Guidance on nitrosamine impurities in

Medications
Evaluating and managing the risks of N-nitrosamine impurities in human
pharmaceutical, biological and radiopharmaceutical
products
Date Adopted: 2024-03-15
Effective Date: 2024-03-15
Health Canada is the federal department responsible for helping the people of Canada maintain and improve
their health. Health Canada is committed to improving the lives of all of Canada's people and to making this
country's population among the healthiest in the world as measured by longevity, lifestyle and effective use of the
public health care system.

Également disponible en français sous le titre :

Lignes directrices sur les impuretés de nitrosamine dans les médicaments
To obtain additional information, please contact:

Health Canada
Address Locator 0900C2
Ottawa, ON K1A 0K9
Tel.: 613-957-2991
Toll free: 1-866-225-0709
Fax: 613-941-5366
TTY: 1-800-465-7735
E-mail: [email protected]

© His Majesty the King in Right of Canada, as represented by the Minister of Health,
Publication date: March 2024

This publication may be reproduced for personal or internal use only without permission provided the source is fully
acknowledged.

Cat. : H164-327/2024E-PDF
ISBN: 978-0-660-70210-0
Pub.: 230750
Table of Contents
Background ..................................................................................................... 1
General........................................................................................................... 2
Scope and responsibilities ............................................................................... 2
1. Drug products that are within the scope of Health Canada’s call for review ...... 2
2. Timelines for completing risk assessments (Step 1), confirmatory testing (Step
2) and changes to the market authorization (Step 3) .................................. 3
3. Outcomes of risk assessments (Step 1) and what is provided to Health Canada
(updated)............................................................................................. 3
4. Determining the priorities and order in which products should be reviewed ..... 4
5. MAHs co-operating with API and drug product manufacturers to perform risk
assessments ......................................................................................... 5
6. Responsibilities of API manufacturers, excipient manufacturers, drug product
manufacturers, MAHs and importers ......................................................... 5
7. Inability to meet specified timelines for risk assessment (Step 1), confirmatory
testing (Step 2) or changes to the market authorization (Step 3) .................. 6
8. Statements or declarations by manufacturers and suppliers in lieu of completing
risk assessments ................................................................................... 6
9. Skipping the risk assessment step (Step 1) and proceeding directly to
confirmatory testing (Step 2) .................................................................. 7
10. Applying the results of a risk assessment and confirmatory testing for a drug
product marketed outside Canada to a drug product authorized for sale in
Canada ................................................................................................ 7
11. Nitrosamine risk assessment applicable to a drug product brought into Canada
under the Special Access Program (SAP) ................................................... 8
12. Confirmatory testing where the risk assessment concludes there is no identified
risk for the presence of nitrosamines ........................................................ 8
13. Managing and submitting Step 3 changes to the market authorization relating to
risk mitigation measures (updated) ........................................................... 9
14. Selling a drug product if changes (specifications, controls) to the market
authorization (Step 3) submitted as a Supplement or Notifiable Change are still
under review ...................................................................................... 10
15. Contacting Health Canada if nitrosamine impurities are detected following the
completion of confirmatory testing .......................................................... 11
16. When information necessary to complete risk assessments is not provided by
the API or drug product manufacturer..................................................... 12
17. Additional expectations of MAHs if nitrosamine impurities are detected in the
API and/or drug product (updated) ........................................................ 13

Guidance on nitrosamine impurities in medications | i

 18. Assessing progress with the request to review the risk of presence of
nitrosamine impurities ......................................................................... 14
19. Approach for drug products that are planned for submission or are already filed
with Health Canada.............................................................................. 14
20. Risk assessments for the potential presence of nitrosamine impurities as part of
the expected content for new submissions ............................................... 15
21. Controls for nitrosamines in APIs purchased for compounding ..................... 17
Communications .......................................................................................... 17
22. Engaging stakeholders and ensuring ongoing communication with industry ... 17
23. Health Canada works with global regulators relating to issues associated with
nitrosamine impurities in drug products .................................................. 18
Safety .......................................................................................................... 18
24. AI limits for nitrosamine impurities that Health Canada considers acceptable . 18
25. AI limits for nitrosamine impurities in drug products that fall within the scope of
the ICH S9 guideline or where the API is genotoxic ................................... 20
26. Communicating if AI limits are revised in the future .................................. 20
27. Acceptable limit if multiple nitrosamines are detected in an API or a drug
product.............................................................................................. 20
28. Application of a less-than-lifetime (LTL) limit by considering the principles in
ICH’s M7 guideline if a nitrosamine impurity is present in a drug product that is
administered for less than a lifetime ....................................................... 21
Quality ......................................................................................................... 22
29. Risk factors and potential root causes to be considered for the presence of
nitrosamine impurities in human pharmaceuticals when performing a risk
assessment ........................................................................................ 22
30. Components of drug products to consider in risk assessments .................... 23
31. Nitrosamine impurities to consider in the Step 1 risk assessment and Step 2
confirmatory testing (updated) .............................................................. 24
32. Testing methodologies provided by Health Canada .................................... 24
33. Validating the limit of quantitation (LOQ) for nitrosamine impurity analytical
procedures (updated) ........................................................................... 25
34. Including routine testing for nitrosamine impurities in the API and/or drug
product specification ............................................................................ 25
35. Potential control options for nitrosamine impurities in the API ..................... 26
36. Confirmatory testing expectations (Step 2) .............................................. 27
37. Analytical laboratories conducting nitrosamine testing and listing on the DEL. 28
38. Number and types of drug product batches as part of confirmatory testing for
marketed products and new market applications ...................................... 28

Guidance on nitrosamine impurities in medications | ii

Appendices .................................................................................................... 30
Appendix 1: Established Acceptable Intake (AI) limits for N-nitrosamine impurities
........................................................................................................ 30
Appendix 2: Guidance with respect to nitrosamine impurities and risk assessments
for Post NOC Changes of new drug products containing chemically synthesized
and semi-synthetic APIs (updated) ......................................................... 31
Appendix 3: Enhanced Ames assay test conditions ......................................... 32
Appendix 4: Carcinogenic Potency Categorization Approach (CPCA) for N-
nitrosamines ...................................................................................... 34

Guidance on nitrosamine impurities in medications | iii

Background
This guidance represents Health Canada's current thinking and recommendations
on issues related to N-nitrosamine impurities (nitrosamine impurities or
nitrosamines). This guidance may be subject to change as new information
becomes available and if further guidance is needed for applicants and market
authorization holders (MAHs).

A questions-and-answers (Q&A) document on nitrosamines was issued to MAHs on

November 26, 2019. This document has undergone a number of revisions and has
been further updated as a guidance document and to provide additional details to
active pharmaceutical ingredient (API) manufacturers, drug product
manufacturers, MAHs and importers of APIs and drug products.

In this guidance document, changes from the previous version are identified with
the descriptors "new" or "updated" (as applicable). Information on a similar theme
is grouped together under general headings (for example, General, Safety and
Quality).

Queries about the Health Canada letters noted below can be directed as follows:

• "Information to MAHs of Human Pharmaceutical Products Regarding

Nitrosamine Impurities: Request to evaluate the risk of the presence of
nitrosamine impurities in human pharmaceutical products containing
chemically synthesized active pharmaceutical ingredients" (October 2, 2019)
• Email to [email protected]
• "Information to MAHs of Human Pharmaceutical Products Regarding
Nitrosamine Impurities: Request to evaluate the risk of the presence of
nitrosamine impurities in biologics and radiopharmaceuticals" (December 15,
2020)
• Email to [email protected]

If you have queries about this guidance document, you may send an email to
[email protected].

Guidance on nitrosamine impurities in medications | 1

General
Scope and responsibilities
1. Drug products that are within the scope of Health Canada’s call for review

The request in Health Canada's call for review to evaluate the risk of the presence
of nitrosamine impurities outlined in the October 2, 2019, letter applies to human
pharmaceutical products with a drug identification number (DIN) containing
chemically synthesized and semi-synthetic APIs. This includes:

• prescription and non-prescription (over-the-counter) drug products

• chemically synthesized excipients and raw materials used in the
manufacturing of drug products

Also considered to be within the scope of Health Canada's call for review are:

• drug products that have been approved but are not yet marketed
• approved drug products with a DIN status reported as "dormant"

The request for conducting risk assessments for the potential presence of
nitrosamine impurities was extended to all biological and radiopharmaceutical
products for human use. This was outlined in Health Canada's letter dated
December 15, 2020.

All human plasma proteins, vaccines and cell-based fermentation products are
classified as biologics. They are, therefore, within the scope of the request for risk
assessment.

Please refer to Health Canada's letter dated December 15, 2020, for further
details.

All non-prescription products with a DIN, such as topical antiseptic products,

grooming and personal hygiene products and sunscreens, are within the scope of
products for assessment if they contain a chemically synthesized or semi-synthetic
API. This is irrespective of the route of administration or any cosmetic properties.

Products that are not within the scope of the October 2, 2019, and
December 15, 2020, letters include cosmetics (which do not have a DIN). The
following categories of drug products are also excluded at this time: antimicrobial
agents, veterinary products (including veterinary health products) and natural
health products. Disinfectant products for use on hard surfaces are also not within
the scope of products for assessment at this time.

Guidance on nitrosamine impurities in medications | 2

2. Timelines for completing risk assessments (Step 1), confirmatory testing (Step 2) and changes
to the market authorization (Step 3)

For drug products containing chemically synthesized and semi-synthetic APIs, the steps
for actions relating to nitrosamines are expected to be completed as follows:

• Step 1: risk assessments by March 31, 2021

• Step 2: confirmatory testing by October 1, 2022
• Step 3: changes to the market authorization by August 1, 2025

For biological and radiopharmaceutical products, the steps for actions relating to
nitrosamines are expected to be completed as follows:

• Step 1: risk assessments by November 30, 2021

• Step 2: confirmatory testing by November 30, 2023
• Step 3: changes to the market authorization by August 1, 2025

3. Outcomes of risk assessments (Step 1) and what is provided to Health Canada (updated)

Risk assessment documentation should be retained by the MAH, unless nitrosamine

impurities are detected in the API, drug product or both during confirmatory testing.
Following the completion of confirmatory testing of the drug product, Health Canada
must be informed if the nitrosamine impurity is detected above the established
Acceptable Intake (AI) limit (refer to Appendix 1 for a listing of established AIs) for the
nitrosamine impurity in question, or above the AI limit established using the
Carcinogenic Potency Categorization Approach (CPCA) (refer to number 24 and
Appendix 4) if an AI limit has not been established by Health Canada. The confirmatory
testing results should be submitted at the same time that Health Canada is informed of
the detection and the details of the risk assessment should be available upon request.
Refer to the information in number 15.

