7 July 2023

EMA/409815/2020 Rev.16

Questions and answers for marketing authorisation

holders/applicants on the CHMP Opinion for the Article
5(3) of Regulation (EC) No 726/2004 referral on
nitrosamine impurities in human medicinal products

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Table of contents
Revision History .......................................................................................... 3
Introduction................................................................................................ 4
1. Should the risk of presence of nitrosamines be considered for all human medicinal
products? ................................................................................................... 5
2. What is the ‘call for review’? ...................................................................... 5
3. For the ‘call for review’ for chemically synthesised and biological medicinal
products, when and how should MAHs report steps 1 and 2 to competent
authorities? ................................................................................................ 6
4. What are the currently identified risk factors for presence of nitrosamines? ....... 8
5. What to do if after submission of step 1 and /or step 2 responses, new
information (e.g. related to new potential risk factors or root causes) is identified?10
6. What factors should be considered in prioritising the risk evaluation? ..............11
7. How should the risk evaluation be performed? .............................................11
8. How should confirmatory tests be conducted by MAHs and manufacturers? ......12
9. What are the requirements of the analytical method(s)? ................................14
10. Which limits apply for nitrosamines in medicinal products? (Updated) ............14
11. What should I do if a nitrosamine is detected in my medicinal product? ..........18
12. Which are the measures to mitigate the risk of presence of nitrosamines? ......19
13. Which changes would be required for Marketing Authorisations? ...................19
14. What is the approach for new and ongoing marketing authorisation applications
(MAA)? .....................................................................................................20
15. When should a test for nitrosamines be included in the MA dossier? ..............21
16. What are the responsibilities of MAHs for APIs with CEPs or ASMFs? ..............22
17. How does the lessons learnt exercise from presence of nitrosamines in sartans
relate to the Article 5(3) Referral Outcome? ....................................................22
18. What about regulatory requirements in other regions? ................................22
19. What is the approach for line extensions and variations applications not linked to
changes required as part of article 5(3) recommendation?.................................23
20. What are the regulatory steps taken by authorities following the identification of
an N-nitrosamine exceeding the AI? ..............................................................23
21. What is the approach to control the presence of nitrosamines until a substance
specific AI is established?.............................................................................24
22. What is the approach to control presence of N-nitrosamine exceeding the AI
during CAPA implementation? ......................................................................24
Annex 1: Decision tree with control options for products containing multiple N-
nitrosamines:.............................................................................................26

Questions and answers for marketing authorisation holders/applicants on the CHMP

Opinion for the Article 5(3) of Regulation (EC) No 726/2004 referral on nitrosamine
impurities in human medicinal products
EMA/409815/2020 Page 2/51
Annex 2: Carcinogenic Potency Categorization Approach for N-nitrosamines .........27
Annex 3: Enhanced Ames Test Conditions for N-nitrosamines ............................50

Revision History
Rev. Summary of changes made Date

0 Replace obsolete Q&A published in 2019 to support the initial “call 03rd August 2020
for review” with a new version reflecting the main principles agreed
as part of the Article 5(3) referral which concluded in July 2020.
1 Update to Q&A 3 in order to clarify products in scope of the call for 29th January 2021
review.
Update to Q&A 4 in order to add the link to the outcome of the
referral under article 3 of Directive 2001/83/EC for ranitidine.
2 Update to Q&A 3 on indicating testing timeline at the time of step 1 24th February 2021
“risk identified” reporting.
3 Update to Q&A 3 on the approach for non-marketed medicines. 15th April 2021
New Q&A 19 on the requirements for line extensions and variation
applications.
4 Update to Q&A 3 on combining step 2 response for multiple products 18th May 2021
from the same MAH.
4* Updates to Q&A 3 on when to perform step 2 confirmatory testing in 29th June 2021
order to meet the established deadline for step 3.
Update and Q&A 10 to add an AI for NMOR.
5 Update to Q&A 10 to add an AI for NNV. 21st September 2021
6 Guidance on confirmatory testing requirements for marketed 14th October 2021
(Q&A 8) and on-going applications (Q&A 14) to include cases where
a potential nitrosamine impurity cannot be synthesised, and when a
product is available in multiple strengths of the same dosage form.
7 Inclusion of additional guidance on control strategies for products 31st January 2022
containing more than one nitrosamine impurity including examples
(Q&A 10) and a decision tree (Annex I).
8 Update to guidance on root causes and risk factors for nitrosamine 24th March 2022
contamination (Q&A 4) and on policy for confirmatory testing (Q&A
8) and dossier requirements (Q&A 15) to allow testing of
intermediates, raw materials or API under certain circumstances.
9 New Q&A 20 providing clarifications on what are the regulatory steps 20th May 2022
for dealing with scenario A cases and update Q&A10 with new AIs
(N-nitrosomethylphenidate, N-nitrosopiperidine, N-nitrosorasagilene,
7-Nitroso-3-(trifluoromethyl)-5,6,7,8-tetrahydro[1,2,4]triazolo-[4,3-
a]pyrazine, N-nitroso-1,2,3,6-tetrahydropyridine, N-
nitrosonortriptyline, N-methyl-N-nitrosophenethylamine) and
guidance on use of Ames test.
10 Update to Q&A 5 to provide clarifications on the expectation for 23rd June 2022
MAHs to continue to re-visit risk evaluations when new information
becomes available with specific reference to API-nitrosamine risk.
Update to Q&A 10 to include newly adopted AI for N-
nitrosodabigatran and to indicate APIs where related nitrosamines
have been identified. Clarification of how to set limits for products
containing salt, hydrate or solvate forms of the API. Update to Q&A
14 to reference the new risk evaluation template for use in
marketing authorisation applications.
11 Update to Q&A 3 on submission of amended step 1 response and 29th July 2022
extension of Step 3 deadline for chemical medicines.

