Nonclinical Testing of Orally Inhaled Nicotine-Containing Drug Products Guidance for Industry

Administrative guidance, not enacted legislation. This captured publisher edition preserves the listed document and attachments; later developments are not independently consolidated.

Nonclinical Testing of
Orally Inhaled
Nicotine-Containing
Drug Products
Guidance for Industry
U.S. Department of Health and Human Services
Food and Drug Administration
Center for Drug Evaluation and Research (CDER)
October 2020
Pharmacology/Toxicology

Nonclinical Testing of
Orally Inhaled
Nicotine-Containing
Drug Products
Guidance for Industry
Additional copies are available from:
Office of Communications, Division of Drug Information
Center for Drug Evaluation and Research
Food and Drug Administration
10001 New Hampshire Ave., Hillandale Bldg., 4th Floor
Silver Spring, MD 20993-0002
Phone: 855-543-3784 or 301-796-3400; Fax: 301-431-6353
Email: druginfo@fda.hhs.gov
https://www.fda.gov/drugs/guidance-compliance-regulatory-information/guidances-drugs
U.S. Department of Health and Human Services
Food and Drug Administration
Center for Drug Evaluation and Research (CDER)
October 2020
Pharmacology/Toxicology

TABLE OF CONTENTS
I. 	INTRODUCTION............................................................................................................. 1
II. 	NONCLINICAL DEVELOPMENT ............................................................................... 3
A. 	Key Considerations ........................................................................................................................ 3
B. 	Recommendations for Nonclinical Development ........................................................................ 4
1. General Principles ........................................................................................................................... 5
2. General Toxicity Studies .................................................................................................................. 6
3. Developmental and Reproductive Toxicology ................................................................................. 7
4. Carcinogenicity ................................................................................................................................ 7
5. Genetic Toxicology .......................................................................................................................... 8
APPENDIX A ................................................................................................................................ 9

Contains Nonbinding Recommendations
1
Nonclinical Testing of Orally Inhaled
Nicotine-Containing Drug Products
Guidance for Industry1
This guidance represents the current thinking of the Food and Drug Administration (FDA or Agency) on
this topic. It does not establish any rights for any person and is not binding on FDA or the public. You
can use an alternative approach if it satisfies the requirements of the applicable statutes and regulations.
To discuss an alternative approach, contact the FDA staff responsible for this guidance as listed on the
title page.
I. 	INTRODUCTION
This guidance provides sponsors with recommendations on the nonclinical information to
support development and approval of orally inhaled nicotine-containing drug products,2
including electronic nicotine delivery systems intended for smoking cessation and related
chronic indications.3
This guidance focuses on novel chemicals of the drug product formulation,4 novel chemicals
generated from any chemical of the drug product formulation by the delivery system5 (e.g., heat-
generated chemicals), and novel impurities from the drug product formulation and delivery
system. This guidance does not address nonclinical toxicity studies that may be requested by the
Center for Devices and Radiological Health to support use of the delivery system (e.g.,
biocompatibility studies).
1 This guidance has been prepared by the Office of Nonprescription Drugs in the Center for Drug Evaluation and
Research at the Food and Drug Administration.
2 The term drug is defined in section 201(g)(1) the Federal Food, Drug, and Cosmetic Act (FD&C Act).
3 An orally inhaled nicotine-containing product can be regulated as either a medical product or a tobacco product
depending on the intended use. See 21 CFR 1100.5, which describes when a product made or derived from tobacco
will be subject to regulation as a drug, device, or combination product.
4 In this guidance, the phrase novel chemicals of the drug product formulation refers to active and inactive
ingredients intentionally added to the drug product that have not been approved by FDA in drugs at an equal or
greater dose, for an equal or greater duration of use, or by a relevant route of administration sufficient to characterize
toxicity via local and systemic exposure.
5 The products addressed by this guidance are generally drug and device combination products with a drug primary
mode of action.