For nitrosamine impurities listed in Appendix 1 that are classified as non-mutagenic,

the submission of the risk assessment and confirmatory testing results is not required,
and these impurities should be controlled according to ICH’s Q3A and Q3B guidelines.

Please note that Health Canada may request to review the MAH's risk assessment for
all products and will request this information directly from the MAH, as necessary.

Canadian importers that received terms and conditions (T&C) on their drug
establishment licence (DEL) for nitrosamine testing of angiotensin II receptor blockers
(known as sartans) may provide supporting information to modify or remove the terms
and conditions. They should submit the API and drug product risk assessments and
testing results completed as per Steps 1 and 2 for consideration. Email to foreign.site-
[email protected].

Guidance on nitrosamine impurities in medications | 3

MAHs may be requested by Canadian importers for a copy of the MAH's risk
assessment and testing results to facilitate this request. Alternatively, MAHs may
provide the requested risk assessment and related information to Health Canada on
behalf of the Canadian importer. In this case, the MAH should specify on whose behalf
the risk assessment and related information is being submitted.

4. Determining the priorities and order in which products should be reviewed

MAHs should use a risk-based approach to determine the order in which their drug
products are reviewed. In order to prioritize the sequence in which products should
be reviewed, MAHs should consider a number of factors, including the following:

• principles set out in the International Council for Harmonisation of Technical

Requirements for Pharmaceuticals for Human Use (ICH) Q9 guideline on
quality risk management
• maximum daily dose of the drug product
• route of administration
• duration of use
• indication and consideration of special populations, such as pregnant women
and children
• toxicological profile of the API
o for example, evaluating the risk of presence of nitrosamine impurities in
cancer therapies in which the API is a potent mutagen could be
considered lower priority and sequenced for review after higher priority
APIs
• market considerations such as the availability of product for sale on the
Canadian market and number of patients being treated with the drug product
• emerging international or domestic information that 1 or more nitrosamine
impurities has been identified in an API (or a structurally similar API) or drug
product
• the presence of structural elements in the API or conditions in the
manufacturing and packaging processes for the API or drug product, which
are conducive to nitrosamine formation (for example, presence of secondary
or tertiary amine groups in the API)

Appendix 1 should be consulted for APIs and drug products that may contain
nitrosamine impurities. Peer-reviewed literature (for example, M.K. Parr, J.F.
Joseph, Journal of Pharmaceutical and Biomedical Analysis 164 (2019) 536–549) and
other sources of information (for example, regulatory communications) should also be
consulted for APIs and drug products known to contain nitrosamine impurities.

Guidance on nitrosamine impurities in medications | 4

5. MAHs co-operating with API and drug product manufacturers to perform risk assessments

After receiving authorization to market in Canada, MAHs are responsible for the
safety, efficacy and quality of their drug products and for carrying out the risk
assessments. They should:

• work with API and drug product manufacturers to review their API and drug
product manufacturing processes to conduct risk assessments
• take into account the API and drug product manufacturers’ knowledge of the
manufacturing processes, potential sources of contamination and other root
causes of the formation and presence of nitrosamine impurities

API and drug product manufacturers should make available to the MAHs the
information necessary for conducting the risk assessments.

If the risk of nitrosamine impurity formation has been assessed during the
development phase of the API or drug product manufacturing processes, the
information from the assessment can be used to support the evaluation.

6. Responsibilities of API manufacturers, excipient manufacturers, drug product manufacturers,

MAHs and importers

After receiving authorization, MAHs are responsible for ensuring the ongoing safety,
efficacy and quality of drug products on the Canadian market. This would include
implementing an ongoing monitoring program to detect trends in quality. Such a
program should be based on appropriate controls for raw materials, all processing
steps, critical process parameters and critical quality attributes.

To complete risk assessments for the potential presence of nitrosamine impurities,

MAHs should complete robust risk evaluations using a holistic approach. A detailed
assessment of all stages of the product's life cycle should be done and would include an
evaluation of the risk factors and potential root causes for the presence of
nitrosamines, including those identified in number 29.

MAHs are responsible for ensuring that personnel with acceptable qualifications and
expertise (for example, relevant training, knowledge and practical experience) have
conducted the risk assessments. Information should be made available to the MAH by
API, excipient and drug product manufacturers.

In the context of control for nitrosamine impurities, manufacturers and importers must
comply with any terms and conditions specified on their DEL. This could include
restrictions or additional specific testing and investigational requirements for
nitrosamine impurities.

Guidance on nitrosamine impurities in medications | 5

 7. Inability to meet specified timelines for risk assessment (Step 1), confirmatory testing (Step
2) or changes to the market authorization (Step 3)

Given the potential risks associated with nitrosamines in drug products, MAHs should
take all necessary measures to complete the 3 steps as soon as possible and within
the designated timelines.

In a follow-up letter issued to MAHs of drug products containing chemically synthesized

active pharmaceutical ingredients on April 14, 2021, Health Canada requested that
affected MAHs indicate their status for completing Step 1, risk assessments. MAHs should
provide a completed Annex 1, Annex 2 or Annex 3, as applicable, as per the instructions
in the April 14, 2021, follow-up letter. If risk assessments have not been completed for
all marketed, approved and dormant products or have been partially completed, Annex 3
should be completed.

MAHs unable to meet the Step 2 or 3 deadlines due to exceptional circumstances may
submit a request for extension to Health Canada. This should be done as soon as
possible. The request should contain relevant information, including the progress to date,
the reasons for not meeting the deadline(s), the remaining work and the expected
timelines for completion.

To prioritize APIs and drug products for the completion of risk assessments, confirmatory
testing or changes to the market authorization, MAHs are reminded to use quality risk
management principles. Consult ICH's Q9 guideline, Health Canada's good manufacturing
practices (GMP) guides 0001 (for drug products) and 0104 (for APIs). Also consult the
information in number 4.

Requests for extensions will be considered on a case-by-case basis. Direct such requests
as follows:

• for drug products containing chemically synthesized or semi-synthetic APIs:

[email protected]
• for biological and radiopharmaceutical products: brdd.nitrosamines.dmbr@hc-
sc.gc.ca

8. Statements or declarations by manufacturers and suppliers in lieu of completing risk

assessments

Statements and declarations provided by manufacturers and/or suppliers are not a

substitute for an overall robust risk assessment by the MAH. While the knowledge
and expertise offered by manufacturers is valuable and is encouraged to support
the risk assessment process, manufacturer/supplier statements or declarations do
not replace a documented risk assessment by the MAH.

Guidance on nitrosamine impurities in medications | 6

9. Skipping the risk assessment step (Step 1) and proceeding directly to confirmatory testing
(Step 2)

The risk assessment step (Step 1) is necessary to identify possible root causes and
the scope of nitrosamine impurities that have the potential to be formed or
introduced into the API or drug product. If a risk of 1 or more nitrosamine
impurities is identified, this knowledge is used to guide the development and
validation of appropriate test methods required for the confirmatory testing stage
(Step 2).

This knowledge may also be useful for the establishment of a suitable control
strategy and changes introduced to prevent the presence of nitrosamines.

As such, it is not appropriate to proceed directly to confirmatory testing (Step 2)

without completing the risk assessment step (Step 1).

10. Applying the results of a risk assessment and confirmatory testing for a drug
product marketed outside Canada to a drug product authorized for sale in Canada

MAHs are responsible for ensuring that risk assessments and, if applicable,
confirmatory testing are relevant to the drug product authorized for sale in
Canada.

If a risk assessment and confirmatory testing have been completed for a drug
product approved for use outside Canada, it may be possible to use that
information for the risk assessment and confirmatory testing of the drug product
authorized for sale in Canada. In this scenario, the 2 drug products must be
identical (for example, composition, strength, manufacturing process, API and
excipient sources, manufacturing sites).

MAHs should prepare a written justification when the risk assessment and
confirmatory testing results of a foreign product will be relied upon. MAHs should be
prepared to provide this justification to Health Canada upon request. This
justification should be included in communications to Health Canada if nitrosamine
impurities are detected in the drug product following confirmatory testing where the
nitrosamine impurity is detected above the established AI limit (refer to Appendix 1)
for the nitrosamine impurity in question, or above the AI limit established using the
CPCA (refer to number 24 and Appendix 4) if an AI limit has not been established by
Health Canada. Refer to the information in number 15.

Guidance on nitrosamine impurities in medications | 7

11. Nitrosamine risk assessment applicable to a drug product brought into Canada under
the Special Access Program (SAP)

Companies may need to conduct nitrosamine risk assessments for drug products
not authorized for sale in Canada that are being made available in Canada through
the SAP. Refer to the approaches described in Health Canada’s October 2, 2019,
and December 15, 2020, letters and in this document.

If the nitrosamine risk assessment or confirmatory testing results (if applicable)

indicate the risk of presence of a nitrosamine impurity, notify the SAP by email at
[email protected].

To protect the health and safety of patients accessing unauthorized drug products,
significant new information on the safety, efficacy and quality of drug products
released under the SAP should be made available to practitioners and the SAP.

12. Confirmatory testing where the risk assessment concludes there is no identified risk for
the presence of nitrosamines

MAHs are expected to conduct a thorough, robust risk assessment. In Health Canada's
letters dated October 2, 2019, and December 15, 2020, Health Canada shared some
potential sources of nitrosamine impurities. Refer to number 29 for more information
on risk factors and potential root causes for nitrosamine impurities.