Questions and answers for marketing authorisation holders/applicants on the CHMP

Opinion for the Article 5(3) of Regulation (EC) No 726/2004 referral on nitrosamine
impurities in human medicinal products
EMA/409815/2020 Page 3/51
 Rev. Summary of changes made Date

12 Update of Q&A 10 to add nitrosoduloxetine and introduction of 10th October 2022

Q&A 21 on approach to control presence of nitrosamine while the AI
is being established.
13 Update of Q&A 10 to add N-nitrosofluoxetine, N-nitrosoparoxetine, 5th December 2022
N-nitrosodiphenylamine, N-nitroso-mefenamic acid, N-
nitrosopyrrolidine and N-nitrosodiethanolamine.
14 Introduction of Q&A 22 on approach to control presence of N- 22nd December 2022
nitrosamine exceeding the AI while CAPAs are being implemented.
Update of Q&A 20 to consider the possibility of an interim limit based
on the LTL approach during CAPA implementation. Update of Q&A 21
for increased clarity on the application of the temporary universal
limit.
15 Amendment of Q&A 22 to indicate that no variation should be 30th March 2023
submitted to implement temporary above AI limits in specifications.
16 Amendment to Q&A 10 to include the Carcinogenic Potency 7th July 2023
Categorization Approach (CPCA) and the enhanced Ames test (EAT)
for establishing AIs for N-nitrosamines. Addition of Appendix 1,
listing the nitrosamines for which AI have been established by the
Non-clinical Working Party (NcWP), including new AIs for N-
nitrosamines determined using the CPCA. Addition of Annex 2,
describing the Carcinogenic Potency Categorization Approach for N-
nitrosamines. Addition of Annex 3, describing the Enhanced Ames
Test Conditions for N-nitrosamines.

Introduction
The assessment report of the CHMP’s Article 5(3) of Regulation (EC) No 726/2004 opinion on
nitrosamine impurities in human medicinal products provides general guidance and recommendations
on mitigating and preventing the presence of nitrosamines in human medicinal products. In this
context all MAHs/Applicants of human medicinal products should work with the manufacturers of their
Active Pharmaceutical Ingredients (APIs) and finished products (FPs) in order to ensure that the
presence of nitrosamine impurities in their medicinal products is mitigated as much as possible and
controlled at or below a limit defined based on ICH M7(R1) principles for substances of the “cohort of
concern” reflected in this guideline and calculated considering a lifetime daily exposure and kept as low
as possible and that appropriate risk mitigating measures are taken.

While the review by CHMP under Article 5(3) was ongoing, the regulatory authorities established in
September 2019 a specific framework (hereinafter ‘call for review’) 1,2 for medicinal products containing
chemically synthesised APIs, to provide details on the reporting to the authorities by the MAHs and set
expectations regarding risk evaluation (step 1), risk assessment/confirmatory testing (step 2) and risk
mitigation measures (step 3) to be carried out. Following the CHMP’s Article 5(3) opinion, a similar
exercise is launched for medicinal products containing a biological API, as further explained in this
document. Further details are provided in Q&A 2 below.

The published CHMP Article 5(3) opinion, supplemented by the current Question and Answer document
on its implementation, will replace the previous letter entitled ‘Information on nitrosamines for
marketing authorisation holders’ (EMA/189634/2019, published on 19 September 2019).

1 https://www.ema.europa.eu/en/documents/referral/nitrosamines-emea-h-a53-1490-information-nitrosamines-marketing-
authorisation-holders_en.pdf
2 https://www.ema.europa.eu/en/documents/referral/nitrosamines-emea-h-a53-1490-questions-answers-information-

nitrosamines-marketing-authorisation_en.pdf

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Opinion for the Article 5(3) of Regulation (EC) No 726/2004 referral on nitrosamine
impurities in human medicinal products
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The terms “nitrosamine” and “N-nitrosamine” are used interchangeably within this Q&A and related
documents and should both be understood to refer to the following structure:

For the purpose of this Q&A please see definitions below:

Risk evaluation: all activities in step 1.

Risk assessment: all activities in step 2.

1. Should the risk of presence of nitrosamines be considered

for all human medicinal products?
MAHs/Applicants of all human medicinal products should ensure that the presence of nitrosamines is
controlled and kept as low as possible, irrespective of marketing status or the type of product (e.g.
generics and over the counter (OTC) products).