Contains Nonbinding Recommendations
2
An adequate nonclinical toxicity assessment can address the potential toxicity of chemicals from
orally inhaled nicotine-containing drug products. Some of these products have already been
associated with toxicity concerns.6,7,8,9,10
Orally inhaled nicotine-containing drug products developed for smoking cessation and related
chronic indications11 are expected to involve continuous use or chronic intermittent use resulting
in 6 months or more exposure over a lifetime. The recommendations for nonclinical toxicity
assessment in this guidance are intended to support the indication of smoking cessation and
related chronic indications in an adult population for either prescription or nonprescription use.
These recommendations for nonclinical testing of orally inhaled nicotine-containing drug
products are based on FDA’s current scientific understanding of toxicity evaluation of orally
inhaled drug products for chronic use. In addition, the recommendations are intended to
complement the recommendations for nonclinical toxicity assessment of drug products in the
ICH guidance for industry M3(R2) Nonclinical Safety Studies for the Conduct of Human Clinical
Trials and Marketing Authorization for Pharmaceuticals (January 2010) and the guidance for
industry Nonclinical Studies for the Safety Evaluation of Pharmaceutical Excipients (May
2005).12
6 Madsen LR, Vinther Krarup NH, Bergmann TK, Bærentzen S, Neghabat S, Duval L, and Knudsen ST, 2016, A
Cancer That Went Up in Smoke: Pulmonary Reaction to e-Cigarettes Imitating Metastatic Cancer, Chest,
149(3):e65–e67.
7 Ghosh A, Coakley RC, Mascenik T, Rowell TR, Davis ES, Rogers K, Webster MJ, Dang H, Herring LE, Sassano
MF, Livraghi-Butrico A, Van Buren SK, Graves LM, Herman MA, Randell SH, Alexis NE, and Tarran R, 2018,
Chronic E-Cigarette Exposure Alters the Human Bronchial Epithelial Proteome, Am J Respir Crit Care Med, epub
ahead of print February 26, 2018, doi: 10.1164/rccm.201710-2033OC.
8 Olmedo P, Goessler W, Tanda S, Grau-Perez M, Jarmul S, Aherrera A, Chen R, Hilpert M, Cohen JE, Navas-
Acien A, and Rule AM, 2018, Metal Concentrations in e-Cigarette Liquid and Aerosol Samples: The Contribution
of Metallic Coils, Environ Health Perspect, 126(2): doi: 10.1289/EHP2175.
9 Rubinstein ML, Delucchi K, Benowitz NL, and Ramo DE, 2018, Adolescent Exposure to Toxic Volatile Organic
Chemicals From E-Cigarettes, Pediatrics, epub ahead of print March 5, 2018, doi: 10.1542/peds.2017-3557.
10 References are not intended to be an exhaustive list of studies that characterize toxicity in e-cigarettes.
11 Nicotine replacement therapy (NRT) drug products can be developed for the following chronic indications:
smoking cessation and reduction in risk of relapse. NRT drug products that first have demonstrated effectiveness
for smoking cessation or reduction in risk of relapse can also include additional information in labeling by
demonstrating effectiveness in the following secondary endpoints: reduction of urge to smoke, relief of cue-induced
craving in former smokers, and relief of withdrawal symptoms not associated with a cessation attempt. See the draft
guidance for industry Smoking Cessation and Related Indications: Developing Nicotine Replacement Therapy Drug
Products (February 2019). When final, this guidance will represent the FDA’s current thinking on this topic. For
the most recent version of a guidance, check the FDA guidance web page at https://www.fda.gov/regulatory-
information/search-fda-guidance-documents.
12 We update guidances periodically. To make sure you have the most recent version of a guidance, check the FDA
guidance web page at https://www.fda.gov/regulatory-information/search-fda-guidance-documents.