MAHs should prepare a report that includes considerations, steps, conclusions and a
rationale. The report should clearly identify which nitrosamine(s) is (are) at risk of
formation, if applicable. If it is concluded that a risk for the presence of nitrosamines is
not identified, then confirmatory testing is not expected.

If a risk of formation or presence of nitrosamines is identified, confirmatory testing

should be carried out using appropriately validated and sensitive methods (refer to
number 36). Following the completion of confirmatory testing of the drug product,
Health Canada must be informed if the nitrosamine impurity is detected above the
established AI limit for the nitrosamine impurity in question, or above the AI limit
established using the CPCA (refer to number 24 and Appendix 4) if an AI limit has not
been established by Health Canada. The reporting addresses are provided in number
15.

Guidance on nitrosamine impurities in medications | 8

13. Managing and submitting Step 3 changes to the market authorization relating to risk mitigation
measures (updated)

Step 3 changes to the market authorization relating to risk mitigation measures

should be submitted to Health Canada in a timely manner in eCTD or non-eCTD
format using the Common Electronic Submission Gateway (CESG).

Step 3 risk mitigation related changes are the result of potential safety concerns
and therefore require Health Canada's critical review of scientific data and
subsequent authorization prior to implementation by the manufacturer. Therefore,
with the exception of certain minor changes (see further details described below in
this section), Step 3 risk mitigation related changes for drug products containing
chemically synthesized and semi-synthetic APIs should be submitted as Level I -
Supplements or Post-Drug Identification Number (DIN) Change submissions, as
applicable. For biological and radiopharmaceutical products, changes should be
submitted as Level I – Supplements or Level II - Notifiable Changes, or Post-Drug
Identification Number (DIN) Change submissions (PDCs), as applicable. For
examples of risk mitigation related changes, refer to number 20 and Appendix 2.

When filing a Supplement, Notifiable Change or Post-DIN Change submission for

the market authorization, applicants should clearly indicate in the covering letter
that the proposed changes are being submitted to address risk mitigation for
nitrosamines (Step 3 of Health Canada’s call for review to evaluate the risk of the
presence of nitrosamine impurities in approved drug products).

A summary of the root cause investigations and the conclusion regarding the confirmed
root cause(s) for nitrosamine presence in the drug product should be included under
section 3.2.P.2.

Where a proposal is made to add individual or cumulative nitrosamine AI limits to

an approved drug substance or drug product specification (based on the use of the
CPCA or other approaches), MAHs should manage these changes as Level I -
Supplements.

For proposed changes unrelated to Step 3 of the call for review of approved drug
products (that is, changes unrelated to risk mitigation for approved drug
products), changes should be managed and, where applicable, submitted
according to the Post-Notice of Compliance (NOC) Quality Document and Post-
Drug Identification Number (DIN) Changes Guidance Document. Refer to number
20 and Appendix 2. Additional guidance is below:

For drug products containing chemically synthesized or semi-synthetic APIs, where

AI limits for individual nitrosamine impurities and, where relevant, limits for
cumulative levels of nitrosamine impurities, are already included in an
approved drug substance or drug product specification the following

Guidance on nitrosamine impurities in medications | 9

 changes can be managed according to Health Canada's Post-Notice of Compliance
(NOC) Quality Document as Level III changes (Annual Notifications):

• tightening of individual or cumulative nitrosamine AI limits in the approved

drug substance or drug product specifications

• relaxation of individual or cumulative AI limits in the approved drug substance

or drug product specifications to adopt AI limits listed in Appendix 1 of this
guidance document

• deletion of a test for a nitrosamine impurity from a drug substance or drug product
specification, with appropriate scientific justification

In addition, the following changes can be managed according to Health Canada's

Post-Notice of Compliance (NOC) Quality Document as Level III changes (Annual
Notifications):

• addition of a test and acceptance criteria to a drug substance specification for

a nitrosamine impurity that is based on a valid Certificate of Suitability (CEP)
issued by the European Directorate of Quality of Medicines and HealthCare
(EDQM)

• addition of a test and acceptance criteria to a drug substance or drug product

specification for a nitrosamine impurity that has been classified as non-
mutagenic in Appendix 1

Where a proposal is made to relax individual or cumulative nitrosamine AI limits

already included in an approved drug substance or drug product specification (based
on the use of the CPCA or other approaches) when an AI limit is not listed in
Appendix 1, MAHs should manage these changes as Level I - Supplements.

14. Selling a drug product if changes (specifications, controls) to the market

authorization (Step 3) submitted as a Supplement or Notifiable Change are still
under review

The ongoing marketing of a drug product depends on the impurity levels and the
risk of nitrosamine impurities that are detected upon notification to Health Canada.
Some of the outcomes of the Health Canada assessment may include recalls or
stop sale requests until the risks are mitigated and suitable corrective and
preventive actions are in place to ensure that all lots being released to market
meet the acceptance criteria for each nitrosamine impurity (and cumulative
nitrosamines, if relevant). Both outcomes would affect the ongoing marketing of
the product while the Supplement or Notifiable Change is being reviewed. Refer to
the information in number 13.

Guidance on nitrosamine impurities in medications | 10

15. Contacting Health Canada if nitrosamine impurities are detected following the completion of
confirmatory testing

Following the completion of confirmatory testing of the drug product, MAHs must
inform Health Canada if nitrosamine impurities are detected above the established AI
limit (refer to Appendix 1) for the nitrosamine impurity in question, or above the AI
limit established using the CPCA (refer to number 24 and Appendix 4) if an AI limit has
not been established by Health Canada. The confirmatory testing results should
accompany the notification to Health Canada by the MAHs.

Health Canada recognizes the challenges faced by MAHs to decrease levels of

nitrosamine impurities in their drug products while maintaining drug supply to
Canadians. To minimize the impacts on drug supply within the Canadian market, MAHs
are requested to engage Health Canada prior to taking any market action for a drug
product due to a nitrosamine impurity issue.

Communications should be directed as follows:

Table 1. Addresses and contact information

Location of firm Reporting address

New Brunswick,
Health Products Compliance & Enforcement Unit East
Newfoundland and
1001 Rue St-Laurent Ouest, Longueuil, QC, J4K 1C7
Labrador, Nova
Phone: 450-646-1353
Scotia,
Toll free: 1-800-561-3350
Prince Edward Email: [email protected]
Island, Québec

Ontario Health Products Compliance & Enforcement Unit Central

2301 Midland Ave., Toronto, ON M1P 4R7
Phone: 416-973-1600
Toll free: 1-800-267-9675
Email: [email protected]

Manitoba,
Health Products Compliance & Enforcement Unit West
Saskatchewan,
Suite 400–4595 Canada Way, Burnaby, BC V5G 1J9
Alberta, British
Phone: 604-666-3350
Columbia, Yukon,
Toll free: 1-800-267-9675
Northwest
Email: [email protected]
Territories, Nunavut

Guidance on nitrosamine impurities in medications | 11

If nitrosamines are not detected during confirmatory testing (for example, less than
the appropriate limit of detection of the validated test method) or are detected
below the established AI limit for the nitrosamine impurity in question or the AI
limit established using the CPCA (refer to number 24 and Appendix 4) if an AI limit
has not been established by Health Canada, MAHs do not need to communicate
this information to Health Canada. However, they should keep the risk assessment,
analytical testing results and analytical method validation documentation on hand
in case Health Canada requests them. Refer to number 13 for information on
communicating changes to market authorization to Health Canada.

16. When information necessary to complete risk assessments is not provided by the API or
drug product manufacturer

MAHs are responsible for ensuring the ongoing safety, efficacy and quality of
products on the Canadian market. When manufacturers do not provide information
that is essential for MAHs to complete the risk assessment due to confidentiality or
other reasons, MAHs may engage a third party (such as a consultant) to work
directly with the manufacturer to complete the risk assessment.

The third-party approach may also be appropriate when the MAH:

• has all of the required information to conduct the risk assessment from the
manufacturer but
• does not have staff with the necessary qualifications (for example, relevant
training and practical experience) to conduct the risk assessment

For additional guidance on outsourced activities, consult the:

• section C.02.012, interpretation 3 to 12 of the Good Manufacturing Practices

Guide for Drug Products (GUI-0001)

Alternatively, MAHs should consider delegating the risk assessment to the API and
drug product manufacturers. In this scenario, MAHs would continue to be
responsible for ensuring the safety, efficacy and quality of their drug products.

MAHs should ensure through internal or third-party audit that:

• risk assessments have been conducted by personnel with acceptable

qualifications (relevant training and practical experience)
• manufacturers have considered all possible risk factors and potential root
causes of nitrosamine impurities (including those in the December 15, 2020,
letter concerning biologics and radiopharmaceuticals, and those identified in
number 29)

Guidance on nitrosamine impurities in medications | 12

 17. Additional expectations of MAHs if nitrosamine impurities are detected in the API
and/or drug product (updated)

Where 1 or more nitrosamine impurities are detected (for multiple nitrosamines,

refer to number 27), in addition to notifying Health Canada, MAHs should have
completed or be completing as necessary:

• a health risk assessment posed by the presence of the nitrosamine(s) along with
intentions related to any actions, as necessary, for the batches on the Canadian
market
o where product recalls are warranted, consult the Drugs and Natural Health
Products Recall Guide (GUI-0039) for procedures
• an assessment to determine if the product is considered to be medically necessary
or medically important and if any disruption to product supply is expected should
market action be taken
• a detailed investigation report assessing all possible root causes of the detected
nitrosamine impurity (or impurities) and describing corrective and preventive
actions
o perform investigations in accordance with written procedures
o evaluate all potential changes to facilities, materials, equipment and/or
process intended to reduce the levels of the nitrosamine impurities through a
formal change control system
• a risk mitigation plan including the establishment of a suitable control strategy for
detected nitrosamine(s) to ensure that, moving forward, nitrosamine impurity
levels will be consistently below the Acceptable Intake (AI) limit at the end of the
retest period for the API or the shelf-life for the drug product (refer to Appendix
1 for a list of established AI limits)

MAHs are reminded to submit changes to the market authorization as per Step 3 of the
October 2, 2019 letter. Refer to number 13 on how changes should be submitted.

