Recommended Acceptable 
Intake Limits for 
Nitrosamine Drug Substance-
Related Impurities (NDSRIs) 
Guidance for Industry 
 
This guidance is for immediate implementation. 
 
 
FDA is issuing this guidance for immediate implementation in accordance with 21 CFR 
10.115(g)(2).  Submit one set of either electronic or written comments on this guidance at any 
time.  Submit electronic comments to https://www.regulations.gov.  Submit written comments to 
the Dockets Management Staff (HFA-305), Food and Drug Administration, 5630 Fishers Lane, 
Rm. 1061, Rockville, MD 20852.  You should identify all comments with the docket number 
listed in the notice of availability that publishes in the Federal Register. 
 
For questions regarding this document, contact (CDER) Jason Bunting (301) 796-1292. 
 
 
 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
 
August 2023 
Pharmacology/Toxicology
   
Recommended Acceptable 
Intake Limits for 
Nitrosamine Drug Substance-
Related Impurities (NDSRIs) 
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) 
 
August 2023 
Pharmacology/Toxicology
   
Preface 
 
The Food and Drug Administration (FDA or Agency) is implementing this guidance without 
prior public comment because the Agency has determined that prior public participation is not 
feasible or appropriate (see section 701(h)(1)(C)(i) of the Federal Food, Drug, and Cosmetic Act 
(21 U.S.C. 371(h)(1)(C)(i)) and 21 CFR 10.115(g)(2) and (g)(3)).  FDA made this determination 
because of the importance of providing additional timely information to manufacturers and 
applicants regarding recommended acceptable intake limits of nitrosamine drug substance-
related impurities (NDSRIs), a class of nitrosamine impurities that have been identified in many 
drug products and also could be present in active pharmaceutical ingredients (APIs).  Since the 
publication of the guidance for industry Control of Nitrosamine Impurities in Human Drugs, 
FDA has received reports of NDSRIs, which share structural similarity to the API and are unique 
to each API.  Based on the chemical structure of the API, there is a risk of NDSRIs forming in a 
substantial number of drug products.  This guidance is being implemented immediately, but it 
remains subject to comment in accordance with the Agencys good guidance practices. 
 
Comments may be submitted at any time for Agency consideration.  Submit written comments to 
the Dockets Management Staff (HFA-305), Food and Drug Administration, 5630 Fishers Lane, 
Rm. 1061, Rockville, MD 20852.  Submit electronic comments to https://www.regulations.gov. 
All comments should be identified with the docket number FDA-2020-D-1530 and complete title 
of the guidance. 
  Contains Nonbinding Recommendations 
TABLE OF CONTENTS 
 
I.  INTRODUCTION............................................................................................................. 1 
II.  SCOPE ............................................................................................................................... 2 
III.  BACKGROUND ............................................................................................................... 3 
A.  Challenges in Determining AI Limits for NDSRIs ..................................................................... 5 
IV.  RECOMMENDATIONS FOR AI LIMITS BASED ON PREDICTED 
CARCINOGENIC POTENCY CATEGORIZATION ................................................. 6 
A.  Introduction to Predicted Carcinogenic Potency Categorization Approach ............................ 6 
B.  Specific Application of the Predicted Carcinogenic Potency Categorization Approach ......... 8 
C.  Determining the Predicted Carcinogenic Potency Categorization and Associated 
Recommended AI Limit ................................................................................................................ 9 
D.  Recommendations for Manufacturers and Applicants Whose Drug Products Contain 
Multiple Nitrosamines ................................................................................................................. 11 
E.  Recommended AI Limits for NDSRIs at Risk of Formation in Certain APIs ....................... 11 
V.  IMPLEMENTATION OF RECOMMENDED AI LIMITS FOR NDSRIS, 
INCLUDING THOSE BASED ON THE PREDICTED CARCINOGENIC 
POTENCY CATEGORIZATION ................................................................................ 12 
A.  Recommended Timeline for Implementing Risk Assessments, Confirmatory Testing, and 
Submission of Required Changes for NDSRIs .......................................................................... 12 
1. Recommended Timeline for Approved or Marketed Drug Products ............................................. 12 
2. Recommended Timeline for Drug Products in Development and Under FDA Review ................. 14 
a.  Pre-submission stage ............................................................................................................... 14 
b.  Applications pending with the Agency ................................................................................... 14 
B.  Recommendations for Manufacturers and Applicants That Have Marketed Products With 
Levels of NDSRIs Above the FDA Recommended AI Limits .................................................. 14 
1. Additional Considerations for Maintaining the Drug Supply for Marketed Products .................. 15 
C.  Approaches to Justify or Qualify a Proposed Alternative AI Limit ....................................... 16 
APPENDIX A: DETERMINATION OF POTENCY SCORE ............................................... 18 
  Contains Nonbinding Recommendations 
Recommended Acceptable Intake Limits for  
Nitrosamine Drug Substance-Related Impurities 
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 manufacturers and applicants of drugs, including prescription and over-
the-counter (OTC) drug products,2,3 with a recommended framework for predicting the 
mutagenic and carcinogenic potential of nitrosamine drug substance-related impurities (NDSRIs) 
that could be present in drug products and recommends acceptable intake (AI) limits for 
NDSRIs.  NDSRIs are a class of nitrosamine impurities that have been identified in many drug 
products4 and also could be present in active pharmaceutical ingredients (APIs).5  These 
impurities share structural similarity to the API (having the API or API fragment in the chemical 
structure) and are therefore unique to each API.  NDSRIs generally form in the drug product 
 
1 This guidance has been prepared by the Office of Regulatory Policy in the Center for Drug Evaluation and 
Research (CDER) at the Food and Drug Administration. 
2 For purposes of this guidance, we use the term drug or drug product to refer to human drug and biological 
products, including drug-led and biologic-led combination products, regulated by CDER, unless otherwise specified.  
See section II (Scope) for full description of the applicability of this guidance document. 
3 For purposes of this guidance, we use the term manufacturers and applicants to refer collectively to new drug 
application, abbreviated new drug application, and biologics license application applicants and application holders, 
sponsors of proposed drug products, drug master file (DMF) holders, owners, manufacturers, or compounders of 
marketed drug products that are not the subject of approved applications (such as drug products compounded by 
outsourcing facilities pursuant to section 503B of the Federal Food, Drug, and Cosmetic Act (FD&C Act) (21 
U.S.C. 353b) or drug products subject to section 505G of the FD&C Act (i.e., OTC monograph drugs)), unless 
otherwise specified, and active pharmaceutical ingredient (API) and drug product manufacturers.   
4 Generally, the presence of high levels of NDSRIs has been associated with drug products rather than APIs because 
NDSRI formation usually results from a reaction between the API or API fragment and nitrosating agents in the 
drug formulation.  However, NDSRIs can potentially form in APIs when nitrosating agents are present in the API 
manufacturing process or when APIs undergo processing steps that can potentially induce their formation such as 
fluid bed drying at an elevated temperature and jet milling because these can create favorable conditions in which 
nitrogen oxides can react with at-risk APIs.  Because the presence of NDSRIs is predominantly associated with drug 
products rather than APIs, for simplicity, we use the term drug product or drug products throughout this guidance.  
However, the recommendations in this guidance also apply to APIs to the extent that NDSRIs are associated with 
APIs.    
5 The term API used throughout this guidance refers to the active ingredient in a drug product.  See 21 CFR 
210.3(b)(7) and 314.3(b) (defining active ingredient and drug substance).  The terms API and drug substance are 
also used interchangeably in this guidance. 
  1  Contains Nonbinding Recommendations 
through nitrosation of APIs (or API fragments) that have secondary or tertiary amines when 
exposed to nitrosating agents such as residual nitrites in excipients used to formulate the drug 
product.  NDSRIs often lack carcinogenicity and mutagenicity study data (typically from animal 
studies) from which an AI limit can be determined.  This guidance provides a recommended 
methodology for AI limit determination that uses structural features of NDSRIs to generate a 
predicted carcinogenic potency categorization and corresponding recommended AI limit that 
manufacturers and applicants can apply, in the absence of other FDA recommended AI limits, in 
their evaluations of approved and marketed drug products as well as products in development or 
under review by FDA.   
 