Contains Nonbinding Recommendations
3
FDA will consider existing clinical and nonclinical information that characterizes the safety of
novel chemicals, to the extent that such data reflect current scientific standards and sponsors own
or have a right of reference13 to the data. In this case, such data should adequately provide the
toxicity information that the FDA-recommended studies (see section II. B., Recommendations
for Nonclinical Development) are designed to provide. Sponsors are encouraged to meet with
FDA to discuss drug development plans before initiating studies.14
In general, FDA’s guidance documents do not establish legally enforceable responsibilities.
Instead, guidances describe the Agency’s current thinking on a topic and should be viewed only
as recommendations, unless specific regulatory or statutory requirements are cited. The use of
the word should in Agency guidances means that something is suggested or recommended, but
not required.
II. 	NONCLINICAL DEVELOPMENT
A. 	Key Considerations
A thorough nonclinical toxicity assessment is integral to the benefit-risk assessment of orally
inhaled nicotine-containing drug products. Sponsors should consider the following:
• 	FDA does not recommend new nonclinical data to characterize the toxicity of nicotine
alone if one of the following applies:
— 	For smoking cessation, sponsors can consider if the nicotine exposure is within the
range of exposure expected from lawfully marketed combustible tobacco products
based on local and systemic exposures relevant to the proposed orally inhaled
nicotine-containing drug product.
— 	Sponsors can rely on the exposure to nicotine in an approved listed drug to inform the
nonclinical toxicity evaluation for this purpose under an abbreviated approval
pathway.15 If sponsors rely on FDA’s finding of safety for an approved listed drug,
that drug should provide exposure16 equal to or higher than the exposure anticipated
from the proposed orally inhaled nicotine-containing drug product, considering the
conditions of use proposed in labeling. For example, a relevant approved drug is one
that has conditions of use similar to the proposed orally inhaled nicotine-containing
drug product, including the dose, duration, route of administration, and the indicated
population.
13 See 21 CFR 314.50(g) regarding right of reference for an NDA under section 505(b) of the FD&C Act.
14 See the draft guidance for industry Formal Meetings Between the FDA and Sponsors or Applicants of PDUFA
Products (December 2017). When final, this guidance will represent the FDA’s current thinking on this topic.
15 See section505(b)(2) of the FD&C Act (21 U.S.C. 355(b)(2)).
16 The pharmacokinetic parameters (e.g., Cmax, Tmax, area under the curve) of the new inhaled product should be
compared with the relied-upon listed drug.

Contains Nonbinding Recommendations
4
• 	Sponsors should submit toxicity information for all chemicals of the drug product
formulation, heat-generated products, and impurities to support clinical use.
— 	In many cases, use of the delivery system will alter chemicals from the formulation or
device, creating new chemicals (e.g., heat-generated products). If an active ingredient
in addition to nicotine is proposed, sponsors should refer to the nonclinical
development recommended in ICH M3(R2) to complement the recommendations
below.
• 	Sponsors should properly characterize the risks from orally inhaled nicotine-containing
drug products. Orally inhaled nicotine-containing drug products result in local and
systemic exposure to nicotine and other chemicals, including heat-generated chemicals,
via the inhalation, buccal, and oral routes of administration. Some chemicals may be
novel, not found in relevant, previously approved drug products, or may not have
adequate toxicity information available. Local and systemic exposure should be
addressed in the nonclinical toxicity assessment. For example, nonclinical toxicity
studies of a chemical administered subcutaneously would not address local effects in oral
or respiratory tract tissues. Consequently, additional information relevant to these tissues
may be warranted.
• 	All drugs have risks. FDA weighs the benefits and risks with respect to the proposed
indication and patient population.17
B. 	Recommendations for Nonclinical Development
The nonclinical toxicity assessment appropriate for marketing approval should include general
toxicity studies, developmental and reproductive toxicity studies, an assessment of carcinogenic
potential, and supporting toxicokinetic and nonclinical pharmacokinetic studies18 in both sexes
(see Appendix A). Whether genetic nonclinical toxicity studies should be conducted depends on
the tobacco use and smoking status of clinical trial subjects. The following recommendations
outline general principles for conducting nonclinical toxicity studies.19
17 For information on benefit-risk assessment, see the guidance for industry Premarketing Risk Assessment (March
2005). See also the FDA Structured Approach to Benefit-Risk Assessment in Drug Regulatory Decision-Making
available at https://www.fda.gov/media/112570/download.
18 See the ICH guidance for industry S3A Toxicokinetics: The Assessment of Systemic Exposure in Toxicity Studies
(March 1995).
19 We support the principles of the 3Rs (reduce/refine/replace) for animal use in testing when feasible. FDA
encourages sponsors to consult with review divisions when considering a nonanimal testing method believed to be
suitable, adequate, validated, and feasible. FDA will consider if the alternative method could be assessed for
equivalency to an animal test method.