Health Canada may use such notifications to request additional actions and/or
information. For example, the origin of nitrosamine impurities may be attributed to the
type of process chemistry used and the risk mitigation plan may necessitate the
establishment of a control strategy by manufacturers for each detected nitrosamine
impurity according to ICH's M7 guideline.

We may request additional actions by other MAHs of the same products to mitigate any
risks identified and protect people's health and safety if necessary.

For more information on the establishment of specifications and controls, refer to number
34 and number 35, respectively.

Guidance on nitrosamine impurities in medications | 13

18. Assessing progress with the request to review the risk of presence of nitrosamine
impurities

On April 14, 2021, Health Canada issued a follow-up letter to the October 2, 2019,
letter. In the letter, we asked MAHs with drug products containing chemically
synthesized and semi-synthetic APIs to provide their progress towards completing
Step 1 (risk assessments).

Health Canada may also:

• verify progress during inspections, proactive risk management projects and

compliance verification upon receipt of a complaint and/or notification or
• request information at such time as changes are made to either the existing
market authorization for a product or for the drug establishment license

We appreciate the significance of this request. We will continue to engage with

stakeholders to look at all options to address the potential risks associated with
nitrosamines.

19. Approach for drug products that are planned for submission or are already filed with
Health Canada

Whenever possible for APIs and drug products that are under development, the
formation or introduction of nitrosamine impurities should be avoided at the outset.

If the formation or introduction of nitrosamine impurities is unavoidable,

manufacturing processes should demonstrate process capability to routinely reduce
the levels of nitrosamine impurities below the AI limit. A control strategy, based on
product and process understanding, should be established for each nitrosamine
impurity present in the API and/or drug product.

For drug products that are planned for submission or have already been submitted,
MAHs and applicants should proactively undertake a risk assessment for the
potential presence of nitrosamine impurities in the drug product (if this has not
already been undertaken) using the considerations and steps provided for approved
products in Health Canada's communications. For planned submissions, the
relevant sections of the Common Technical Document (CTD) in the drug application
should include information on these risk assessments.

A summary and discussion of the risk assessment for nitrosamine impurities in the
drug product should be placed in section 3.2.P.2 of the CTD. This summary is
expected to include sufficient detail to allow Health Canada to assess the adequacy
and robustness of the risk assessment. Expectations for the content of the
summary and discussion of risk assessments are found under number 20.

Guidance on nitrosamine impurities in medications | 14

Confirmatory testing results and updated control strategy (where warranted) should
also be included in the drug application (for example, under sections 3.2.S.2, 3.2.S.4,
3.2.S.7, 3.2.P.3, 3.2.P.4, 3.2.P.5, 3.2.P.8).

For submitted applications currently under review, MAHs and applicants may be
asked to provide the risk assessment and confirmatory testing results as part of the
assessment procedure. For further information, refer to number 20.

20. Risk assessments for the potential presence of nitrosamine impurities as part of the
expected content for new submissions

Risk assessments for the potential presence of nitrosamine impurities should be

conducted routinely during API and drug product development. The outcome of the risk
assessment for nitrosamine impurities in the drug product and the justification for the
proposed control strategy for nitrosamine impurities should be made available for
assessment in New Drug Submissions (NDSs), Abbreviated New Drug Submissions
(ANDSs), applications for a Drug Identification Number for a Pharmaceutical Product
(DINAs) (with Chemistry & Manufacturing (C&M) data), applications for a Drug
Identification Number for a Biological Product (DINBs), Supplements, Notifiable
Changes and Post-DIN Changes submissions (refer to number 13). For more
information on mutagenic impurity considerations and quality risk management
principles, consult the following:

• Good Manufacturing Practices Guide for Drug Products (GUI-0001)

• Good Manufacturing Practices Guidelines (GMP) for Active Pharmaceutical
Ingredients (GUI-0104)
• ICH's M7 guideline (PDF format)
• ICH's Q9 guideline (PDF format)

All NDSs, ANDSs, DINAs (with C&M data), and DINBs and all Supplements, Notifiable
Changes and Post-DIN Change submissions (for quality changes that may impact the
potential presence of nitrosamine impurities in the API or drug product) should include
a summary and discussion of the risk assessment for the potential formation/presence
of nitrosamine impurities in the drug product. This is required as follows:

• as of April 1, 2021, for NDSs, ANDSs, and Supplements for pharmaceutical

products containing chemically synthesized and semi-synthetic APIs
• as of April 1, 2021, for DINAs (with C&M data) including Post-DIN Change
Submissions for quality changes for pharmaceutical products containing
chemically synthesized and semi-synthetic APIs
• as of November 30, 2021, for NDSs, Supplements and Notifiable Changes for
biological and radiopharmaceutical products
• as of November 30, 2021, for DINBs and Post-DIN Change (PDC) submissions
for biological and radiopharmaceutical products

Guidance on nitrosamine impurities in medications | 15

The summary and discussion of the risk assessment for the drug product is expected to
include sufficient detail to allow Health Canada to assess the adequacy and robustness
of the risk assessment. It should include a discussion of the risk factors and potential
root causes considered in relation to specific knowledge of the drug product and its
components (including the API). Checklists lacking sufficient discussion and detail
should be avoided. The summary and discussion should include the following:

• identification of any third parties (for example, suppliers, manufacturers,

consultants) who have been authorized to perform the risk assessment on
behalf of the applicant
• identification of intrinsic and extrinsic risk factors related to formation or
introduction of nitrosamine impurities originating from all drug product
components as well as quality/compliance considerations
• identification of those nitrosamines potentially formed and/or introduced
• information on the established process and/or analytical controls and how they
may mitigate risk
• supporting scientific data (for example, confirmatory testing results) and
calculations
• an overall conclusion on the risk of presence of nitrosamines in the drug product
together with an appropriate scientific rationale/justification

For Supplements, Notifiable Changes and Post-DIN Change submissions (for quality
changes that may impact the potential presence of nitrosamine impurities in the API or
drug product), the summary and discussion of the risk assessment need only address
the impact of the proposed change(s) on nitrosamine impurities relative to the
approved drug product. Examples of changes that may impact the potential presence
of nitrosamine impurities relative to an approved drug product include, but are not
limited to, changes in drug substance or drug product manufacturing processes,
changes to the drug product composition (API, excipients), introduction of a new
dosage form, and changes to the container closure system.

Refer to Appendix 2 for additional guidance with respect to nitrosamine impurities and
risk assessments for Post NOC Changes of new drug products containing chemically
synthesized and semi-synthetic APIs.

For Clinical Trial Applications (CTAs) (as described in section 9.1 of ICH's M7
guideline):

• For Phase 1 clinical trials of 14 days or less, include a description of efforts to

mitigate risks of mutagenic impurities focused on Class 1 and Class 2 impurities
and those in the cohort of concern (for example, nitrosamine impurities).
• For Phase 1 clinical trials greater than 14 days and for Phase 2 and 3 clinical
trials, also include Class 3 impurities that require analytical controls.

Failure to include this information could result in requests for additional information,
delays in the review process, and potentially the issuance of negative decisions.

Guidance on nitrosamine impurities in medications | 16

21. Controls for nitrosamines in APIs purchased for compounding

The Policy on Manufacturing and Compounding Drug Products in Canada (POL-

0051) provides a policy framework to help distinguish between compounding and
manufacturing activities of drug products in Canada. In Canada, the compounding
of drugs is done mainly by pharmacists as an integral part of their profession. It’s
regulated by the respective regulatory authorities in each province/territory.

This policy indicates that compounded products should be either:

• produced from an authorized API used in an authorized drug product for use
in Canada or
• listed in a recognized Pharmacopoeia (for example, USP/NF, Ph. Eur., Ph.
Int., BP, Codex - Schedule B, Food and Drugs Act)

Health Canada recommends, therefore, that principles outlined in the October 2,

2019, letter and in this guidance should be considered when purchasing APIs and
producing compounded products.

Health care professionals and compounding firms are encouraged to access the
nitrosamines webpage to stay informed on affected medications and recalls due to
the presence of nitrosamines. This page is updated regularly and also includes
general information on nitrosamine impurities, what Health Canada is doing to
address the issue and our testing results.

Communications
22. Engaging stakeholders and ensuring ongoing communication with industry

Health Canada is committed to sharing information with stakeholders and

maintaining transparency as we continue to analyze and better understand this
evolving, global situation.

To date, we have shared information openly with stakeholders, including

information on the potential sources of nitrosamine impurities, root causes and new
findings. We hosted stakeholder sessions in January 2020, February 2021 and
October 2021, and may host more sessions in the future if necessary.

We also established a dedicated webpage on nitrosamine impurities in medications.

The webpage includes the following:

• summaries of drug products that have been affected or recalled due to the
presence of nitrosamines
• analytical testing results of several products for levels of nitrosamine
impurities

Guidance on nitrosamine impurities in medications | 17

Discussions are ongoing to determine the most appropriate and effective methods
to continue to engage stakeholders as new information becomes available to ensure
a coordinated and consistent approach in dealing with this complex issue.

23. Health Canada works with global regulators relating to issues associated with
nitrosamine impurities in drug products

Health Canada regularly collaborates with international regulatory partners,

including those in Europe, the United States, Japan, Switzerland, Singapore,
Australia and Brazil, as well as the World Health Organization. Through
collaboration, we hope to increase the understanding of the issues associated with
nitrosamine impurities, align requirements and actions as appropriate, and share
information under the terms of our confidentiality agreements.

Discussions amongst the consortium of regulatory authorities continued, with the

"Nitrosamines International Strategic Group" (NISG), which was formed in 2018. The
NISG began meeting regularly with a focus on knowledge sharing on market and other
regulatory actions. Due to a mutual interest of the participating regulatory agencies of
the NISG to have greater in-depth discussions on technical issues and scientific
developments, a sub-group of the same regulators, the "Nitrosamines International
Technical Working Group" (NITWG), was established in late 2020.

