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.  Special Toxicity and Other Types of Toxicity Screens Eye irritation studies A reconstructed human cornea-like epithelium model can replace rabbit tests. Special Toxicity and Other Types of Toxicity Screens Dermal irritation studies A 3D reconstructed human epidermis model is accepted for human pharmaceuticals. Special Toxicity and Other Types of Toxicity Screens Skin sensitization The murine local lymph node assay (LLNA) is a validated sensitization assay that reduces the number of animals used and refines their treatment. CDER accepts negative LLNA data without further testing in the guinea pig test. The LLNA can also be used as part of a WoE evaluation to discriminate between strong and weak sensitizers. 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): 

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. 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. 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. 

When an animal dosimetry study is needed, the sponsor should evaluate radioactivity in organs over time post-administration.  Duration of data collection can be adjusted as needed or alternative approaches and modeling can be considered.

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. 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. 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. 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.  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. 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. 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. 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. Applied Regulatory Examples CD3 bispecifics

A 2022 research project showed that for the CD3 bispecific products examined, absence of chronic (i.e., three-month) toxicology studies did not have adverse regulatory impact. When three-month studies were conducted, they were not informative; toxicities were predicted based on the available information (pharmacology and short-term toxicology, target biology, and phase 1 human data). Thus, a WoE assessment may predict the outcome of chronic dosing. 

Data collected (2017) shows common toxicities in animals from CD3 bispecifics (e.g., cytokine release syndrome and inflammatory response) and challenges associated with conducting three-month toxicology (e.g., anti-drug antibody formation, confounding the results).

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.

Applied Regulatory Examples PD-(L)1 blocking antibodies For these anti-cancer products, a WoE risk assessment may be considered in lieu of a three-month toxicology study. 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.

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