Contains Nonbinding Recommendations
5
1. 	General Principles
Following are FDA recommendations for general principles that apply to developing orally
inhaled nicotine-containing drug products:
• 	We recommend a full chemical analytical characterization of the aerosol, including heat-
generated chemicals, using the proposed delivery system, to identify the substances that
can be generated by an orally inhaled nicotine-containing drug product. The device and
system used to generate the aerosol should be fully described, including the settings used.
• 	FDA does not recommend pharmacology studies to address the mechanism of action if
nicotine is the only active ingredient. FDA recommends that sponsors provide
justification that any proposed inactive ingredients do not have pharmacological activity.
If an additional active ingredient is proposed, sponsors should characterize its
pharmacology as recommended in ICH M3(R2).
• 	To inform the benefit-risk assessment, nonclinical toxicity studies can benefit from the
inclusion of a testing group or groups exposed to aerosol from the proposed formulation
or formulations heated in a relevant delivery system, compared with a reference testing
group exposed to cigarette smoke. Adequate negative controls should be included in all
studies. See section II. B. 1. 2. for adequate negative control groups (e.g., sham air
control group, vehicle control group).
• 	Heat-generated chemicals should be evaluated as a mixture in nonclinical toxicity studies.
Novel chemicals (e.g., heat-generated products) and chemicals that are a safety concern
should be identified by quantitative dosing analysis and measurement of exposure (e.g.,
toxicokinetics) in nonclinical toxicity studies. Quantitative dosing analysis in nonclinical
toxicity studies should measure the level of chemicals as they are being administered to
animals. For example, the dose is measured at the site of administration (e.g., the nose
for rats) in nonclinical inhalation studies. The resulting systemic exposure is determined
based on toxicokinetic data.
• 	In general, FDA recommends inhalation studies to support the use of novel chemicals
because systemic toxicity studies by other routes do not sufficiently model drug
deposition in the lung (i.e., bronchi, bronchioles, and alveoli) that occurs following oral
inhalation exposure. Sponsors should conduct inhalation studies with an aerosol
generated using the proposed delivery system.
• 	Toxicokinetic measurements are usually obtained during ongoing nonclinical toxicity
studies rather than through separate studies.
• 	FDA recognizes that metabolism may affect toxicity. Sponsors should collect
toxicokinetic information for active ingredients in nonclinical toxicity studies. Regarding
nicotine, full characterization of absorption, distribution, metabolism, and excretion
(ADME) is generally not necessary because of existing information. However, for other
active ingredients, sponsors should conduct full ADME characterization as recommended