When determining appropriate regulatory measures to address the presence of

nitrosamine impurities that exceed the AI limit in human drug products, individual
jurisdictions must determine timelines and actions that will best protect patient
safety and work within the relevant regulatory framework.

Safety
24. AI limits for nitrosamine impurities that Health Canada considers acceptable

AI limits have been derived for several nitrosamines (Appendix 1). These AI limits are
considered appropriate for all routes of administration and should be applied to the
maximum daily dose (MDD) of the drug product.

AI limits can be established using several approaches.

In cases where there is reliable compound-specific data for a nitrosamine impurity,

MAHs and applicants may:

Establish an AI limit based on reliable compound-specific data

1. Linearly extrapolate from the dose giving a 50% tumour incidence (TD50) to a 1
in 105 excess cancer risk, using the most relevant TD50 value from a sufficiently
robust carcinogenicity study (refer to ICH’s M7 Addendum for guidance on
selecting an appropriate carcinogenicity study).

Guidance on nitrosamine impurities in medications | 18

 2. Provide a negative, GLP-compliant, enhanced Ames test using the enhanced
Ames test conditions described in Appendix 3 to justify a limit of 1.5 µg/day.
3. Provide negative in vivo mutagenicity data (for example, a negative in vivo
mutagenicity assay conducted per the Organisation for Economic Co-operation
and Development (OECD)’s Test Guideline No. 488 “Transgenic Rodent Somatic
and Germ Cell Gene Mutation Assays”) to justify controlling a nitrosamine
impurity per the recommendations in ICH’s Q3A and Q3B guidelines.

In cases where there is insufficient reliable compound-specific data available for a

nitrosamine impurity, MAHs and applicants may:

Establish an AI limit based on a structure-activity relationship (SAR)

assessment and read-across to a surrogate with sufficient compound-specific
data

To justify an appropriate surrogate for read-across, the SAR assessment should take
into consideration structural similarity (both overall and at the local site of activation),
similarity of physicochemical characteristics, steric and electronic factors impacting
reactivity and metabolic similarity (for example, metabolic pathway, stability/reactivity
of metabolites).

If an appropriate surrogate for read-across is identified, to calculate an AI limit, the

TD50 should be derived from a sufficiently robust carcinogenicity study. Parameters to
consider include adequate description of the study design and appropriate
histopathological analysis, number of dose groups (i.e., single-dose studies are not
considered appropriate), number of animals per dose group, duration of exposure,
route of administration, observed dose-response relationship. Refer to ICH’s M7
Addendum for guidance on selecting an appropriate carcinogenicity study.

Consistent with international regulatory practices, Health Canada will continue to use,
and expect applicants and MAHs to use, mass-based calculations (rather than molar-
based) to derive AI limits for nitrosamine impurities when a surrogate is selected for
read-across.

Establish an AI limit using the Carcinogenic Potency Categorization Approach

(CPCA)

The Carcinogenicity Potency Categorization Approach (CPCA) is an approach for

assigning a nitrosamine to a predicted carcinogenic potency category.

A total of five carcinogenic potency categories are available, each with a corresponding
AI limit that ranges from 18 ng/day to 1500 ng/day.

A nitrosamine is assigned to a predicted carcinogenic potency category based on an

assessment of alpha hydrogen atoms and activating or deactivating structural features
present in the nitrosamine. Refer to Appendix 4 for a description of the approach which
also includes case examples to illustrate application of the CPCA.

Guidance on nitrosamine impurities in medications | 19

MAHs and applicants should also refer to the following items for more information:

• number 27 on the presence of multiple nitrosamines

• number 28 on applying a less-than-lifetime limit
• number 31 on which nitrosamines should be included in risk assessments and
confirmatory testing

25. AI limits for nitrosamine impurities in drug products that fall within the scope of the ICH
S9 guideline or where the API is genotoxic

If a nitrosamine impurity is identified in a pharmaceutical, biologic or

radiopharmaceutical product that is intended for advanced cancer indications
(defined in the scope of the ICH S9 guideline), the impurity can be controlled per
the recommendations in the ICH S9 questions-and-answers document.

If a nitrosamine impurity is identified in a drug product where the API is genotoxic

at therapeutic concentrations, the impurity can be controlled at limits for non-
mutagenic impurities. Refer to the ICH Q3A and Q3B guidelines.

26. Communicating if AI limits are revised in the future

Health Canada continues to work with international regulatory agencies to

determine acceptable limits for nitrosamine impurities. We will communicate any
changes to the acceptable limits for nitrosamine impurities to MAHs and applicants
in a timely manner.

Interim AI limits were originally communicated to MAHs for 5 nitrosamine impurities

in angiotensin II receptor blockers (also known as “sartans”). These were in place
until September 30, 2020, and will not be reduced to a lower level.

27. Acceptable limit if multiple nitrosamines are detected in an API or a drug product

If an API or drug product has the risk of containing more than 1 actual or potential
nitrosamine impurity, total (cumulative) daily exposure should be limited to the
nitrosamine with the most conservative AI limit at the maximum daily dose of the
drug product.

Examples:

• If a drug product contains both NDMA and NMBA, the total/cumulative daily
exposure of the 2 nitrosamines should be limited to 96.0 ng/day.
• If a drug product contains both NDMA and NDEA, the total/cumulative daily
exposure of the 2 nitrosamines should be limited to 26.5 ng/day.

Guidance on nitrosamine impurities in medications | 20

If an applicant or MAH proposes to control multiple nitrosamine impurities in an API
or drug product using an alternative methodology, Health Canada will assess the
acceptability of the approach on a case-by-case basis. Any proposed alternative
methodology should ensure that excess cancer risk does not exceed 1 in 100,000.

For nitrosamine impurities that are considered non-mutagenic, the

recommendations provided in ICH’s Q3A and Q3B guidelines apply and such
impurities do not need to be included in a limit for total nitrosamines.

28. Application of a less-than-lifetime (LTL) limit by considering the principles in ICH’s M7

guideline if a nitrosamine impurity is present in a drug product that is administered for
less than a lifetime

Considering the risk profiles of nitrosamines and the possibility of an additive biological
effect, the AI limits outlined in Appendix 1 are considered appropriate for lifetime and
LTL administration of a drug product.

If a nitrosamine impurity cannot be controlled at the AI limit, Health Canada may

consider an interim limit higher than the AI limit. We will do so on a case-by-case basis
and only in exceptional circumstances (for example, to avoid a drug shortage of a drug
product that is considered medically necessary or medically important).

Where an applicant or MAH proposes an interim limit higher than the AI limit for a
nitrosamine impurity, Health Canada will consider:

• the medical necessity or medical importance of the drug product

• levels of impurity observed in representative batches
• other risk management considerations (for example, the availability of
alternative medications on the Canadian market)
We will consider an interim limit higher than the AI limit for a nitrosamine impurity as a
transitory measure only, until appropriate changes to reduce the level of the
nitrosamine impurity to at or below the AI limit have been implemented.

For nitrosamine impurities that are considered non-mutagenic, the recommendations

provided in ICH’s Q3A and Q3B guidelines apply and such impurities do not need to be
included in a limit for total nitrosamines.

Guidance on nitrosamine impurities in medications | 21

Quality
29. Risk factors and potential root causes to be considered for the presence of
nitrosamine impurities in human pharmaceuticals when performing a risk assessment

Knowledge of risk factors and potential root causes for nitrosamine impurities continues
to evolve. Applicants and MAHs should stay up-to-date on risk factors and potential
root causes that Health Canada and other regulators have identified in their guidances
and peer-reviewed publications. The collaboration of the quality technical experts of the
international regulatory partners of the NITWG has led to the development and
publication of the paper "Regulatory Experiences with Root Causes and Risk Factors for
Nitrosamine Impurities in Pharmaceuticals" (Horne et al. J. Pharm. Sci. 2023, 112,
1166-1182). This publication is designed to share current information and experiences
from a quality perspective on root causes, risk factors, and risk mitigation measures
relating to nitrosamine impurities in pharmaceuticals for human use.

Inadequate process design and/or process controls, as well as gaps in quality and
compliance oversight, may contribute to the presence of nitrosamine impurities in APIs
and drug products above AI limits. Applicants and MAHs should consider both intrinsic
and extrinsic factors when conducting risk assessments for nitrosamine impurities.

Potential and confirmed root causes and risk factors for the presence of nitrosamines in
drug products include the following:

• Nitrosation of a secondary or tertiary amine during API or drug product

manufacturing, with insufficient downstream purge of the nitrosamine formed,
and/or during API or drug product storage (common nitrosation conditions
involve the combination of amines and nitrite ion under acidic conditions)
o Sources of amines include APIs, API intermediates, starting materials,
reagents, solvents, catalysts, reaction by-products and degradation
products. Certain non-medicinal ingredients may also contain amines as
part of their structure. Amines leading to stable nitrosamines include
secondary amines and tertiary amines. Quaternary ammonium salts are
also potential precursors to nitrosamines. Primary and tertiary amines
may contain secondary amines as impurities. Amines may be present as
impurities in amides or formed through the degradation of amides (for
example, via hydrolysis). Tertiary amines may be nitrosated by a
dealkylative pathway to produce one or more secondary amines, which
may subsequently undergo nitrosation to produce multiple nitrosamines.
o Nitrosating agent precursors and sources include:
▪ nitrite ion intentionally used in a manufacturing process (for
example, as used in diazotization chemistry or as a reducing agent
for azide ion)
▪ nitrite present as an impurity in reagents (for example, sodium
azide), common non-medicinal ingredients (for example,
microcrystalline cellulose, magnesium stearate)