For details on the approach required, please refer to Q&A 10 on the limits for nitrosamines and Q&A 12
on the measures to mitigate the risk of presence of nitrosamines.

MAHs/Applicants are reminded of their obligations to ensure that, in accordance with Article 23 and
Annex I of Directive 2001/83/EC and Article 16 of Regulation (EC) No 726/2004, their medicinal
products are manufactured and controlled by means of processes and methods in compliance with the
latest state of scientific and technical progress.

Therefore, MAH/ Applicants shall:

• design their manufacturing processes and controls to prevent if possible or mitigate as much as
possible the presence of N-nitrosamines in their API and FP(s);

• assess the risk of presence nitrosamine impurities in their API(s) and FP(s) and introduce any
resultant changes to the dossier as needed (e.g. changes to their manufacturing processes);

• ensure that active substances and excipients used in their FPs are manufactured in compliance
with good manufacturing practices in line with Article 46(f) of Directive 2001/83/EC.

Compliance of the MAHs/Applicants with the above-mentioned obligations is subject to regular controls
by competent authorities including during GMP inspections.

While the Article 5.3. recommendations on controlling nitrosamine impurities apply to all human
medicinal products, the call for review applies only to human medicines containing chemically
synthesised APIs or biological APIs, as further explained in Q&A 2 below.

2. What is the ‘call for review’?

In September 2019, a ‘call for review’ was launched for medicinal products containing chemically
synthesised APIs to request MAHs to review their manufacturing processes in order to identify and, if
necessary, mitigate the risk of presence of nitrosamine impurities and report the outcome back to
authorities. This exercise was started while the review by CHMP under Article 5(3) for Nitrosamine
impurities in human medicinal products was ongoing.

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Opinion for the Article 5(3) of Regulation (EC) No 726/2004 referral on nitrosamine
impurities in human medicinal products
EMA/409815/2020 Page 5/51
Following the conclusion of the review under Article 5(3), the CHMP considered that there is also a risk
of presence of nitrosamines in biological medicinal products, in particular for the biological medicines
with the following risk factors:

• biologicals containing chemically synthesised fragments, where risk factors similar to chemically
synthesised active substances are present;

• biologicals using processes where nitrosating reagents are deliberately added;

• biologicals packaged in certain primary packaging material, such as blister packs containing
nitrocellulose.

For the above reasons the current call for review has been extended to cover also all
biological medicinal products for human use. For further reference on what is considered to be a
biological medicinal product for the purpose of this exercise, please consult the CMDh Questions &
Answers on Biologicals.

The call for review consists of 3 steps:

• Step 1: MAHs to perform a risk evaluation to identify if APIs and/or FPs could be at risk of presence
of nitrosamine;

• Step 2: if a risk is identified, MAHs to proceed with confirmatory testing in order to confirm or
refute the presence of nitrosamines. MAHs should report outcomes as soon as possible;

• Step 3: if the presence of nitrosamine(s) is confirmed, MAHs should implement effective risk
mitigating measures through submission of variation.

Please refer to Q&A 3 for further details on the ‘call for review’ including the timelines for chemicals
and the timelines for biologicals.

For the specific case of sartans with a tetrazole ring that have been subject to a review under Article
31 of Directive 2001/83/EC, further guidance will be published soon.

3. For the ‘call for review’ for chemically synthesised and

biological medicinal products, when and how should MAHs
report steps 1 and 2 to competent authorities?
Submission of step 1 outcome

Products that have been approved after 26 September 2019 but for which a risk evaluation was not
assessed within the MAA procedure should comply with the call for review deadlines, if not already
done so.

For product containing chemically synthesised APIs, the step 1 risk evaluation should be concluded
and reported at the latest by 31st March 2021.

For product containing biological APIs, step 1 risk evaluation should be concluded and reported at the
latest by 01st July 2021.

The risk assessment has to be performed for all products for which a potential risk has been identified
in step 1, irrespective of the marketing status of the product or whether any registered manufacturers
are actively used in supply. However, it is recognised that step 2 may not be possible for medicines
that are not marketed, including the case of manufacturers not actively used in supply, since there
may be no finished product batches available for confirmatory testing. In these cases, i.e. where no

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impurities in human medicinal products
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batches of finished products are available, it would be acceptable to submit a written commitment that
step 2 confirmatory testing will be conducted once finished product has been manufactured and/or the
product is launched. The outcome of step 2 testing as well as any necessary variation(s) as part of step
3 will therefore need to be submitted and approved before the product can be placed on the market or
the manufacturer can be actively used in supply, even if this is after the step 2 and 3 deadlines.
MAHs’/Applicants’ compliance with the above-mentioned obligations is subject to regular controls by
competent authorities including during inspections.

All MAHs should inform the concerned Competent Authorities of the outcome of their risk evaluation
(step 1) using the dedicated templates.

If a risk has been identified, the expected timeline for the testing activities should also be provided as
foreseen in the dedicated template. No additional documentation is required at this stage. However,
the risk review should be adequately documented, and related documentation should be made
available upon request.

Step 2 should be started as soon as a risk is identified in API and/or FP and in accordance with product
prioritisation (see Q&A 6).