Uncertainty about the presence and acceptability of the level of an NDSRI in drug products raises 
regulatory challenges and has led to some applicants conducting unnecessary studies or, in some 
cases, discontinuing drug products from the market.  Because nitrosamine impurities have been 
identified in many drug products, disruptions in supply and access have increased, sometimes 
resulting in drug shortages.  These challenges can impact patient access to medications, 
particularly with respect to drug products that are considered medically necessary.   
 
This guidance recommends a risk-based safety assessment of NDSRIs and can be used by 
manufacturers and applicants to identify AI limits for NDSRIs in their drug products and APIs in 
conjunction with the recommendations in the guidance for industry Control of Nitrosamine 
Impurities in Human Drugs (February 2021) (Nitrosamine Guidance).6  FDA recognizes that the 
AI limits recommended by the Agency and those generated by manufacturers and applicants may 
evolve with advances in the science and generation of data for nitrosamines.  As industry groups 
and consortia perform investigations that provide insights into mutagenicity and carcinogenicity 
risks of nitrosamine impurities, FDA encourages sharing such information to help expand the 
available knowledge base. 
 
In general, FDAs guidance documents do not establish legally enforceable responsibilities. 
Instead, guidances describe the Agencys 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.  SCOPE 
 
This guidance applies to drugs, including prescription and OTC drug products that are the subject 
of an approved or pending new drug application (NDA) or abbreviated new drug application 
(ANDA), as well as products7 not marketed under a drug application, including nonprescription 
drugs subject to section 505G of the Federal Food, Drug, and Cosmetic Act (FD&C Act) (i.e., 
OTC monograph drugs), or otherwise subject to current good manufacturing practice (CGMP).  
This guidance also applies to prescription and OTC drug products in clinical development.  In 
 
6 FDA published the Nitrosamine Guidance on September 3, 2020 (85 FR 55017) and updated it on February 24, 
2021.  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.  
7 See footnote 2.  
  2  Contains Nonbinding Recommendations 
addition, this guidance applies to biological products8 that contain chemically synthesized 
fragments or biologic-led combination products that contain a drug constituent part whether such 
products are in development or the subject of an approved or pending biologics license 
application (BLA).  The recommendations in this guidance apply to both drug product and drug 
substance manufacturers.9  
 
The recommendations in this guidance do not apply to NDSRIs that are detected in products 
indicated for use in patients with advanced cancers (i.e., drug products falling under the scope of 
International Council for Harmonisation (ICH) guidance for industry S9 Nonclinical Evaluation 
for Anticancer Pharmaceuticals (March 2010) (ICH S9)).  Principles of ICH S9, ICH guidance 
for industry Q3A(R2) Impurities in New Drug Substances (June 2008), and ICH guidance for 
industry Q3B(R2) Impurities in New Drug Products (August 2006) may be applied to set 
impurity specifications for these products. 
 
 
III.  BACKGROUND 
 
FDA has been addressing the evaluation and management of nitrosamine impurities in certain drug 
products since 2018 when FDA was informed of the presence of an impurity identified as N-
nitrosodimethylamine (NDMA) in the angiotensin receptor blocker valsartan.10  The initial reports 
of nitrosamine impurities in drug products were of small molecule nitrosamines such as NDMA, 
compounds that form by a nitrosating reaction between amines (secondary, tertiary, or quaternary 
amines) and nitrous acid (nitrite salts under acidic conditions), generally due to API synthesis 
conditions that introduce nitrous acid, nitrite and amide solvents.  In 2020, FDA issued guidance to 
industry, the Nitrosamine Guidance, recommending that manufacturers of APIs and drug products 
take steps to detect and prevent unacceptable levels of nitrosamine impurities in drug products, or 
to avoid their presence when feasible.  The Nitrosamine Guidance introduced a three-step process 
that manufacturers and applicants should take to mitigate nitrosamine impurities in their drug 
products: (1) conduct risk assessments for nitrosamines in their APIs and drug products; (2) 
conduct confirmatory testing if risks are identified; and (3) report changes implemented to prevent 
or reduce the presence of nitrosamine impurities in APIs and drug products in approved and 
pending NDAs and ANDAs. 
 
 
8 As explained in footnote 2, we use the term biological products in this guidance to refer only to biological products 
regulated by CDER. 
9 See footnote 5.  
10 See the FDA Statement on FDAs Ongoing Investigation Into Valsartan Impurities and Recalls and an Update on 
FDAs Current Findings, available at https://www.fda.gov/news-events/press-announcements/fda-statement-fdas-
ongoing-investigation-valsartan-impurities-and-recalls-and-update-fdas-current. 
  3  Contains Nonbinding Recommendations 
As explained in the Nitrosamine Guidance, manufacturers of APIs and drug products should take 
appropriate measures to prevent unacceptable levels of nitrosamines impurities in their drug 
products.11  Because nitrosamine compounds have the potential to be potent genotoxic agents in 
several animal species and some are classified as probable or possible human carcinogens,12 they 
are included in a group of high potency mutagenic carcinogens referred to as cohort of concern 
compounds in the ICH guidance for industry M7(R2) Assessment and Control of DNA Reactive 
(Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk (July 2023) 
(ICH M7(R2)).  The ICH M7(R2) guidance provides recommendations on control of any known 
or potential mutagenic carcinogens, such as nitroso-compounds at or below a level associated 
with negligible human cancer risk associated with exposure to these impurities.13  
 
An AI limit is a level that approximates an increased cancer risk of one additional case in 
100,000 people based on a conservative assumption of daily exposure to the impurity over a 
lifetime (70 years).  To phrase it another way, an AI limit is a daily exposure to a compound such 
as NDMA that approximates a 1:100,000 cancer risk after 70 years of exposure.14  This risk level 
represents a small theoretical increase in risk when compared to human overall lifetime 
incidence of developing any type of cancer, which is greater than 1 in 3.  If a potential risk for a 
nitrosamine impurity has been identified, and appropriate testing has confirmed the nitrosamine 
level, then an appropriate control strategy leveraging process understanding and analytical 
controls should be developed to ensure that the nitrosamine impurity is at or below the AI 
limit.15  As recommended in ICH M7(R2), an AI limit is generally determined based on database 
and literature searches for carcinogenicity and bacterial mutagenicity data.   
 