Guidance on nitrosamine impurities in medications | 22

 ▪ nitrogen oxides (for example, NO, N2O3)
▪ nitric acid
▪ nitrosyl halides
▪ alkyl nitrites and nitro compounds (for example, nitromethane)
▪ potable and/or purified water containing nitrite
• Using a nitrosamine as a starting material or synthetic intermediate, with
incomplete conversion of the nitrosamine and/or insufficient downstream purge
• Reaction of nitrite ion and an amine under process conditions with pH >7 under
catalysis by a carbonyl compound, with insufficient downstream purge
• Oxidation of a hydrazine functional group in an API, starting material,
intermediate or a reagent to produce a nitrosamine, with insufficient
downstream purge
• Using certain materials in container closure components, such as:
o nitrocellulose, found in certain lidding foils used for blister packaging
o certain types of vulcanisation accelerators (for example, dithiocarbamate,
thiourea, thiruams) which are used in rubber manufacturing
• Using recycled materials (for example, solvents, reagents, catalysts)
contaminated with nitrosamines and/or nitrosamine precursors
• Cross-contamination of materials with nitrosamines/nitrosamine precursors in
multi-product facilities (for example, through the use of shared equipment)
• Poor operation of a process step (for example, during liquid-liquid phase
separations), which is intended to purge nitrosamines
• Using certain manufacturing operations that could facilitate contact between
nitrosamine precursors (for example, wet granulation) or introduce nitrosating
agents/precursors (for example, nitrogen oxides during fluid bed drying)

30. Components of drug products to consider in risk assessments

All components of the finished drug product should be considered as potential

sources of nitrosamine impurities, or their precursor nitrosating agents and amines,
in the context of the designated process and storage conditions. For example, some
excipients may contain residual levels of nitrite (Wu, Y. et al. AAPS PharmSciTech
2011, 12(4), 1246-1263) or reactive amines as part of their molecular structure.
Under certain manufacturing process or storage conditions, this may lead to the
formation of nitrosamine impurities.

Refer to number 29 for more information on risk factors and potential root causes
to take into consideration.

Guidance on nitrosamine impurities in medications | 23

31. Nitrosamine impurities to consider in the Step 1 risk assessment and Step 2
confirmatory testing (updated)

Each API and drug product manufacturing process is unique. Thus, the
nitrosamines listed in Appendix 1 of this guidance are not exhaustive and do not
represent all nitrosamines potentially present in APIs and drug products.
Conversely, the nitrosamines listed in Appendix 1 may not be potential impurities in
all APIs and drug products.

MAHs and applicants should ensure that the risk assessments consider and identify
the possibility of any nitrosamine impurity that may be formed or introduced. All
nitrosamines that have been determined to be potentially formed or introduced
should be included within the program for confirmatory testing (Step 2).

For nitrosamines not included Appendix 1, MAHs and applicants should refer to
number 24 for guidance on how to establish an AI limit.

32. Testing methodologies provided by Health Canada

Several regulators, including Health Canada, Europe’s network of Official Medicines

Control Laboratories (OMCLs) and the U.S. Food and Drug Administration (FDA),
have published and shared testing methodologies. These methods may be used,
although there is no requirement to do so.

In all cases, companies should use appropriately sensitive, validated analytical

methods and conduct the testing at a GMP-compliant facility. If other
methodologies are used, there is no need to verify the method with Health Canada
prior to use.

Analytical methods should be quantitative in nature (as opposed to limit-based

tests) and should be fully validated before confirmatory testing begins. If limit-
based tests are used, ensure that the appropriate scientific justification is provided
in the risk assessment documentation. For example:

• demonstration that the limit test is valid at or lower than the AI limit
• supporting evidence that indicates there is no increase in the concentration
of nitrosamine impurities over time

Unless otherwise justified, method validation should be performed using the drug
product that is authorized for use in Canada.

Where multiple strengths of a drug product exist and the validation is to cover
multiple strengths, the justification for the choice of product strength used for
validation should be described in the validation protocol.

Guidance on nitrosamine impurities in medications | 24

33. Validating the limit of quantitation (LOQ) for nitrosamine impurity analytical procedures
(updated)

The LOQ for analytical procedures that are intended for quantitation of individual
nitrosamine impurities in APIs and drug products should be equal to or less than
the established AI limit (Appendix 1), the AI limit established using the CPCA (refer
to number 24 and Appendix 4) if an AI limit has not been established by Health
Canada, or the relevant ICH Q3A or Q3B limit for a nitrosamine impurity that has
been classified as non-mutagenic in Appendix 1.

Analytical procedures should be validated with a LOQ which is less than or equal to
10% of the acceptable limit for an individual nitrosamine, if a proposal to not
routinely test for the nitrosamine in the drug product specification is anticipated
(refer to number 34).

Analytical procedures should be validated with a LOQ which is less than or equal to
30% of the acceptable limit for an individual nitrosamine, if a proposal for periodic
(skip) testing is anticipated.

34. Including routine testing for nitrosamine impurities in the API and/or drug
product specification

The API specification should include a test and acceptance criterion for each
nitrosamine impurity when:

• the risk for nitrosamine presence is considered to be high and/or

• the concentration of any nitrosamine is found to be at significant levels (for
example, greater than 30% of the AI limit) during confirmatory testing

Examples where the risk for nitrosamines is considered high:

• potential for nitrosamine formation on storage

• presence of nitrosamine precursor functional groups in the API
• late-stage formation/introduction of a nitrosamine impurity in the
manufacturing process

Where multiple nitrosamines are detected in an API, a cumulative limit should also be
included in the specification using one of the approaches outlined in number 27.

Routine testing for nitrosamine impurities should be included in the drug product
specification when:

• the potential for nitrosamine introduction during drug product manufacturing,

packaging and storage is identified and/or
• a nitrosamine impurity is detected in the drug product during confirmatory
testing and the root cause is unknown

Guidance on nitrosamine impurities in medications | 25

Where such a risk is identified, a test and acceptance criteria for both release and shelf
life specifications should be included. Where multiple nitrosamines are detected,
control for total nitrosamines using one of the approaches outlined in number
27 should be included in the specification. Alternatively, control limits expressed on an
individual impurity basis (for example, a limit for each nitrosamine set at a percentage
of its AI limit such that the sum of the % AI limits for each specified nitrosamine does
not exceed 100%) may be proposed with appropriate justification. Other approaches
for establishing a suitable specification when multiple nitrosamines are concerned may
be acceptable if appropriately justified.

The presence of one or more nitrosamines at <10% of their individual AI limits in a

drug product constitutes a negligible toxicological risk; if the root cause for the
presence of such nitrosamine impurities is understood and appropriate controls have
been established to ensure such impurities will consistently be <10% of their individual
AI limits, then such impurities do not need to be specified in the drug product
specifications. Nitrosamines present below 10% of their respective AI limit do not need
to be factored into the calculation of limits for total nitrosamines.

MAHs should test all new lots of drug product for nitrosamines and only release lots
that meet the acceptance criteria for individual (and multiple nitrosamines, if relevant).
Continue routine testing of all drug product lots until the root cause is identified and
alternative controls/risk mitigation measures (such as process controls, raw material
specifications) have been implemented. Ensure that nitrosamine impurities will be
routinely below the AI limit in the future.

35. Potential control options for nitrosamine impurities in the API

Control options for nitrosamine impurities include:

• routine testing in the API (ICH M7 option 1)

• control in upstream intermediate specifications at the acceptable limit (ICH
M7 option 2) (when the route cause, or causes, of nitrosamine presence have
been established unequivocally)
• control in upstream intermediate specifications at acceptance criteria that
exceed the acceptable limit (ICH M7 option 3) (when the root cause, or
causes, have been established unequivocally and justification of the proposed
limit is supported by demonstrated process capability (for example, spike and
purge studies))

Proposals for an ICH M7 option 4 control strategy for nitrosamine impurities in a

new market authorization application will be evaluated on a case-by-case basis. An
option 4 control strategy proposal may not be appropriate when the concentration
of any nitrosamine impurity in an API is greater than 30% of the AI limit. However,

Guidance on nitrosamine impurities in medications | 26

such a strategy may be acceptable when process understanding has been
demonstrated by fate-purge studies, identification of process parameters that
impact nitrosamine impurity levels and when supported by appropriate analytical
data. Predicted purge factor calculations should be supported by appropriate
analytical data.

This information should be provided along with copies of analytical procedures and
method validation reports in the new market authorization application.

Refer also to number 34 for information on routine testing for nitrosamine

impurities in the API and/or drug product specification.

36. Confirmatory testing expectations (Step 2)

During confirmatory testing, MAHs should test the drug product to determine the
levels of nitrosamine impurities.

Testing the API is also recommended if the risk assessment indicated that the API is
a potential source of nitrosamine impurities in the drug product. The API testing
results may be used to support root cause investigations and the development of a
justified control strategy for nitrosamine impurities in the API.

If a drug product is available in multiple strengths of the same dosage form with
the same risk factors applicable to each, then testing could be rationalized by
testing only the worst-case scenario strength. The worst-case approach should be
justified by the MAH on a case-by-case basis. The justification should be
documented in the risk assessment in the MAH's pharmaceutical quality system.

If, despite extensive efforts, it becomes apparent that a nitrosamine impurity

cannot be synthesized or isolated and purified, then this could be an indication that
the nitrosamine either does not exist, is unstable, or that there is no risk of it being
formed. In such cases, it may not be necessary to conduct confirmatory testing.
This should be justified thoroughly on a case-by-case basis according to appropriate
scientific principles. A scientific justification including experimental data which
summarizes the efforts made to synthesize and/or isolate and purify the impurity
should be included in the summary and discussion of the risk assessment in the
regulatory submission and documented in the MAH's pharmaceutical quality
system.

Guidance on nitrosamine impurities in medications | 27

37. Analytical laboratories conducting nitrosamine testing and listing on the DEL

The analytical lab used for nitrosamine confirmatory testing (Step 2) does not have
to be listed on the DEL at this time. However, in all cases, the confirmatory testing
must be conducted at a GMP-compliant facility. A foreign analytical lab, if used for
conducting the nitrosamine confirmatory testing, must either:

• have been deemed GMP-compliant by Health Canada or

• have a valid GMP inspection by a competent or qualified regulatory authority
demonstrating compliance with current GMP standards

Ethical drugs are those that do not require a prescription, but are generally
prescribed by a medical practitioner as professional use products (for example,
hemodialysis solutions, nitroglycerine). For testing ethical and over-the-counter
drugs, if no inspection reports by regulatory/qualified authorities are available, a
corporate or consultant audit report to demonstrate GMP compliance is acceptable.