If a risk has been identified for the API, the MAH is advised to report this outcome by using step 1
response template and to proceed directly to step 2 confirmatory testing of the FP. If no risk has been
identified in the API, the MAH is advised to proceed with the risk evaluation of the FP and to present
the result of Step 1 when a final conclusion has been reached on both the API and the FP. MAHs should
inform the concerned Competent Authorities of the outcome of their risk evaluation (step 1) even if no
risk has been identified in the API or FP.

It is acceptable for the submission of the outcome of step 1 to submit one email notification grouping
products with identical outcome under the following provisions:

• For those Member States that have a dedicated portal, the MAH should submit the notification via
this portal;

• If the outcome of step 1 is “risk identified”, it is possible to provide a response by grouping these
products. MAHs are still required to indicate the expected testing timeline on the related “Step 1
risk identified response template” excel file.

In specific cases it may be possible to correct a former step 1 outcome from “risk” to “no risk” by using
the “Step 2 no nitrosamine detected response template”. This template now contains a tick box for
such cases. The possibility to amend the step 1 outcome may only be used in those cases where data
was missing at the March 2020 deadline and is now available.

Submission of step 2 outcome

The step 2 confirmatory testing should be conducted in accordance with product prioritisation (see
Q&A 6).

For product containing chemically synthesised APIs, confirmatory testing activities at Step 2 are
expected to be finalised at the latest by 26th September 2022. MAHs should refrain from submitting
incomplete step 2 outcomes.

The deadline for the submission of any changes required to Marketing Authorisations (Step 3, see
Q&A 13) is by 1st October 2023.

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Opinion for the Article 5(3) of Regulation (EC) No 726/2004 referral on nitrosamine
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For product containing biological APIs, confirmatory testing activities at Step 2 and submission of any
changes required to Marketing Authorisations (Step 3, see Q&A 13), are expected to be finalised at the
latest by 1st July 2023.

In order to meet the above deadlines for submission of any changes required to Marketing
Authorisations at Step 3 for products containing chemically synthesised or biological APIs, it would be
expected that confirmatory testing activities at Step 2 are finalised in advance of these deadlines.

MAHs should forthwith inform the competent authorities if tests confirm the presence of nitrosamine,
irrespective of the amount detected and by utilising the dedicated reporting templates. The immediate
risk to patients should be assessed based on the limits defined in Q&A 10 and appropriate action
proposed to avoid or minimise the exposure of patients to nitrosamines.

For the submission of the outcome of step 2 confirmatory testing several products can be combined
when the outcome is “no nitrosamines detected”. When the outcome is “nitrosamines detected” all
strengths and pharmaceutical forms of one marketing authorisation can be combined in one response
template when the supporting documentation is completely identical for all products concerned; if not
the response has to be submitted separately.

In case one or more nitrosamines are identified that exceed the limit defined in Q&A 10, the following
supportive documentation is required at the time of reporting:

• testing results expressed in ng and ppm;

• interim investigation report including (preliminary) root cause, risk mitigating plan and benefit/risk
assessment.

For their responses, MAHs are required to use dedicated templates and contact points as outlined on
the EMA and CMDh websites.

4. What are the currently identified risk factors for presence

of nitrosamines?
N-Nitrosamines can be formed when an amine and nitrosating agent are combined under favourable
conditions although other generation pathways are also possible, such as e.g. oxidation and reduction
processes from hydrazine-type compounds and N-nitro derivatives. 3,4 Root causes for N-nitrosamines
in medicinal products identified to date can be grouped as risk factors linked exclusively with the
manufacturing process and storage of active substance and/or as risk factors associated with
manufacture and storage of the finished product. Moreover, there are risk factors specifically linked to
GMP aspects. Currently identified risk factors for N-nitrosamine impurities in medicinal products are
listed below, along with some identified in the literature. However, the list is not exhaustive and
further root causes may also be applicable – it is up to MAHs to determine if there is a risk with their
product:

Risk factors related to the manufacture of the active substance:

1. Use of nitrite salts and esters (e.g. NaNO2, alkyl nitrites), or other nitrosating agents (e.g.
nitroso halides, nitrosonium salts, nitrogen oxides, nitro alkanes, halogenated nitro alkanes,
Fremy’s salt, nitroso sulfonamides),3,4 in the presence of secondary or tertiary amines within
the same or different steps of the manufacturing process. Sources for secondary or tertiary
amines can also be starting materials, intermediates, reagents, solvents (e.g. DMF, DMAc and

3 Lessons learnt from presence of N-nitrosamine impurities in sartan medicines EMA/526934/2019.

4 Org. Process Res. Dev. 2020, 24 (9), 1558–1585

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 NMP) and catalysts, which contain amine functionality, amine impurities (e.g. quaternary
ammonium salts) or which are susceptible to degradation to reveal amines.