Contains Nonbinding Recommendations
6
in ICH M3(R2) and the guidance for industry Safety Testing of Drug Metabolites
(November 2016). ADME of excipients should be characterized as described in the
guidance for industry Nonclinical Studies for the Safety Evaluation of Pharmaceutical
Excipients. Sponsors should also address the safety of metabolites from heat-generated
chemicals, following the principles in the excipients guidance and the drug metabolites
guidance.
• 	Sponsors should follow available guidance on assessing drug substance and drug product
impurities20 and consider if the nicotine derived from plant-based products may be
associated with genotoxic impurities. Nicotine-specific impurities that are present at
higher levels than in approved drug products, considering the route of administration,
population, dose, and duration, are a concern if the impurities also exceed relevant ICH-
recommended limits. FDA will assess such impurities on a case-by-case basis.
• 	To support marketing approval, sponsors should submit a nonclinical toxicity assessment
of extractables and leachables of the delivery system and any container/closure system.
Sponsors should consider the level of these impurities under different conditions,
including when overheating occurs to produce a dry puff.
2. 	General Toxicity Studies
Following are FDA recommendations for a general toxicity assessment:
• 	For general toxicity studies to address novel chemicals, FDA recommends studies in
rodent and nonrodent species (see Appendix A), consistent with international standards
for pharmaceutical development.21 FDA strongly recommends that both species be dosed
by the inhalation route of administration, provided that this route of administration results
in systemic exposure in at least one species sufficient to assess toxicity compared with
the anticipated clinical systemic exposure. Inhalation studies should include adequate
control groups (e.g., sham air control group, vehicle control group) and a full panel of
tissues, not just tissues of the respiratory tract, to address route-dependent systemic
toxicity. For information about a positive control group exposed to cigarette smoke, see
section 1, General Principles.
— 	If systemic exposure is not sufficient after inhalation, we recommend that:
 	The rodent species be dosed by a noninhalation route to allow for systemic
toxicity assessment. Rodents are primarily nose breathers and may not receive
20 For impurities and degradants of the drug substance and drug product, see the ICH guidances for industry
Q3A(R2) Impurities in New Drug Substances (June 2008), Q3B(R2) Impurities in New Drug Products (July 2006),
and M7(R1) Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential
Carcinogenic Risk (March 2018). For solvents and elemental impurities, see the ICH guidances for industry Q3C
Impurities: Residual Solvents (December 1997) and Q3D Elemental Impurities (September 2015).
21 See ICH M3(R2).

Contains Nonbinding Recommendations
7
adequate buccal and oral exposure to the drug relevant to clinical use of orally
inhaled nicotine-containing drug products.
 	Because the nonrodent species, in contrast, are not exclusive nose breathers, they
should be dosed by the inhalation route of exposure using a method (e.g., a face
mask) that allows for oral and nasal inhalation of chemicals, resulting in buccal
and oral exposure to the drug, to model oral inhalation in humans.
 	The relevant mucosa be evaluated macroscopically and microscopically.
3. 	Developmental and Reproductive Toxicology
FDA recommends developmental and reproductive toxicity studies22 for novel chemicals for
which adequate toxicity data are not available. Sponsors should conduct these studies using a
route of administration that results in systemic exposure and exposure to the reproductive organs.
Following are FDA recommendations for a developmental and reproductive toxicity assessment:
• 	Sponsors should consider FDA’s general recommendations (see Appendix A) and refer to
ICH M3(R2) for more specific recommendations on the timing of developmental and
reproductive toxicity studies.
• 	Timing of developmental and reproductive toxicity studies can also be affected by
findings that are a cause for concern (e.g., when male reproductive organs are identified
as target organs in general toxicity studies).
• 	ICH M3(R2) also describes nonclinical data recommended to minimize the risk of
unintentional exposure of the embryo or fetus when including women of childbearing
potential in clinical trials.
4. 	Carcinogenicity
Following are FDA recommendations for a carcinogenicity assessment:
• 	FDA recommends that sponsors conduct carcinogenicity studies in two rodent species for
novel chemicals for which adequate toxicity data are not available, consistent with
international standards for pharmaceutical development.23 In general, sponsors should
conduct a carcinogenicity study that involves administering novel chemicals by the
inhalation route to mice or rats for 2 years. Sponsors should also conduct a second
carcinogenicity study by a route that produces adequate systemic exposure. This study
22 See the ICH guidance for industry S5(R2) Detection of Toxicity to Reproduction for Medicinal Products Toxicity
to Male Fertility (November 2005).
23 See the ICH guidance for industry S1A The Need for Long-Term Rodent Carcinogenicity Studies of
Pharmaceuticals (March 1996).