As FDAs understanding of NDRSIs is evolving, this guidance recommends a risk-based safety 
assessment of NDSRIs.  The predicted carcinogenic potency categorization approach 
recommended in this guidance complements the approach recommended in ICH M7(R2).  It is 
based on principles of ICH M7(R2) that recommend the use of structure-activity relationship 
(SAR) concepts to assess and classify the mutagenic and carcinogenic risk of impurities to limit 
potential carcinogenic risk in drug products.  The science on structure-activity relationships of N-
nitroso compounds has progressed, so that at this time, FDA recommends that this predictive 
methodology be used to assess the carcinogenic potency of NDSRIs and more accurately predict 
their mutagenic potential.  This is important because it is currently unknown if all or some 
NDSRIs are in fact high potency mutagenic carcinogens.  This guidance considers that not all 
NDSRIs have the same carcinogenic potency, as reflected by the different recommended AI 
limits associated with each potency category. 
 
 
11 Nitrosamine Guidance at 9. 
12 See the International Agency for Research on Cancer (IARC) Monographs on the Identification of Carcinogenic 
Hazards to Humans web page at https://monographs.iarc.who.int/list-of-classifications.  See, e.g., NDMA, N-nitroso 
piperidine (NPIP), 4-(methylnitrosoamino)-1-(3-pyridinyl)-1-butanone (NNK), N-nitroso pyrrolidine (NPYR), and 
N-nitroso morpholine (NMOR). 
13 See ICH M7(R2), available at https://www.fda.gov/regulatory-information/search-fda-guidance-documents. 
14 See Nitrosamine Guidance at 10 and Appendix B (FDA Determination of Acceptable Intake Limits).   
15 An AI limit can be converted into a parts per million (ppm) control limit for a manufacturer or applicant.  The 
conversion varies by product and is calculated based on a drugs maximum daily dose (MDD) as reflected in the 
drug labeling (ppm = AI (nanograms (ng))/MDD (milligrams)).  Nitrosamine Guidance at 10. 
  4  Contains Nonbinding Recommendations 
A.  Challenges in Determining AI Limits for NDSRIs 
 
Determining an AI limit for NDSRIs is often more challenging than determining AI limits for 
small molecule nitrosamines, primarily because NDSRIs are unique to each API and, therefore, 
there are usually limited or no existing safety data (e.g., rodent carcinogenicity data) on 
NDSRIs.16  FDA has communicated recommended AI limits for a limited number of NDSRIs, 
but unlike more commonly known nitrosamines (such as those identified in the Nitrosamine 
Guidance), a recommended AI limit has not previously been determined for most NDSRIs.   
 
A recommended AI limit is based on a safety assessment that includes evaluation of the 
mutagenic and carcinogenic potential of the impurity and represents the level at or below which 
FDA has determined that the impurity or impurities would not pose a safety concern for patients 
taking the drug product.  A compound-specific AI can be calculated based on rodent 
carcinogenic potency data such as TD  values (doses giving a 50 percent tumor incidence 
50
equivalent to a cancer risk probability level of 1:2) identified in the published scientific 
literature.  Carcinogenicity data may be available from the Carcinogenic Potency Database 
(CPDB) or Lhasa Carcinogenicity Database (LCDB).17  TD  values for approximately 140 
50
nitrosamines, mostly small molecule nitrosamines, were originally reported in the CPDB and 
have been incorporated into the LCDB.  The carcinogenic potency values range over four orders 
of magnitude, and some nitrosamines have been found to be non-carcinogenic.18  However, 
many studies are not robust and cannot solely be relied on for calculating AI limits. 
 
When the mutagenic potential of an NDSRI is not adequately characterized, FDA and applicants 
have used (quantitative) SAR methods to support the identification of a robustly-tested surrogate 
that is similar in structure and reactivity to the NDSRI to generate an estimate of carcinogenic 
potency from which an AI limit can be scientifically determined.  In this scenario, surrogates are 
compounds containing an N-nitroso structural alert in the same chemical environment as an 
NDSRI and for which robust carcinogenicity data are available.19  The rationale for the choice of 
surrogate (similar in structure and reactivity) is significant because test data from the identified 
surrogate are then used to generate an estimate, either quantitatively or qualitatively, for the data-
poor compound (commonly referred to as a read-across analysis).20  
 
The structural environment surrounding the N-nitroso group of the NDSRI is an important factor 
when selecting appropriate reference compounds for a read-across analysis.  It may include 
 
16 See ICH M7(R2) at 9 to 11 and Note 4 on calculating a compound-specific AI limit.   
17 Lhasa Carcinogenicity Database (LCDB) is available at https://carcdb.lhasalimited.org.  As indicated in the Field 
Descriptions from LCDB, LCDB includes data from Carcinogenic Potency Database (CPDB).  CPDB is available at 
https://files.toxplanet.com/cpdb/index.html.  
18 For example, see Thresher A, Gosling JP, and Williams R, 2019, Generation of TD50 Values for Carcinogenicity 
Study Data, Toxicol Res, 8:696-703; Thresher A, Foster R, Ponting DJ, et al., 2020, Are All Nitrosamines 
Concerning? A Review of Mutagenicity and Carcinogenicity Data, Regul Toxicol Pharmacol, 116:104749. 
19 For example, see Cross KP and Ponting DJ, 2021, Developing Structure-Activity Relationships for N-Nitrosamine 
Activity, Comput Toxicol, 20:100186; Thomas R, Tennant RE, Oliveira AAF, and Ponting DJ, 2022, What Makes a 
Potent Nitrosamine? Statistical Validation of Expert-Derived Structure-Activity Relationships, Chem Res Toxicol, 
35:19972013; and Ponting DJ, Dobo KL, Kenyon MO, and Kalgutkar AS, 2022, Strategies for Assessing 
Acceptable Intakes for Novel N-Nitrosamines Derived From Active Pharmaceutical Ingredients, J Med Chem, 
65:1558415607. 
20 See section IV. 
  5  Contains Nonbinding Recommendations 
consideration of the degree of substitution, steric bulk, electronic influences, potential for 
metabolic activation, stability/reactivity of the resulting metabolites, and overall molecular 
weight.   
 
 
IV.  RECOMMENDATIONS FOR AI LIMITS BASED ON PREDICTED 
CARCINOGENIC POTENCY CATEGORIZATION  
 
A.  Introduction to Predicted Carcinogenic Potency Categorization Approach 
 
This section describes a recommended methodology that uses predicted carcinogenic potency 
categorization to assign a recommended AI limit to an NDSRI based on the NDSRIs activating 
and deactivating structural features.  In the context of this guidance, activating or deactivating 
features are defined as molecular substructures that are associated with an increase or decrease, 
respectively, in carcinogenic potency.  The predicted carcinogenic potency categorization 
approach to determine a recommended AI limit incorporates SAR concepts described in recent 
scientific publications for nitrosamine compounds.21  The approach assumes that the -
hydroxylation mechanism of metabolic activation22 is responsible for the mutagenic and highly 
potent carcinogenic response observed for many nitrosamines.  Structural features that directly 
increase or decrease the favorability of the activation mechanism or that increase the clearance of 
the nitrosamine by other biological pathways will have a corresponding effect on carcinogenic 
potency.  Therefore, a prediction of the mutagenic potential and carcinogenic potency of an 
NDSRI can be generated based on its structural features. 
 