2. Nitrite formation by oxidation of hydroxylamine or nitrite release from nitro-aromatic precursors

(e.g. by fluoro de-nitration), in the presence of secondary or tertiary amines within the same or
different steps of the manufacturing process (see 1). 5

3. Use of disinfected water (chlorination, chloro-amination, ozonisation) in the presence secondary

or tertiary amines within the same or different steps of the manufacturing process (see 1). 6,7,8,9

4. Oxidation of hydrazines, hydrazides and hydrazones by hypochlorite, air, oxygen, ozone and
peroxides in the manufacturing process or during storage.4 Use of contaminated raw materials
in the API manufacturing process (e.g. solvents, reagents and catalysts).

5. Use of contaminated recovered or recycled materials (e.g. solvents, reagents and catalysts).

6. Use of contaminated starting materials and intermediates supplied by vendors who use
processes or raw materials which may contain residual nitrosamines or nitrosating agents.

7. Carry-over of nitrosamines deliberately generated (e.g. as starting materials or intermediates)

during the manufacturing process.

Risk factors also related to the finished product:

8. Reaction of nitrosatable nitrogen functionality in APIs or their impurities/degradants with

nitrosating agents present in components of the FP during formulation or storage. A particular
risk of formation of nitrosamines should be noted for active substances that contain a
nitrosatable amine functional group. Several examples have been reported where the amine
functionality was shown to be vulnerable to nitrosation and formation of the corresponding N-
nitroso impurity (i.e. NO-API). Secondary amines appear particularly vulnerable to this reaction
although some cases with tertiary amines have also been observed. Vulnerable amines could
also be formed by degradation (e.g. hydrolysis) during formulation or storage. Nitrites have
been identified as impurities in many common excipients. 10 MAHs and/or applicants should be
aware that N-nitroso API impurities can form at levels exceeding the AI even if nitrite levels in
the excipients are very low. The overall nitrite content will also depend on the relative
composition in terms of the excipients. As it has been reported that N-nitroso impurities can
form from APIs or their impurities/degradants (containing amine functionality or susceptible to
degradation to reveal amines) during manufacture of the finished product, as well as during
storage, MAHs should give consideration to the stability of the finished product and should
ensure that the AI of any N-nitrosamine impurity is not exceeded until the end of shelf life of
the FP. For further information, please refer to the assessment report of the CHMP’s Article 5(3)
opinion on nitrosamine impurities in human medicinal products.

9. Degradation processes of active substances, including those induced by inherent reactivity (e.g.
presence of nitro-alkyl, oxime, or other functionality311,4) or by the presence of an exogenous
nitrosating agent. This could potentially occur during both active substance and finished product
manufacturing processes or during storage and could be influenced by crystal structure, crystal

5
Chem. Rev. 2016, 116, 422−518
6 Crit. Rev. in Environ. Sci. 2017, 47, (24), 2448-2489
7 J. Pharm. Biomed., 2019, 164, 536-549
8 Water Research, 2011, 45 (2), 944-952
9 J. Org. Chem. 2021, 86, 2037−2057
10 AAPS Pharm. Sci. Tech. 2011, 12 (4), 1248- 1263
11 Org. Process Res. Dev. 2020, 24 (12), 2915–2926

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 habit and storage conditions (temperature, humidity etc.). For more details, refer to page 6 of
Referral under Article 31 of Directive 2001/83/EC for ranitidine and published literature.11,12

10. Oxidation of hydrazine or other amine-containing functional groups present in active substances
or their impurities/degradants (e.g. from hydrazones and hydrazides), either in active
substance manufacturing processes or during storage.4 This root cause has also been observed
during manufacture and storage of finished products containing such functional groups.
Potential oxidants include oxygen and peroxides (common impurities in some excipients).10

11. Use of certain packaging materials. Relevant nitrosamine contamination has been observed in
primary packaging of finished products in blister with lidding foil containing nitrocellulose.
During the blister heat-sealing process, nitrogen oxides can be generated thermally from
nitrocellulose. Under these conditions, nitrosamines have been shown to form from low
molecular weight amines present either in printing ink or in the finished product and to transfer
to the product and/or to the cavity via evaporation and condensation.

12. Reaction of amines leaching from quaternary ammonium anion exchange resins (e.g. used for
purification steps) with nitrosating agents present in the liquid phase. A recent example of this
was in the production of water for injections where residual chloramine used to disinfect
incoming water reacted with dimethylamine leaching from the anion exchange resin used in the
demineralisation step to form NDMA. In addition, disinfection procedures such as e.g.
chlorination, chloro-amination and ozonisation can lead to significant N-nitrosamine generation
as by-products in case vulnerable amines are present.6,7,8,9 Given the source of contamination,
risk is related to the concentration of the reactive agent(s) and thus to the volume of water in
or used to dilute a particular product. The same risks could be associated with active substances
or finished products manufactured using water purified using similar resins.

Risk factors related to GMP aspects:

13. Cross-contamination due to different processes being run successively on the same
manufacturing line.

14. Carry-over of impurities between process steps due to operator-related errors or insufficiently
detailed batch records such as inadequate phase separations during work-up procedures.

15. Use of contaminated recovered or recycled materials (e.g. solvents, reagents and catalysts)
where the recovery is outsourced to third parties who are not aware of the content of the
materials they are processing. Recovery processes carried out in non-dedicated equipment
should also be considered.