For more information about acceptable GMP evidence and regulatory requirements,
refer to the following guidance:

• How to Demonstrate Foreign Building Compliance with Drug Good Manufacturing

Practices (GUI-0080)

However, analytical labs must be listed on the applicable annex of the DEL if they
are conducting:

• nitrosamine testing used to release APIs and drug products for the Canadian
market or
• tests that are part of the API or drug product specification
o includes testing imposed through the sartan terms and conditions

For guidance on Health Canada’s expectations for testing facilities or for other
related questions, please send an email to [email protected].

38. Number and types of drug product batches as part of confirmatory testing for
marketed products and new market applications

For marketed products, the number of batches to be tested should be

commensurate with the risk. Examples of high risk include:

• late-stage formation/introduction of a nitrosamine impurity in a

manufacturing process
• presence of nitrosamine precursor functional groups in the API
• potential for nitrosamine formation on storage

Guidance on nitrosamine impurities in medications | 28

MAHs and manufacturers should test a representative number of batches of the
drug product as appropriate based on the risk assessment (for example, batches
that are representative of sources of components, manufacturing processes/sites,
manufacturing dates).

If the root cause for nitrosamine risk has been identified and scientifically
demonstrated, and impurity levels are expected to be consistent from batch-to-
batch (for example, as demonstrated by spike-purge studies), testing should be
conducted on 10% of annual batches, or 3 per year, whichever is highest. Testing
should include both newly produced batches as well as retained samples of batches
still within the expiry date. If fewer than 3 batches are manufactured annually, then
all batches within the expiry date should be tested.

Testing plans or protocols (for example, a protocol for the number and type of
batches to be tested) do not need to be submitted to Health Canada for assessment
and approval before initiating confirmatory testing.

If nitrosamine impurities are detected at significant levels (approaching, at or above

AI limits), additional batches of the drug product on the Canadian market and
within the expiry date should undergo confirmatory testing. In such cases, MAHs
may be requested to test all lots on the Canadian market that are within the expiry
date.

For NDSs, ANDSs, Supplements and Notifiable Changes (for quality changes that
may impact the potential presence of nitrosamines in the drug substance or drug
product, refer to number 13), at least 6 pilot or 3 commercial-scale batches should
undergo confirmatory testing where a risk of nitrosamines has been identified. A
higher number of batch results should be submitted for assessment where the risk
of nitrosamine presence is high. Examples include:

• the late-stage formation/introduction of a nitrosamine impurity

• nitrosamine precursor functional groups in the API
• stability concerns exist for nitrosamine formation over the retest period/shelf
life

Testing results of stability batches for a nitrosamine impurity should be conducted

where:

• a risk has been identified that nitrosamine levels could increase in the API or
drug product over time or
• the potential for increases over time is unclear

Where applicable, testing for the nitrosamine impurity for a minimum of 6 months
of accelerated and long-term stability data in the proposed container closure
system(s) should be provided in the drug application.

Guidance on nitrosamine impurities in medications | 29

Appendices
Appendix 1: Established Acceptable Intake (AI) limits for N-nitrosamine impurities

• See separate Appendix 1.

Guidance on nitrosamine impurities in medications | 30

 Appendix 2: Guidance with respect to nitrosamine impurities and risk assessments for Post NOC
Changes of new drug products containing chemically synthesized and semi-synthetic APIs (updated)

*For example, changes to drug substance or drug product manufacturing processes,

changes to the drug product composition (API, excipients), changes to a dosage form,
changes to the container closure system.

Guidance on nitrosamine impurities in medications | 31

Appendix 3: Enhanced Ames assay test conditions

The Organisation for Economic Co-operation and Development (OECD)’s Test Guideline
No. 471 “Bacterial Reverse Mutation Test” provides standard recommendations for the
conduct of the bacterial reverse mutation test (also known as the Ames assay) to
assess the mutagenic potential of a test compound. For N-nitrosamines, enhanced
testing conditions for the Ames assay are recommended due to the reported reduced
sensitivity of the assay under standard conditions for some N-nitrosamines such as N-
nitroso-dimethylamine (NDMA). Moreover, very little is known about the sensitivity of
the Ames assay to N-nitrosamine drug substance related impurities (NDSRIs), which
are a recently recognized class of N-nitrosamine impurities structurally related to the
drug substance. NDSRIs generally have a wider variety of functional groups present
than typically found in low molecular weight N-nitrosamines (such as NDMA)
historically studied.

If a standard Ames assay is conducted and produces a positive result, there is no need
to conduct an additional assay using enhanced testing conditions.

The enhanced Ames assay test conditions presented below are informed by work
conducted by FDA’s National Center for Toxicological Research (NCTR) (Li et. al.,
2023), as well as other groups, and have been evaluated for a variety of N-
nitrosamines including NDSRIs. Evaluation of Ames assay test conditions for N-
nitrosamines is ongoing with a goal to identify the most robust Ames testing
conditions. The enhanced Ames assay test conditions described below will be updated
as warranted. Deviations from the recommended conditions should be justified.

Tester strains: S. typhimurium TA98, TA100, TA1535, TA1537, and E. coli WP2 uvrA
(pKM101) tester strains should be included.

Type of assay and preincubation time: The pre-incubation, and not plate
incorporation, method should be used. The recommended pre-incubation time is 30
minutes.

Species and concentration of S9: Ames assays should be conducted in the absence
of a post-mitochondrial fraction (S9), and also in the presence of 30% rat liver S9, as
well as 30% hamster liver S9. The rat and hamster post-mitochondrial fractions (S9s)
should be prepared from rodents treated with inducers of cytochrome P450 enzymes
(for example, a combination of phenobarbital and β-naphthoflavone).

Negative (solvent/vehicle) control: Solvents need to be compatible with the Ames

assay as per the OECD 471 guideline. Solvents can include, but are not limited to:

• water
• organic solvents such as acetone, methanol and DMSO

When an organic solvent is used, the lowest possible volume should be included in the
pre-incubation mixture with justification to indicate that the volume of solvent does not

Guidance on nitrosamine impurities in medications | 32

interfere with metabolic activation of the N-nitrosamine.

Positive controls: Concurrent strain-specific positive controls should be included per

the OECD 471 guideline.

Two N-nitrosamines that are known to be mutagenic in the presence of S9 should also
be included as positive controls.

The choice of the N-nitrosamine positive controls needs to be justified based on the
anticipated metabolism of the N-nitrosamine and the cytochrome P450 enzymes most
likely involved. In addition, if an organic solvent is used to dissolve the test compound,
it is recommended that the volume of organic solvent employed to dissolve the N-
nitrosamine positive controls results in a similar concentration as for the test compound
in the pre-incubation mix, if possible.

N-Nitrosamine positive controls to consider include:

1. NDMA (CAS # 62-75-9)

2. 1-Cyclopentyl-4-nitrosopiperazine (CAS # 61379-66-6)
3. An NDSRI

All other recommendations for the Ames assay should follow the OECD 471 guideline.

References:

OECD Test Guideline No. 471 “Bacterial Reverse Mutation Test”. 2020

Li et al. Revisiting the mutagenicity and genotoxicity of N-nitroso propranolol in

bacterial and human in vitro assays. Regulatory Pharmacology and Toxicology. 2023

Guidance on nitrosamine impurities in medications | 33

Appendix 4: Carcinogenic Potency Categorization Approach (CPCA) for N-nitrosamines

This document describes an approach for assigning an N-nitrosamine impurity

(including nitrosamine drug substance-related impurities [NDSRIs]) to a predicted
carcinogenic potency category, with a corresponding Acceptable Intake (AI) limit,
based on an assessment of activating or deactivating structural features present in the
molecule. In the context of this document, activating or deactivating features are
defined as molecular substructures that are associated with an increase or decrease,
respectively, in carcinogenic potency.

The Carcinogenic Potency Categorization Approach is based on structure-activity

relationship (SAR) concepts described in recent scientific publications for N-nitrosamine
compounds1 and also used a set of approximately 80 N-nitrosamines with either rat
TD50 values from the Carcinogenic Potency Database (CPDB) and/or the Lhasa
Carcinogenicity Database (LCDB)2, relative potency classifications as defined by Rao et
al. (1979)3, and/or AI limits based on previously-conducted surrogate analyses4. The
approach assumes that the α-hydroxylation mechanism of metabolic activation5 is
responsible for the mutagenic and highly potent carcinogenic response observed for
many N-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, are expected to have a corresponding effect
on carcinogenic potency. Therefore, a prediction of the mutagenic potential and
carcinogenic potency of an N-nitrosamine can be generated based on its structural
features.

It is recognized that the science is evolving in the prediction of mutagenic potential and
carcinogenic potency based on SAR concepts. Therefore, the predicted Carcinogenic
Potency Categorization Approach described in this document 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. This may include refinement of
the AI limits associated with predicted carcinogenic potency categories and changes to
the structural features and their associated activating and deactivating feature scores.

1
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, 35:1997–2013;
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, 65:15584–15607.
2
See Lhasa Carcinogenicity Database at https://carcdb.lhasalimited.org/.
3
Rao TK, Young JA, Lijinsky W and Epler JL, 1979. Mutagenicity of Aliphatic Nitrosamines in Salmonella typhimurium,
Mutat Res, 66:1-7.
4
Questions and answers for marketing authorisation holders / applicants on the CHMP opinion for the Article 5(3)
referral.
5
Li Y, Hecht SS, 2022. Metabolic Activation and DNA Interactions of Carcinogenic N-Nitrosamines to Which Humans Are
Commonly Exposed, Int J Mol Sci, 23:4559.