The predicted carcinogenic potency categorization approach described in this guidance applies to 
NDSRIs bearing a carbon atom on both sides of the N-nitroso group, where the carbon is not 
directly double bonded to a heteroatom (i.e., N-nitrosamides, N-nitrosoureas, N-
nitrosoguanidines and other related structures are excluded).  Additionally, the predicted 
carcinogenic potency categorization approach does not apply to NDSRIs where the N-nitroso 
group is within an aromatic ring (e.g., nitrosated indoles).    
 
The predicted carcinogenic potency categorization approach enables manufacturers and applicants 
to identify the appropriate potency category and associated recommended AI limits for NDSRIs in 
APIs and drug products and to facilitate development of methods for confirmatory testing of 
impurity levels in drug batches.  Potency categorization offers a science-based predictive solution 
to recommending AI limits for NDSRIs.  FDA scientists, in collaboration with other international 
drug regulatory authorities, developed an approach to evaluate the predicted carcinogenic potency 
of NDSRIs based on the structural features of these compounds.  The approach is based on 
information from published data on small molecule nitrosamines that are available through CPDB 
and LCDB, combined with chemical and biological mechanistic considerations described in recent 
nitrosamine scientific publications.23  The goal is to set recommended AI limits by predicting the 
potency of the substances using activating and deactivating structural features present in 
 
21 See footnote 18. 
22 Li Y and Hecht SS, 2022, Metabolic Activation and DNA Interactions of Carcinogenic N-Nitrosamines to Which 
Humans Are Commonly Exposed, Int J Mol Sci, 23:4559. 
23 See footnote 18. 
  6  Contains Nonbinding Recommendations 
nitrosamines.  In developing the model, relative weights were assigned to activating and 
deactivating features based on SAR trends reported in the published literature, SAR trends 
observed in the CPDB and LCDB, and a general scientific understanding of the chemical and 
biological mechanistic pathways of the compounds.  The approach based on structural features can 
then be applied to an NDSRI to predict the relative carcinogenic potency of the NDSRI.  This 
approach considers the effects of multiple features in the molecule and leverages a larger body of 
carcinogenicity data in the published scientific literature than is available for establishing an AI 
based on a surrogate nitrosamine molecule.  FDA anticipates that the analysis of these structural 
features, and the weighting that has been assigned to these features, may change over time as the 
science advances and additional data are generated. 
 
The predicted carcinogenic potency categorization and resulting recommended AI limit approach 
described in this guidance should not be applied to NDSRIs in circumstances in which FDA 
otherwise recommends an AI limit (e.g., based on compound-specific assessments or read-across 
analysis from a surrogate).24  Generally, FDA has communicated recommended AI limits directly 
to an applicant or manufacturer or through an FDA guidance (the Nitrosamine Guidance).  FDA 
may continue to do so, including in connection with this guidance.25  When FDA communicates a 
recommended AI limit based on compound-specific assessments or read-across analysis from a 
surrogate, manufacturers and applicants should apply that recommended AI limit rather than using 
the predicted carcinogenic potency categorization approach in this guidance to identify a 
recommended AI limit.    
 
FDA continues to work with international regulators in the development and use of this modeling 
approach to identify recommended AI limits for NDSRIs to minimize the potential impact of 
nitrosamine impurities on the drug supply.  
 
The Agency recognizes that the science is evolving in the prediction of mutagenic potential and 
carcinogenic potency based on SAR concepts.  Therefore, the predicted carcinogenic potency 
categorization method described in section IV.B is a conservative approach that represents the 
best available science at this time and is expected to be further refined and expanded as new data 
become available.  Additionally, as with all guidance documents, manufacturers and applicants 
can use an alternative approach if it satisfies the requirements of the applicable statutes and 
regulations. 
 
 
24 In corresponding with certain manufacturers and applicants, FDA previously recommended that certain NDSRIs, 
for which there is no compound-specific data and no suitable surrogate for a read-across analysis, be controlled at 
levels that do not exceed a conservative AI limit of 26.5 ng/day.  For these NDSRIs, we now recommend that 
manufacturers and applicants use the framework recommended in this guidance to determine an appropriate 
predicted carcinogenic potency category and corresponding recommended AI limit. 
25 To reflect the evolving and highly technical nature of the relevant information, FDA intends to provide certain 
updated information on recommended AI limits for NDSRIs, in connection with this guidance, at the CDER web 
page at https://www.fda.gov/regulatory-information/search-fda-guidance-documents/updated-information-
recommended-acceptable-intake-limits-nitrosamine-drug-substance-related.  This associated information will be 
updated periodically.  For the most recent version, check the web page. 
  7  Contains Nonbinding Recommendations 
B.  Specific Application of the Predicted Carcinogenic Potency Categorization 
Approach 
 
FDA recommends the following AI limits based on the predicted carcinogenic potency 
categorization approach for NDSRIs.  
 
Table 1 shows the predicted carcinogenic potency categories and associated recommended AI 
limits.  Notably, the associated recommended AI limits generally represent the conservative 
lower limit for the range of potencies falling within each category.   
 
Table 1. The Five Predicted Carcinogenic Potency Categories and Associated 
Recommended AI Limits for NDSRIs 
 
 Potency  Recommended AI 
Comments 
Category  (ng/day) 
The recommended AI limit of 26.5 ng/day* is equal to the class-specific limit for 
nitrosamine impurities based on the most potent, robustly tested nitrosamine, N-
nitrosodiethylamine (NDEA).** NDSRIs assigned to Category 1 are predicted to have 
1   26.5 
carcinogenic  potency  no  higher  than  the  class-specific  limit  for  nitrosamine 
impurities. 
 
The recommended AI limit of 100 ng/day is representative of two potent, robustly 
tested nitrosamines, N-nitrosodimethylamine (NDMA) and 4-(methylnitrosamino)-
1-(3-pyridyl)-1-(butanone) (NNK), which have recommended AI limits of 96 ng/day 
2   100 
and 100 ng/day, respectively. NDSRIs assigned to Category 2 are predicted to have 
carcinogenic potency no higher than NDMA and NNK. 
 
Compared to Potency Category 2, NDSRIs in this category have lower carcinogenic 
potency due to, for example, the presence of a weakly deactivating structural feature. 
3   400  The recommended AI limit was set to reflect a 4-fold decrease in carcinogenic 
potency from Category 2.    
 
NDSRIs assigned to Category 4 may be metabolically activated through an alpha-
hydroxylation pathway but are predicted to be of low carcinogenic potency, for 
example, because the pathway is disfavored due to steric or electronic influences, or 
4   1500 
because clearance pathways are favored.  The recommended AI limit of 1500 ng/day 
is set at the TTC per ICH M7(R2).*** 
 
NDSRIs assigned to Category 5 are not predicted to be metabolically activated via 
an -hydroxylation pathway due to steric hindrance or the absence of -hydrogens, 
5   1500  or are predicted to form unstable species that will not react with DNA. The 
recommended AI limit of 1500 ng/day is set at the TTC per ICH M7(R2).*** 
 
 
AI = acceptable intake; ng = nanogram; NDSRI = nitrosamine drug substance-related impurities; TTC = threshold of 
toxicological concern. 
* For products intended for marketing in the United States, FDA recommends an AI limit of 26.5 ng/day for 
Category 1, even if a different limit is recommended in other regulatory regions.  
** See the guidance for industry Control of Nitrosamine Impurities in Human Drugs (February 2021).   
  8  Contains Nonbinding Recommendations 
*** See the International Council for Harmonisation guidance for industry M7(R2) Assessment and Control of DNA 
Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk (July 2023) (ICH 
M7(R2)).  The threshold of toxicological concern (TTC) of 1.5 micrograms/day (1500 ng/day) as explained in ICH 
M7(R2), represents an AI for any unstudied chemical that poses a negligible risk of carcinogenicity or other toxic 
effect.    
 