5. What to do if after submission of step 1 and /or step 2

responses, new information (e.g. related to new potential
risk factors or root causes) is identified?
MAHs together with API and FP manufacturers are expected to maintain the quality of their product
throughout its lifecycle. Therefore, once step 1 and/or 2 responses are submitted, MAHs are expected
to continue to review and re-visit the outcome of the risk evaluation as and when new information
becomes available. MAHs are advised to routinely check this Q&A document and in particular Q&A 4
which will be kept up to date as regards newly identified risk factors for formation of nitrosamines, and
also Q&A 10 concerning limits for nitrosamines.

12 Chem. Pharm. Bull., 2021, 69, 872–876

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In particular, MAHs should note the risk of formation of nitrosamine impurities from active substances
(or their related impurities) containing a vulnerable amine during finished product formulation and/or
storage due to the presence of traces of nitrites. This has been recently elaborated as a risk factor to
Q&A 4 (bullet 8) based on understanding gained during the call for review. MAHs that did not take into
account this risk as part of step 1 response for their products containing active substances with
vulnerable amines should reconsider their original step 1 risk evaluations in light of this new
information and proceed to step 2 confirmatory testing as appropriate (see also see also Q&A 10, table
1, column 3).

Appropriate timelines for reviewing the previous risk evaluation and for conducting confirmatory
testing (if needed), should be followed depending on the risk identified.

The same approach should be followed for medicinal products granted a positive opinion and marketing
authorisation during the call for review.

6. What factors should be considered in prioritising the risk

evaluation?
When conducting the risk evaluation and risk assessment, MAHs should use a risk-based approach to
prioritise products for evaluations and confirmatory testing. MAHs may consider factors such as the
maximum daily dose taken for the concerned medicinal product, duration of treatment, therapeutic
indication and number of patients treated. For example, medicinal products with higher daily dose and
those for chronic use may take priority.

In order to undertake the analysis of the identified medicinal products at risk, MAHs can also use tools
such as Failure Mode Effects Analysis (FMEA) and Failure Mode, Effects and Criticality Analysis (FMECA)
as outlined in the ICH Q9 guideline on quality risk management.

7. How should the risk evaluation be performed?

MAHs/Applicants in collaboration with API, FP manufacturers and their raw material suppliers are
required to perform risk evaluations using quality risk management principles, as outlined in ICH Q9
guideline. The principles described in ICH M7 guideline and in the Assessment report of the CHMP’s
Article 5(3) opinion on nitrosamine impurities in human medicinal products in relation to the toxicology
assessment, control strategy and changes to the manufacturing processes for active substances should
also apply.

Manufacturers of active substances and FP and their raw material suppliers should provide
MAHs/applicants with all information necessary for a comprehensive risk evaluation. If the risk of
nitrosamine impurity formation was assessed during the development phase of the API/FP
manufacturing processes, the information from this assessment can be used to support the risk
evaluation.

MAHs/Applicants and manufacturers should consider as part of the risk evaluation all potential sources
of contamination or formation of nitrosamine, notably the root causes listed under Q&A 4.

As MAHs/Applicants and manufacturers for products containing biological APIs should consider the
following aspects that may increase the risks of nitrosamine presence in their products:

• biologicals containing chemically synthesised fragments, where risk factors similar to chemically
synthesised active substances are present;

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• biologicals using processes where nitrosating reagents are deliberately added;

• biologicals packaged in certain primary packaging material, such as blister packs containing
nitrocellulose.

For further information on root causes, please refer also to the assessment report of the CHMP’s Article
5(3) opinion on nitrosamine impurities in human medicinal products.

If, after completion of the risk evaluation, a risk is identified in the API and/or the FP, MAHs/applicants
must notify the competent authorities of the identified risk, proceed without further delay with
confirmatory tests (see Q&A 8) and introduce any necessary changes to the dossier.

All MAHs should inform the concerned Competent Authorities of the outcome of their risk evaluation
(step 1) even if a risk has not been identified, please see Q&A 3 for further details.

8. How should confirmatory tests be conducted by MAHs and

manufacturers?
For the purpose of confirmatory testing as part of step 2 of the call for review to MAHs, testing should
generally be carried out on the FP. Testing of the API, its intermediates, starting materials, solvents,
reagents, excipients or any other raw materials for nitrosamines, amines, nitrites or other compounds
with potential to generate nitrosamines is also recommended, if the risk assessment indicates that
they are a potential source of nitrosamine impurities in the FP. In such cases, the results of testing
API, intermediates or other relevant materials may be used to support root cause investigations and
the development of a justified control strategy for nitrosamine impurities.

However, some root causes may only be linked to the API manufacturing process (see Q&A 4). In
these cases, testing of the API or intermediates upstream of the active substance could be used as a
surrogate for testing the finished product, provided that the risk assessment performed on the FP
concluded no additional risk factors for formation of nitrosamine impurities in the finished product (see
Q&A 4, risk factors related to the finished product). If testing is carried out on an intermediate, then
there should also be no risk factors associated with subsequent steps in the API manufacturing process
or the finished product. The confirmatory testing strategy is the responsibility of the MAH and should
be justified based on the risk assessment for the finished product and documented in the MAH’s
pharmaceutical quality system. It should be clearly justified why testing of the active substance or
intermediate is appropriate and why further risk of nitrosamine formation in the finished product or
subsequent API manufacturing steps can be excluded. If nitrosamines are detected, then an
appropriate control strategy should be implemented in the dossier.