Guidance on nitrosamine impurities in medications | 34

 The Carcinogenic Potency Categorization Approach applies to N-nitrosamines bearing a
carbon atom on both sides of the N-nitroso group, and where the carbon is not directly
double bonded to a heteroatom (that is, N-nitrosamides, N-nitrosoureas, N-
nitrosoguanidines and other related structures are excluded). Additionally, the potency
categorization approach does not apply to N-nitrosamines where the N-nitroso group is
within an aromatic ring (for example, nitrosated indole). For N-nitrosamines containing
two N-nitroso groups, the group with the highest predicted carcinogenic potency (that
is, the group with the lowest numerical potency category) defines the AI for the entire
molecule6. The α- and β-carbons are defined relative to the N-nitroso group, as
illustrated in Figure 1.

Figure 1. Structural Representation of α- and β-carbons on an N-nitrosamine

The process for predicting the appropriate carcinogenic potency category is described
in Figure 2. Table 1 summarizes the five predicted carcinogenic potency categories
and their associated AI limits. Supporting tables to calculate the Potency Score
referenced in Figure 2 are in Appendix A and example calculations are presented in
Appendix B.

6
For N-nitrosamines containing more than two N-nitroso groups, the applicant or manufacturer should contact the
applicable drug regulatory authority for further guidance.

Guidance on nitrosamine impurities in medications | 35

Figure 2. Flowchart to Predict the Potency Category of an N-nitrosamine

*
A tertiary α-carbon is defined as an α-carbon atom in an sp3 hybridization state,
bonded to three other carbon atoms.
**
To calculate Potency Score, see Appendix A.

Guidance on nitrosamine impurities in medications | 36

 Table 1. The Five Predicted Potency Categories and Associated AI Limits for N-
Nitrosamines

Recommended
Potency
AI Limit Comments
Category
(ng/day)

1 18 The recommended AI limit of 18 ng/day is equal to the class-

specific TTC for N-nitrosamine impurities.* N-nitrosamines
assigned to Category 1 are predicted to have high carcinogenic
potency; however, the class-specific TTC for N-nitrosamine
impurities is considered sufficiently protective to patients.

2 100 The recommended AI limit of 100 ng/day is representative of two

potent, robustly tested N-nitrosamines, N-nitrosodimethylamine
(NDMA) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-(butanone)
(NNK), which have recommended AI limits of 96 ng/day and 100
ng/day, respectively. N-nitrosamines assigned to Category 2 are
predicted to have carcinogenic potency no higher than NDMA and
NNK.

3 400 Compared to Potency Category 2, N-nitrosamines in this category

have lower carcinogenic potency due to, for example, the
presence of a weakly deactivating structural feature. The
recommended AI limit was set to reflect a 4-fold decrease in
carcinogenic potency from Category 2.

4 1500 N-Nitrosamines assigned to Category 4 may be metabolically

activated through an α-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 because
clearance pathways are favored. The recommended AI limit of
1500 ng/day is set at the TTC per ICH M7.**

5 1500 N-Nitrosamines assigned to Category 5 are not predicted to be

metabolically activated via an α-hydroxylation pathway due to
steric hindrance or the absence of α-hydrogens, 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.**

* Assessment report Procedure under Article 5(3) of Regulation EC (No) 726/2004 Nitrosamine
impurities in human medicinal products Procedure number: EMEA/H/A-5(3)/1490
** See the International Council for Harmonisation guidance for industry M7 Assessment and Control of
DNA Reactive (Mutagenic) Impurities in Pharmaceuticals To Limit Potential Carcinogenic Risk. Threshold
of Toxicological Concern (TTC) of 1.5 µg/day (1500 ng/day) as explained in ICH M7, represents an AI
for any unstudied chemical that poses a negligible risk of carcinogenicity or other toxic effect.

Guidance on nitrosamine impurities in medications | 37

Appendix A. Calculation of potency score

For N-nitrosamines not assigned to Potency Category 5, the Potency Score is calculated
as the sum of the α-Hydrogen Score (Table 2), Deactivating Feature Score (Table 3) and
Activating Feature Score (Table 4) based on selected structural features present in the N-
nitrosamine. The N-nitrosamine structure is expected to match exactly one of the α-
hydrogen definitions in Table 2, but it may contain multiple or no structural features
identified in Tables 3 and 4. In cases where one or more features from Tables 3 and 4 are
contained in the N-nitrosamine, the Potency Score should be calculated as outlined in the
box below. In cases where the N-nitrosamine contains no features from Tables 3 and 4,
the Potency Score will be equal to the α-Hydrogen Score.

Potency Score = α-Hydrogen Score + Deactivating Feature Score (sum all

scores for features present in the N-nitrosamine) + Activating Feature
Score (sum all scores for features present in the N-nitrosamine)

Table 2. Count of hydrogen atoms on each α-carbon (lowest count first) and
corresponding α-Hydrogen Score. Examples are intended to be illustrative only and
are not intended to be exhaustive.

α-
Count of Hydrogen Atoms on Each
Example Hydrogen
α-Carbon, Lowest First
Score

0,2 3*

0,3 2

1,2 3

1,3 3

2,2 1

2,3 1

*A score of 3 applies when the methylene α-carbon is not part of an ethyl group. If the
methylene α-carbon is part of an ethyl group, a score of 2 should be applied.

Guidance on nitrosamine impurities in medications | 38

Table 3. List of deactivating features and associated scores. To calculate
Deactivating Feature Score, sum the individual scores for all listed features present in
the N-nitrosamine structure. Each deactivating feature row in the table may only be
counted once. For N-nitrosamines 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

+2
ring*

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 side of acyclic N-nitroso group. +1
Not more than 4 atoms in each chain
may be in the same ring.

Electron-withdrawing group** bonded

to α-carbon on only one side of N- +1
nitroso group (cyclic or acyclic)

Guidance on nitrosamine impurities in medications | 39

 Electron-withdrawing groups** bonded
to α-carbons on both sides of N-nitroso +2
group (cyclic or acyclic)

Hydroxyl group bonded to β-

carbon*** on only one side of N- +1
nitroso group (cyclic or acyclic)

Hydroxyl group bonded to β-carbon 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 “β-carbon electron withdrawing groups.”
***β-Carbon must be in an sp3 hybridization state for this feature to apply

Table 4. List of activating features and associated scores. To calculate Activating

Feature Score, sum the individual scores for all listed features present in the N-
nitrosamine 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 α-carbon (i.e.,
benzylic or pseudo-benzylic substituent -1
on N-nitroso group)

Methyl group bonded to β-carbon

-1
(cyclic or acyclic)

Guidance on nitrosamine impurities in medications | 40

Appendix B. Example carcinogenic potency categorization approach calculations based on flow chart

Example 1 – N-Nitroso-felodipine

Example 1 shows how the potency categorization approach flow chart (Figure 2) can be
applied to the N-nitrosamine, N-nitroso-felodipine. N-Nitroso-felodipine is placed in
Potency Category 5 with an associated AI limit of 1500 ng/day.

Guidance on nitrosamine impurities in medications | 41

Guidance on nitrosamine impurities in medications | 42
Example 2 – N-Nitroso-enalapril

Example 2 shows how the potency categorization approach flow chart (Figure 2) can be
applied to the N-nitrosamine, N-nitroso-enalapril. N-Nitroso-enalapril is placed in Potency
Category 5 with an associated AI limit of 1500 ng/day.

Guidance on nitrosamine impurities in medications | 43

Guidance on nitrosamine impurities in medications | 44
Example 3 – N-Nitroso-ketamine

Example 3 shows how the potency categorization approach flow chart (Figure 2) can be
applied to the N-nitrosamine, N-nitroso-ketamine. N-Nitroso-ketamine is placed in
Potency Category 5 with an associated AI limit of 1500 ng/day.

Guidance on nitrosamine impurities in medications | 45

Guidance on nitrosamine impurities in medications | 46
Example 4 – N-Nitroso-nebivolol

Example 4 shows how the potency categorization approach flow chart (Figure 2) can be
applied to the N-nitrosamine, N-nitroso-nebivolol. A Potency Score of 4 is calculated for
N-nitroso-nebivolol, resulting in its placement in Potency Category 4 with an associated
AI limit of 1500 ng/day.

Guidance on nitrosamine impurities in medications | 47

Guidance on nitrosamine impurities in medications | 48
Example 5 – N-Nitroso-meropenem

Example 5 shows how the potency categorization approach flow chart (Figure 2) can be
applied to the N-nitrosamine, N-nitroso-meropenem. A Potency Score of 4 is calculated
for N-nitroso-meropenem, resulting in its placement in Potency Category 4 with an
associated AI limit of 1500 ng/day.

Guidance on nitrosamine impurities in medications | 49

Guidance on nitrosamine impurities in medications | 50
Example 6 – N-Nitroso-desloratadine

Example 6 shows how the potency categorization approach flow chart (Figure 2) can be
applied to the N-nitrosamine, N-nitroso-desloratadine. A Potency Score of 3 is calculated
for N-nitroso-desloratadine, resulting in its placement in Potency Category 3 with an
associated AI limit of 400 ng/day.

Guidance on nitrosamine impurities in medications | 51

Guidance on nitrosamine impurities in medications | 52
Example 7 – N-Nitroso-sertraline
Example 7 shows how the potency categorization approach flow chart (Figure 2) can be
applied to the N-nitrosamine, N-nitroso-sertraline. A Potency Score of 2 is calculated
for N-nitroso- sertraline, resulting in its placement in Potency Category 2 with an
associated AI limit of 100 ng/day.

Guidance on nitrosamine impurities in medications | 53

Guidance on nitrosamine impurities in medications | 54
Example 8 – N-Nitroso-lorcaserin

Example 8 shows how the potency categorization approach flow chart (Figure 2) can be
applied to the N-nitrosamine, N-nitroso-lorcaserin. A Potency Score of 1 is calculated for
N-nitroso-lorcaserin, resulting in its placement in Potency Category 1 with an associated
AI limit of 18 ng/day.

Guidance on nitrosamine impurities in medications | 55

Guidance on nitrosamine impurities in medications | 56