C.  Determining the Predicted Carcinogenic Potency Categorization and 
Associated Recommended AI Limit 
 
The flowchart shown in Figure 1 describes the recommended process of assigning an NDSRI to 
a predicted carcinogenic potency category, with a corresponding recommended AI limit, based 
on an assessment of activating and deactivating structural features present in the NDSRI.  Two 
example calculations for determining the Potency Score and corresponding potency category are 
provided in Appendix A.  The - and -carbons in the NDSRI referenced in the flowchart are 
defined relative to the N-nitroso group, as illustrated in Figure 2.   
 
As noted above, the predicted carcinogenic potency categorization and recommended AI limit 
approach described in this guidance applies to NDSRIs bearing a carbon atom on both sides of 
the N-nitroso group, where the carbon is not directly double bonded to a heteroatom (i.e., N-
nitrosamides, N-nitrosoureas, N-nitrosoguanidines and other related structures are excluded).  
Additionally, the potency categorization approach does not apply to NDSRIs where the N-nitroso 
group is within an aromatic ring (e.g., nitrosated indoles).  Figure 1 illustrates how to predict the 
potency categorization of an NDSRI, and crosswalk to the recommended AI limit.  For NDSRIs 
containing two N-nitroso groups, the group with the highest predicted carcinogenic potency 
category should be used to determine the recommended AI limit for the entire molecule.26  
 
26 For NDSRIs containing more than two N-nitroso groups, the applicant or manufacturer should contact the Agency 
for further guidance.  
  9  Contains Nonbinding Recommendations 
Figure 1. Flowchart to Predict the Carcinogenic Potency Category of an NDSRI and 
Identify an Associated Recommended AI Limit 
 
 
 
 
NDSRI = nitrosamine drug substance-related impurities; AI = acceptable intake; ng = nanogram. 
* A tertiary -carbon is defined as an -carbon atom in an sp3 hybridization state, bonded to three other carbon atoms.  
**To calculate Potency Score, see Appendix A. 
 
  10  Contains Nonbinding Recommendations 
Figure 2. Structural Representation of - and -carbons on an NDSRI. 
 
 
 
D.  Recommendations for Manufacturers and Applicants Whose Drug Products 
Contain Multiple Nitrosamines 
 
As discussed above, the recommended AI limits for NDSRIs correspond to a single NDSRI in a 
drug product.  If more than one nitrosamine impurity (NDSRI(s) and/or small molecule 
nitrosamine(s)) is identified and the total level of nitrosamines exceeds the recommended AI 
limit for the most potent nitrosamine in the drug product based on the maximum daily dose, the 
manufacturer or applicant should contact the Agency.  FDA also recognizes that if the 
recommended AI limits for the individual nitrosamines vary greatly, basing the total nitrosamine 
limit on the most potent individual nitrosamine limit might not be practical.  In such cases, FDA 
encourages manufacturers to contact the Agency if proposing a nitrosamine limit other than the 
recommended AI of the most potent nitrosamine impurity detected.27 
 
E.  Recommended AI Limits for NDSRIs at Risk of Formation in Certain APIs 
 
FDA has identified a number of APIs that have secondary or tertiary amines and are therefore at 
risk for forming NDSRIs.  Hypothetically, under certain conditions related to the formulation 
and manufacturing process for the drug product, such as residual nitrites in excipients used to 
formulate the drug product, these APIs could form NDSRIs.  FDA applied the predicted 
carcinogenic potency categorization to these APIs.  More specifically, based on scientific 
analysis of chemical structures, we have concluded that when an API contains hypothetical 
nitrosated forms of secondary amine- and dimethyl tertiary amine- groups, these recommended 
 
27 Applicants can discuss with FDA or propose an AI limit of total nitrosamine impurities through the following 
mechanism, as appropriate: Type C meeting requests for NDAs and 351(a) BLAs, pre-ANDA meetings, and pre-
submission meetings for ANDAs as well as supplements, amendments, controlled correspondences for NDAs, 
ANDAs, and BLAs. 
  11  Contains Nonbinding Recommendations 
AI limits based on predicted carcinogenic potency should be applied to the NDSRIs to the extent 
that they form in these APIs.28   
 
 
V.  IMPLEMENTATION OF RECOMMENDED AI LIMITS FOR NDSRIs, 
INCLUDING THOSE BASED ON THE PREDICTED CARCINOGENIC 
POTENCY CATEGORIZATION 
 
A.  Recommended Timeline for Implementing Risk Assessments, Confirmatory 
Testing, and Submission of Required Changes for NDSRIs 
 
FDA expects manufacturers and applicants to ascertain the presence of nitrosamine impurities, 
including NDSRIs, using the three-step mitigation strategy described in sections III and V of the 
Nitrosamine Guidance, but is clarifying here that the recommended timelines in that guidance do 
not apply to risk assessments, confirmatory testing, and submission of required changes related 
to NDSRIs.29  For NDSRIs, FDA recommends different implementation timelines for 
manufacturers and applicants depending on the regulatory status of the drug product.  
 
1.  Recommended Timeline for Approved or Marketed Drug Products  
 
FDA has learned that some manufacturers and applicants have considered the nitrosamine 
impurities originally identified in the Nitrosamine Guidance but have not considered NDSRIs in 
their risk assessments.  FDA recommends that if NDSRIs were not considered in previous risk 
assessments, manufacturers and applicants reevaluate the risk within 3 months of publication of 
this guidance, with a recommended completion date by November 1, 2023, as part of overall risk 
management.  Confirmatory testing using sensitive and appropriately validated methods should 
start as soon as the risk of an NDSRI is identified and should begin immediately for drug 
products considered to be at high risk.  If NDSRIs are detected in the drug product at levels that 
exceed the recommended AI limit (e.g., associated with their predicted carcinogenic potency 
category), then FDA recommends that manufacturers and applicants develop control strategies 
and/or design approaches to control NDSRIs within acceptable levels.30   
 
To enable manufacturers and applicants to complete the three-step mitigation strategy based on 
increased awareness of NDSRIs, FDA recommends conclusion of NDSRI confirmatory testing 
 