In any case, if the control point of nitrosamines is not in the finished product, the responsibility for
quality lies with the MAH.

The number of batches to be tested should be commensurate with the risk. MAHs and manufacturers
should test a representative number of batches of FP and the relevant starting materials,
intermediates, API or raw materials as applicable. If the source of risk has been identified and is well
understood (e.g. by spike and purge studies) such that impurity levels are expected to be consistent
from batch to batch, testing should be conducted on 10% of annual batches, or 3 per year, whichever
is highest. This includes testing not only of newly produced batches but also retained samples of
batches still within expiry date. If fewer than 3 batches are manufactured annually, then all batches
should be tested.

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If multiple manufacturers, manufacturing processes and/or sources of at-risk raw materials are used,
(or were used historically for batches still within expiry date), then testing of additional batches would
be necessary to cover these risk factors.

If a product is available in multiple strengths of the same dosage form with the same risk factors
applicable to each, then testing could be rationalised 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.

During development of an analytical method, a reference standard of the relevant nitrosamine impurity
is generally needed. If, despite extensive efforts, it becomes apparent that the relevant nitrosamine
impurity cannot be synthesised, then this could be an indication that the nitrosamine either does not
exist 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. The justification could include relevant literature, information on
structural/stereo-electronic features and reactivity of the parent amine, stability of the nitrosamine and
experimental data to illustrate the efforts made to synthesise and to analyse the impurity. The
justification should be documented in the risk assessment in the MAH's pharmaceutical quality system.

Methods for determination of various nitrosamines in sartans with a tetrazole ring, metformin and
ranitidine have already been developed by the Official Medicines Control Laboratories and are available
for reference on the European Directorate for the Quality of Medicines & HealthCare (EDQM) website.
These may serve as a starting point for the development and validation of analytical methods for
testing other APIs/FPs.

Appropriately sensitive analytical methods for determination of specific nitrosamines in other medicinal
products should be developed and validated accordingly before testing. The limit of quantification
(LoQ) should be at or below the acceptable limit for the respective nitrosamine impurity. If the same
analytical method is used to test for multiple nitrosamines, then the selectivity of the method should
be demonstrated at the LoQ for each nitrosamine.

Given the trace levels of nitrosamines to be measured, the following technical aspects should be
considered when developing analytical methods:

• Interference caused by presence of trace amounts of nitrosamines in testing materials utilised (e.g.
water, airborne sources, plastics products and rubber/elastomeric products);

• Contamination during sample preparation (avoiding cross contaminations from gloves, membranes,
solvents etc.) which could lead to false positive results;

• In situ formation of nitrosamines during analysis;

• Use of accurate mass techniques are required (MS/MS or high-resolution accurate mass systems)
in order to overcome interference in the identification of the specific peak of a certain nitrosamine
(e.g. false positives have been observed from DMF co-eluting with NDMA).

As a result of the above considerations, control experiments should be conducted such as analysing
samples by orthogonal analytical methods.

Further details in relation to analytical methodology can be found on EDQM website and in the CHMP
assessment report of the CHMP’s Article 5(3) opinion on nitrosamine impurities in human medicinal
products.

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9. What are the requirements of the analytical method(s)?
The analytical methods need to be sufficiently sensitive in order to adequately detect and quantify
trace levels of nitrosamine impurities. The following principles apply:

• The limit of quantification (LoQ) provides the minimum level at which an analyte can be quantified
with acceptable accuracy and precision and should thus be used for impurity testing and decision-
making;

• If quantitative testing is performed as a routine control, the LoQ should be ≤ of the acceptable
limit based on the relevant acceptable intake (AI) for the respective nitrosamine impurity;

• If quantitative testing is performed to justify skip testing, the LoQ of the analytical procedure
employed should be ≤ 30% of the acceptable limit based on the AI;

• If quantitative testing is performed to justify omission of specification, the LoQ of the analytical
method employed should be ≤ 10% of the acceptable limit based on the AI;

• Exceptions are anticipated for medicinal products used at high daily doses (AI may be below
technical feasibility of the method), or in case more than one nitrosamine is anticipated or
identified in a given medicinal product.

Different analytical methods may be used for determination of multiple nitrosamines. If the same
analytical method is used for multiple nitrosamines, the selectivity of the method should be
demonstrated for each nitrosamine.

10. Which limits apply for nitrosamines in medicinal

products? (Updated)
ICH M7(R1) guideline defines N-nitrosamines as substances of the “cohort of concern” for which limits
in medicinal products refer to the so-called substance-specific acceptable intake (AI) (the Threshold of
Toxicological Concern, TTC, value of 1.5 ug/day cannot be routinely applied) which is associated with a
negligible risk (theoretical excess cancer risk of <1 in 100,000 over a lifetime of exposure). The
calculation of AI assumes a lifelong daily administration of the maximum daily dose of the medicinal
product and is based on the approach outlined in the ICH M7(R1) guideline as well as the principles
described in relation to the toxicological evaluation in the assessment report of the CHMP’s Article 5(3)
opinion on nitrosamine impurities in human medicinal products.