Contains Nonbinding Recommendations
8
can be a 2-year study or a shorter (usually 6 months) alternative carcinogenicity model
accepted by FDA, but either study should be conducted in a species different from that
used in the inhalation carcinogenicity study.24 Therefore, at a minimum, one species
should be dosed by the inhalation route, and the second species should have systemic
exposure to adequately assess systemic toxicity. Regardless of the route of
administration, all carcinogenicity studies should address a full panel of tissues.25 See
section II. B. 1. 2. for adequate negative control groups (e.g., sham air control group,
vehicle control group).
— 	Carcinogenicity studies by the oral route in two different rodent species (e.g., mouse
and rat) can be sufficient (i.e., no inhalation carcinogenicity study) for novel
chemicals when proliferative or preneoplastic changes in the respiratory tract are not
observed in chronic inhalation toxicity studies and when adequate local buccal and
airway exposure by the oral route is demonstrated.26
5. 	Genetic Toxicology
Following are FDA recommendations for a genetic toxicology assessment:
• 	FDA’s recommendation for genetic toxicity studies of novel chemicals depends on the
tobacco use and smoking status of subjects in the proposed clinical trials because of the
differential cancer risks in these populations.
— 	FDA recommends genetic toxicity studies, as described in ICH M3(R2), to assess the
toxicity of novel chemicals if clinical trials are conducted in subjects who are not
current smokers.
— 	In general, FDA does not recommend genetic toxicity studies to support clinical trials
in current smokers because this population is already at risk for cancer, and genetic
toxicity studies do not provide organ-specific risk assessment for cancer relevant to
current smokers.
24 See the ICH guidance for industry S1B Testing for Carcinogenicity of Pharmaceuticals (July 1997).
25 FDA recommends submitting the carcinogenicity study protocol or protocols for review in concurrence with the
Center for Drug Evaluation and Research’s Executive Carcinogenicity Assessment Committee before initiating the
studies. For further guidance regarding carcinogenicity studies, see the guidance for industry Carcinogenicity Study
Protocol Submissions (May 2002).
26 This is consistent with the guidance for industry and review staff Nonclinical Safety Evaluation of Reformulated
Drug Products and Products Intended for Administration by an Alternate Route (October 2015).

Contains Nonbinding Recommendations
9
APPENDIX A
Table 1: Milestones and Pivotal Nonclinical Toxicity Studies Recommended for Novel
Chemicals1,2,3
Milestones and
toxicity studies
Drug product development phase
Phase 1 	Phase 2 	Phase 3 	Phase 4
Clinical
Characteristics
Small number of
subjects and
short duration of
treatment
Larger number
of patients and
longer duration
of treatment
Larger number
of patients and
long-term
duration of
treatment
Large number of
patients and
limited control
on dose and
duration
General toxicity
Short-term
studies in rodent
and nonrodent
species
(adequate
dose/duration)
Maximum 6-
month rodent,
9-month
nonrodent
studies
(adequate
dose/duration
Chronic studies
(6-month rodent,
9-month
nonrodent)
Toxicity-specific
mechanistic
studies, if
recommended
Developmental
and
reproductive
toxicity
Not necessary 	Not necessary
Effects on
fertility and early
embryonic
development
(rodent study)
and embryofetal
development
(rodent and
nonrodent
studies)
Effects on pre-
and post-natal
development
(rodent study)
continued
1 Section II. B.1. of this guidance provides additional recommendations regarding the assessment of impurities,
including assessment of extractables and leachables of the delivery system of any container/closure system.
2 If an active ingredient other than nicotine is proposed, sponsors should refer to the nonclinical development
recommended in the ICH guidance for industry M3(R2) Nonclinical Safety Studies for the Conduct of Human
Clinical Trials and Marketing Authorization for Pharmaceuticals (January 2010) to complement the
recommendations above.
3 Timing of metabolite characterization follows recommendations in ICH M3(R2).

Contains Nonbinding Recommendations
10
Table 1, continued
Milestones and
toxicity studies
Drug product development phase
Phase 1 	Phase 2 	Phase 3 	Phase 4
Carcinogenicity 	Not necessary 	Not necessary 	Not necessary
Carcinogenicity
assessment (e.g.,
carcinogenicity
studies in two
rodent species)
Genetic toxicity
Depends on
tobacco
use/smoking
status of clinical
trial subjects
Depends on
tobacco
use/smoking
status of clinical
trial subjects
Depends on
tobacco
use/smoking
status of clinical
trial subjects
Addressed
earlier in
development, if
recommended