28 To reflect the evolving and highly technical nature of the relevant information, FDA intends to provide a list of 
APIs that hypothetically could be at risk of forming NDSRIs due to the presence of secondary or tertiary amines.  
This list will include associated recommended AI limits for these hypothetical NDSRIs based on predicted 
carcinogenic potency categorization, in connection with this guidance, at the CDER web page at 
https://www.fda.gov/regulatory-information/search-fda-guidance-documents/updated-information-recommended-
acceptable-intake-limits-nitrosamine-drug-substance-related.  This is not an all-inclusive list of APIs that have the 
potential to form NDSRIs.  In addition, the recommended AI limits for the APIs on this website do not necessarily 
correspond to the presence or level of any NDSRI(s) found in any particular drug product.  This associated 
information will be updated periodically.  For the most recent version, check the web page.    
29 See, e.g., 21 CFR 314.60, 314.70, 314.96, 314.97, and 601.12. 
30 See section V.B.1.  FDA intends to issue additional guidance on approaches to reduce or prevent NDSRI 
formation in drug products. 
  12  Contains Nonbinding Recommendations 
of drug products and submission of required changes in drug applications by August 1, 2025.31  
By August 1, 2025, manufacturers and applicants should ensure that any NDSRIs in their drug 
products meet the FDA-recommended AI limit (e.g., associated with their predicted carcinogenic 
potency category).32  
 
FDA acknowledges that the implementation timeline includes investigating the root causes of 
NDSRI impurities, identifying effective changes as appropriate (e.g., changes in manufacturing 
process, component supplier, or product formulation),33 and confirming that any proposed 
changes will minimize the presence of NDSRIs without otherwise adversely affecting drug 
product quality.  FDA may request an expedited risk assessment, confirmatory testing, or other 
regulatory action based on information available to the Agency. 
 
If a risk of NDSRIs in a drug product is identified, confirmatory testing of batches should be 
conducted using sensitive and appropriately validated methods.34  If a nitrosamine impurity is 
detected, manufacturers should investigate the root cause and implement changes in the 
manufacturing process or product formulation to eliminate, mitigate, or reduce nitrosamine 
impurities consistent with the recommendations in the Nitrosamine Guidance.35  Manufacturers 
must implement any changes in accordance with appropriate requirements.36   In addition, 
applicants must submit proposed formulation changes through supplements or amendments to their 
applications.37  Generally, FDA considers reformulation of an approved drug product a major 
change requiring a prior approval supplement (PAS).38    
 
If an NDSRI falls at or below the FDA-recommended AI limit (e.g., associated with its predicted 
carcinogenic potency category),39 the manufacturer or applicant should still develop an 
appropriate control strategy to ensure that the nitrosamine level is reliably at or below that AI 
 
31 See, e.g., 21 CFR 314.60, 314.70, 314.96, 314.97, and 601.12. 
32 In certain circumstances, consistent with those described in section V.C., manufacturers or applicants may provide 
a scientifically justified rationale to pursue an AI limit different than the FDA-recommended limit.  In those cases, 
we recommend that manufacturers complete this process in time to submit any relevant changes to applications by 
August 1, 2025. 
33 See, e.g., FDA, Updates on Possible Mitigation Strategies To Reduce the Risk of Nitrosamine Drug Substance-
Related Impurities in Drug Products, available at https://www.fda.gov/drugs/drug-safety-and-availability/updates-
possible-mitigation-strategies-reduce-risk-nitrosamine-drug-substance-related-impurities.  Last accessed June 4, 
2023. 
34 To reflect the evolving and highly technical nature of the relevant information, FDA may provide certain updated 
information on testing methods for NDSRIs, in connection with this guidance, at the CDER web page at 
https://www.fda.gov/regulatory-information/search-fda-guidance-documents/updated-information-recommended-
acceptable-intake-limits-nitrosamine-drug-substance-related.  This associated information will be updated 
periodically.  For the most recent version, check the web page. 
35 See Nitrosamine Guidance at 14. 
36 See, e.g., requirements related to manufacturing changes in section 506A of the FD&C Act (21 U.S.C. 356a).  See 
also 21 CFR 314.70, 21 CFR 314.97, and 21 CFR 601.12.  Regardless of the change, the methods used in, and the 
facilities and controls used for, the manufacture, processing, packing, or holding of a product, including packaging 
and labeling operations, testing, and quality control of products, must comply with CGMP (see sections 501 of the 
FD&C Act (21 U.S.C. 351) and 21 CFR parts 210, 211, 212, and 600 through 680.)  All manufacturing changes 
must be reviewed and approved by the quality control unit (see 21 CFR 211.100(a) and 211.160(a)). 
37 21 CFR 314.70, 314.97, and 601.12 
38 See 21 CFR 314.70(b); see also 21 CFR 314.97(a) and 601.12(b).   
39 In certain circumstances, consistent with those described in section V.C., manufacturers or applicants may provide 
a scientifically justified rationale to pursue an AI limit different than the FDA-recommended limit. 
  13  Contains Nonbinding Recommendations 
limit.  Where applicable, applicants should report changes to an approved application.40  If an 
NDSRI level is detected above the FDA-recommended AI limit (e.g., associated with its 
predicted carcinogenic potency category),41 see the recommendations in section V.B.  
 
2.  Recommended Timeline for Drug Products in Development and Under FDA 
Review 
 
a.  Pre-submission stage 
 
FDA recommends that an applicant conduct a risk assessment for NDSRIs and conduct 
confirmatory testing as appropriate prior to submission of an original application.  However, the 
risk assessment and submission of confirmatory testing, if appropriate, and changes to the drug 
master file or application may be submitted in an amendment if these are not available at the 
time of the original application submission.  Such an amendment should be submitted as quickly 
as possible after the original application submission to minimize any potential adverse impact on 
the application assessment timeline.42 
 
b.  Applications pending with the Agency 
 
Applicants with pending applications should conduct the risk assessment expeditiously and 
inform FDA if confirmatory testing finds NDSRI levels above the AI limits recommended in this 
guidance.43  If an NDSRI is detected above the recommended AI limit, the applicant should 
amend the application as appropriate.  The Agency will work with the applicant in an effort to 
resolve issues during the review cycle.  
 
FDA intends to review applications consistent with the performance goals and procedures agreed 
to by FDA and industry in the respective commitment letters that accompany the Prescription 
Drug User Fee Act reauthorization for fiscal years 2023 to 2027 (PDUFA VII) and the Generic 
Drug User Fee Amendments reauthorization for fiscal years 2023 to 2027 (GDUFA III). 
 
B.  Recommendations for Manufacturers and Applicants That Have Marketed 
Products With Levels of NDSRIs Above the FDA Recommended AI Limits 
 
Generally, any drug product batch found to contain levels of an NDSRI above the FDA-
recommended AI limit (e.g., associated with its predicted carcinogenic potency category),44 
should not be released by the drug product manufacturer for distribution and may warrant 
removal from the market, because such drug products may be considered adulterated under 
 
40 See 21 CFR 314.70(b)(2) and the guidance for industry Changes to an Approved NDA or ANDA (April 2004); see 
also 21 CFR 314.97 and 21 CFR 601.12. 
41 In certain circumstances, consistent with those described in section V.C., manufacturers or applicants may provide 
a scientifically justified rationale to pursue an AI limit different than the FDA-recommended limit.   
42 For NDA submissions, applicants should discuss the need for an amendment with the Agency at the pre-NDA 
stage.  
43 For NDAs, the applicant should contact the review regulatory project manager for the specific product.  For 
ANDAs, the applicant should contact the project manager specified for the ANDA.  
44 In certain circumstances, consistent with those described in section V.C., manufacturers or applicants may provide 
a scientifically justified rationale to pursue an AI limit different than the FDA-recommended limit. 
  14  Contains Nonbinding Recommendations 
section 501 of the FD&C Act,45 for example if they are not manufactured, processed, packed, or 
held in conformity with CGMP.  As discussed further below, FDA may exercise enforcement 
discretion when warranted to prevent or mitigate a shortage of a drug.46  Manufacturers should 
contact the Agency if a recall is initiated.47   
 
1.  Additional Considerations for Maintaining the Drug Supply for Marketed 
Products 
 
If drug product batches already in distribution contain levels of NDSRIs above the FDA- 
recommended AI limit (e.g., associated with their predicted carcinogenic potency category),48 
and manufacturing changes or recalls are likely to lead to a disruption in the drug supply, then 
manufacturers and applicants should immediately contact the Center for Drug Evaluation and 
Researchs Drug Shortage Staff at drugshortages@fda.hhs.gov.  Contacting the Drug Shortage 
Staff can assist manufacturers in meeting any obligations to report discontinuances or 
interruptions in their drug manufacture under section 506C of the FD&C Act and implementing 
regulations under 21 CFR 314.81(b)(3)(iii).  Timely contact with the Drug Shortage Staff also 
enables FDA to consider, as soon as possible, what actions, if any, may be needed to avoid 
shortages and protect the health of patients who depend on the affected products.49 
 
When contacted about a potential disruption in the drug supply, FDA intends to evaluate each 
circumstance on a case-by-case basis.  FDA may work directly with a specific manufacturer or 
applicant of the marketed drug and intends to consider whether it is appropriate or not 
appropriate to recommend an interim AI limit for a temporary period.  If FDA recommends an 
interim AI limit, it generally does not intend to object, for example based on applicable 
underlying CGMP violations, to distribution of such drug product batches that contain NDSRI 
levels at or below the recommended interim AI limit during the specified period under certain 
circumstances on a case-by-case basis.   
 
In certain cases where FDA does not intend to object to the distribution of drug products from 
multiple drug manufacturers that contain NDSRI levels at or below the recommended interim AI 
limit, FDA intends to post such recommended interim AI limit on the FDA website in connection 
with this guidance.50 
 
 
45 See 21 U.S.C. 351. 
46 FDA, Drug Shortages Management, Manual of Policies and Procedures 4190.1 Rev.3 (Nov. 30, 2018).  MAPPs 
can be found on the Manual of Policies and Procedures web page at https://www.fda.gov/about-fda/center-drug-
evaluation-and-research/cder-manual-policies-procedures-mapp.  See also section 506D(b) and (c) of the FD&C 
Act.   
47 Manufacturers can contact the recall coordinator assigned to the product type and location. Contact information 
for recall coordinators is available at https://www.fda.gov/safety/industry-guidance-recalls/ora-recall-coordinators. 
48 In certain circumstances, consistent with those described in section V.C., manufacturers or applicants may provide 
a scientifically justified rationale to pursue an AI limit different than the FDA-recommended limit.  
49 See footnote 46.  
50 To reflect the evolving and highly technical nature of the relevant information, FDA intends to provide updated 
information on these recommended interim AI limits, in connection with this guidance, at the CDER web page at 
https://www.fda.gov/regulatory-information/search-fda-guidance-documents/updated-information-recommended-
acceptable-intake-limits-nitrosamine-drug-substance-related.  This associated information will be updated 
periodically.  For the most recent version, check the web page.   
  15  Contains Nonbinding Recommendations 
A decision to release lots remains solely the firms responsibility.  Firms are responsible for 
ensuring that their drugs are manufactured in compliance with all applicable requirements, 
including CGMP, and FDA expects them to vigilantly monitor and promptly report to FDA any 
adverse drug experiences or other findings that may affect product quality or safety.51 
 
C.  Approaches to Justify or Qualify a Proposed Alternative AI Limit 
 
If the observed level of an NDSRI in a drug product exceeds the FDA-recommended AI limit 
(e.g., associated with the predicted carcinogenic potency category for that NDSRI), the Agency 
recommends that manufacturers and applicants pursue mitigation efforts to reduce or remove the 
NDSRI.52  A manufacturer or applicant should submit a scientifically justified rationale to pursue 
an AI limit higher than the FDA-recommended limit associated with the predicted carcinogenic 
potency category for that NDSRI.  Alternative approaches using safety data, such as obtaining 
compound-specific data or using read-across assessment to a suitable surrogate, could be used to 
support a higher AI limit.  Importantly, manufacturers and applicants should note that the 
Agency may request additional safety data, beyond what is described here, to support alternative 
AI limits.  If compound-specific data or a read-across approach is pursued, we recommend the 
following:   
 
a.  Mutagenicity assessment:53,54  When testing the NDSRI using a bacterial 
mutagenicity assay (Ames assay), FDA recommends use of the full complement of 
testing strains described in Organisation for Economic Co-operation and 
Development (OECD) 471,55 utilizing the preincubation method and using both rat 
and hamster S9 at concentrations of 30 percent.  FDA recommends using S9 fractions 
typically prepared from animals treated with an inducer of CYP450s, like a 
combination of phenobarbital and beta naphthyl flavone.  Manufacturers and 
applicants should use the recommended pre-incubation time of 30 minutes as part of 
the optimal conditions for detecting a signal for mutagenicity. 
 
b.  Read-across assessment based on surrogate:  An NDSRI may have a well-justified AI 
limit based on a surrogate that maintains a 1 in 100,000 cancer risk estimate and is 
higher than the recommended AI limit associated with the predicted carcinogenic 
 
51 See, e.g., 21 CFR 314.80(a) and (c), and 314.81(b)(1). 
52 FDA, Updates on Possible Mitigation Strategies To Reduce the Risk of Nitrosamine Drug Substance-Related 
Impurities in Drug Products, available at https://www.fda.gov/drugs/drug-safety-and-availability/updates-possible-
mitigation-strategies-reduce-risk-nitrosamine-drug-substance-related-impurities.  Last accessed June 4, 2023. 
53 For nitrosamines, enhanced testing conditions for the Ames assay are recommended due to the reported reduced 
sensitivity of the assay under standard conditions for some nitrosamines.  FDA is investigating experimental 
conditions that improve Ames assay sensitivity for nitrosamines and NDSRIs.  See also Li X et al., 2023, Revisiting 
the Mutagenicity and Genotoxicity of N-nitroso Propranolol in Bacterial and Human In Vitro Assays, Regul Toxicol 
Pharmacol, 141:105410.   
54 To reflect the evolving and highly technical nature of the relevant information, FDA intends to provide certain 
updated information on safety testing methods for NDSRIs, in connection with this guidance, at the CDER web page 
at https://www.fda.gov/regulatory-information/search-fda-guidance-documents/updated-information-recommended-
acceptable-intake-limits-nitrosamine-drug-substance-related.  This associated information will be updated 
periodically.  For the most recent version, check the web page.  
55 See OECD (2020), Test No. 471 Bacterial Reverse Mutation Test, OECD Guidelines for the Testing of 
Chemicals, Section 4, OECD Publishing, Paris, available at https://doi.org/10.1787/9789264071247-en . 
  16  Contains Nonbinding Recommendations 
potency category for that NDSRI in certain cases.  When conducting a read-across 
assessment based on a surrogate, manufacturers and applicants can perform a (Q)SAR 
analysis to predict mutagenic potential by following recommendations described in 
ICH M7(R2).  To calculate an AI limit, the Agency recommends selection of 
surrogates with robust carcinogenicity data.56  The Agency recognizes that a robustly 
tested surrogate nitrosamine that adequately represents the structural and mechanistic 
characteristics of the NDSRI is frequently unavailable.  Therefore, when 
appropriately justified, the following compounds should be considered for use as 
surrogates due to the availability of robust mutagenicity and carcinogenicity data: 
NDMA, N-nitroso piperidine (NPIP), 4-(methylnitrosoamino)-1-(3-pyridinyl)-1-
butanone (NNK), N-nitroso pyrrolidine (NPYR), and N-nitroso morpholine 
(NMOR).57   
 
Approaches to justify or qualify a proposed alternative AI limit, as described in this section, 
should be submitted to FDA in a supplement or amendment, as appropriate, before 
implementation.58 
 
 
56 See also section III. 
57 See footnote 12.  
58 Generally, changes in specifications from those in the approved application must be submitted in a prior approval 
supplement unless otherwise exempted by regulation or guidance (see 21 CFR 314.70(b)(2) and 314.97(a) and the 
guidance for industry Changes to an Approved NDA or ANDA; see also 21 CFR 601.12(b)(2)). 
  17  Contains Nonbinding Recommendations 
APPENDIX A: DETERMINATION OF POTENCY SCORE 
 
For nitrosamine drug substance-related impurities (NDSRIs) not assigned to Potency Category 5, 
the Potency Score is calculated as the sum of the -Hydrogen Score (Table A), Deactivating 
Feature Score (Table B), and Activating Feature Score (Table C) based on selected structural 
features present in the NDSRI.  An NDSRI structure is expected to match exactly one of the -
hydrogen definitions in Table A, but it may contain multiple or no structural features identified 
in Tables B and C.  In cases where one or more features from Tables B and C are contained in 
the NDSRI, the Potency Score should be calculated as outlined in the box below. In cases where 
the NDSRI contains no features from Tables B and C, the Potency Score will be equal to the -
Hydrogen Score.  
 
 
Potency Score = -Hydrogen Score + Deactivating Feature Score (sum all scores for 
features present in NDSRI) + Activating Feature Score (sum all scores for features present in 
NDSRI) 
 
 
Table A. Count of hydrogen atoms on each -carbon (lowest count first) and corresponding -
Hydrogen Score. Examples are intended to be illustrative only and are not intended to be 
exhaustive. 
 
Count of Hydrogen Atoms on Each - -Hydrogen 
Example 
Carbon, Lowest First  Score 
0,2  3$ 
 
0,3  2 
 
1,2  3 
 
1,3  3 
 
2,2  1 
 
  18  Contains Nonbinding Recommendations 
2,3  1 
 
$A score of 3 applies when the methylene -carbon is not part of an ethyl group. If the methylene -carbon is part of 
an ethyl group, a score of 2 should be applied. 
 
Table B. List of deactivating features and associated scores. To calculate Deactivating Feature 
Score, sum the individual scores for all listed features present in the NDSRI structure.  Each 
deactivating feature row in the table may only be counted once.  For NDSRIs where the N-
nitroso group is within more than one ring, the feature score for only the smallest matching ring 
should be applied.  Examples are intended to be illustrative only and are not intended to be 
exhaustive. 
 
Individual 
Deactivating 
Deactivating Feature  Example 
Feature 
Score 
Carboxylic acid group anywhere on 
+3 
molecule 
 
N-nitroso group in a pyrrolidine ring  +3 
 
N-nitroso group in a 6-membered ring 
+3 
containing at least one sulfur atom 
 
N-nitroso group in a 5- or 6-membered ring*  +2 
 
N-nitroso group in a morpholine ring  +1 
 
N-nitroso group in a 7-membered ring  +1 
 
Chains of 5 consecutive non-hydrogen 
atoms (cyclic or acyclic) on both sides of 
+1 
acyclic N-nitroso group. Not more than 4 
atoms in each chain may be in the same ring. 
 
  19  Contains Nonbinding Recommendations 
 
Electron-withdrawing group** bonded to -
carbon on only one side of N-nitroso group  +1 
(cyclic or acyclic) 
 
Electron-withdrawing groups** bonded to -
carbons on both sides of N-nitroso group  +2 
(cyclic or acyclic) 
 
Hydroxyl group bonded to -carbon*** on 
only one side of N-nitroso group (cyclic or  +1 
acyclic) 
 
Hydroxyl group bonded to -carbons*** on 
both sides of N-nitroso group (cyclic or  +2 
acyclic) 
 
* Excludes examples where N-nitroso group is in a pyrrolidine ring, a 6-membered ring containing at least one sulfur 
atom, or a morpholine ring (all counted separately). 
**Excludes carboxylic acid and aryl (counted separately), and ketone (conflicting data).  Additional electron 
withdrawing group examples are limited to those described in Cross KP and Ponting DJ, 2021, Developing 
Structure-Activity Relationships for N-Nitrosamine Activity, Comput Toxicol, 20:100186, where they are referred to 
as -carbon electron withdrawing groups. 
***-Carbon must be in an sp3 hybridization state for this feature to apply. 
 
Table C. List of activating features and associated scores. To calculate Activating Feature Score, 
sum the individual scores for all listed features present in the NDSRI structure.  Each activating 
feature row in the table may only be counted once.  Examples are intended to be illustrative only 
and are not intended to be exhaustive. 
 
Individual 
Activating 
Activating Feature  Example 
Feature 
Score 
Aryl group bonded to -carbon (i.e., benzylic 
or pseudo-benzylic substituent on N-nitroso  -1 
group) 
 
Methyl group bonded to -carbon (cyclic or 
-1 
acyclic) 
 
 
  20  Contains Nonbinding Recommendations 
 
Example 1 below shows how the predicted carcinogenic potency categorization approach flow 
chart (Figure 1) can be applied to the hypothetical NDSRI, N-nitroso-l-nebivolol.  A Potency 
Score of 4 is calculated for N-nitroso-l-nebivolol, resulting in its placement in Potency Category 
4 with an associated recommended acceptable intake limit of 1500 nanograms (ng)/day.   
 
Example 1  N-Nitroso-l-nebivolol 
 
 
***-Carbon must be in an sp3 hybridization state for this feature to apply. 
 
  21  Contains Nonbinding Recommendations 
 
NDSRIs = nitrosamine drug substance-related impurities; AI = acceptable intake; ng = nanogram. 
* A tertiary -carbon is defined as an -carbon atom in an sp3 hybridization state, bonded to three other carbon atoms.  
**To calculate Potency Score, see Appendix A. 
 
  22  Contains Nonbinding Recommendations 
Example 2 below shows how the predicted carcinogenic potency categorization approach flow 
chart (Figure 1) can be applied to the hypothetical NDSRI, N-nitroso-ketamine. N-nitroso-
ketamine is placed in Potency Category 5 with an associated recommended acceptable intake 
limit of 1,500 ng/day.   
 
Example 2  N-Nitroso-ketamine 
 
 
 
 
  23  Contains Nonbinding Recommendations 
 
NDSRI = nitrosamine drug substance-related impurities; AI = acceptable intake; ng = nanogram. 
* A tertiary -carbon is defined as an -carbon atom in an sp3 hybridization state, bonded to three other carbon atoms.  
**To calculate Potency Score, see Appendix A. 
 
  24