The ‘less than lifetime’ (LTL) approach should not be applied in calculating the limits as described
above but can only be considered after consultation with competent authorities as a temporary
measure until further measures can be implemented to reduce the contaminant at or below the limits
defined above.

For products intended for advanced cancer only as defined in the scope of the ICH S9 guideline, N-
nitrosamine impurities should be controlled according to ICH Q3A(R2) and ICH Q3B(R2) guidelines, as
specified in the Q&A document to ICH S9 guideline. If the active substance itself is mutagenic or
clastogenic at therapeutic concentrations, N-nitrosamine impurities should be controlled at limits for
non-mutagenic impurities according to ICH M7(R1).

The same risk approach is applicable to all routes of administration. Corrections to limits are generally
not acceptable unless route-specific differences are justified by data.

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Establishment of the AIs

Two scenarios are foreseen for detection of new nitrosamines:

A. If N-nitrosamines are identified with sufficient substance specific animal carcinogenicity data, the
TD50 should be calculated and used to derive a substance specific limit for lifetime exposure as
recommended in ICH M7(R1) guideline.

B. If N-nitrosamines are identified without sufficient substance specific data to derive a substance
specific limit for lifetime exposure as recommended in ICH M7(R1) guideline,

1. The Carcinogenic Potency Categorization Approach (CPCA) for N-nitrosamines (Annex 2) should
be used to establish the AI, unless other robust data are available that would override this AI.

2. A negative result in an GLP-compliant enhanced Ames test (EAT, Annex 3) allows control of the
N-nitrosamine at 1.5 µg/day. For substances testing positive, the AI should be established
using options 1 or 3.

3. If a surrogate nitrosamine is available with sufficiently robust carcinogenicity data, the TD50
from the surrogate substance can serve as a point of departure for derivation of AI by SAR and
read across.

4. A negative result in a relevant well-conducted in vivo mutagenicity study can allow control of
the N-nitrosamine as a non-mutagenic impurity, i.e. according to Q3A/B limits, irrespective of
the limit calculated through option 1, 2 or 3. For substances testing positive, the AI should be
established using options 1 or 3.

The risk approach is applicable to all routes of administration. Corrections to limits are generally not
acceptable unless data justify route-specific differences.

Appendix 1 lists the nitrosamines for which acceptable intakes have been established by the Non-
clinical Working Party.

Calculation of the limit when a single known nitrosamine is identified

The conversion to a specification limit in ppm for a particular medicinal product is calculated by
dividing the respective above limit (ng) by the maximum daily dose (mg) of a given product as
reflected in the SmPC.

The maximum daily dose is defined in line with the definition of the product strength in the Guideline
on the SmPC. Therefore, the limit in ppm should usually be expressed per active moiety (free base,
free acid or anhydrous/non-solvated material) for control point in the FP. Exceptions to this are active
substances in medicinal products where the strength has traditionally been expressed in the form of a
salt or hydrate and active substances present in the formulation as ester or pro-drug.

For a control point in the API only, the limit should be expressed in general per drug substance (i.e.
relating to form of salt, hydrate, solvate etc. where relevant).

Calculation of limit when more than one nitrosamine is identified in the same product

Please also refer to the decision tree in Annex 1 for further guidance.

For determining limits in the case of presence of more than one nitrosamine, two approaches are
considered acceptable in order not to exceed the acceptable risk level of 1:100,000 as outlined in ICH
M7(R1) guideline:

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1. The total daily intake of all identified N-nitrosamines not to exceed the AI of the most potent N-
nitrosamine identified, or

2. Total risk level calculated for all identified N-nitrosamines not to exceed 1 in 100,000.

The approach chosen needs to be duly justified by the MAH/Applicant.

Specifications for individual N-nitrosamines should generally include an AI limit expressed in ppm or
ppb. The conversion to an AI limit in ppm/ppb for a particular medicinal product is calculated by
dividing the respective above AI (in ng/d) by the maximum daily dose (in mg) of a given product as
reflected in the SmPC. The calculation of the specification limit does not take into account the
molecular weight of the N-nitrosamine.

It is considered that the presence of one or more N-nitrosamines at <10% of their respective AI
constitutes a negligible toxicological risk, and as such, they do not need to be specified. N-
Nitrosamines present below 10% of their respective AI do not need to be factored into the calculation
of limits for individual or total N-nitrosamine(s).

However, the overall principle of the Article 5(3) referral should still be considered, notably that “the
presence of N-nitrosamines in human medicinal products shall be mitigated as much as possible.”
Therefore, manufacturers are encouraged to improve their processes, even if they result in only very
small amounts (<10% AI) of multiple nitrosamines, as processes and controls should be designed to
prevent if possible or mitigate as much as possible the presence of N-nitrosamines in APIs and FPs
(see Q&A 1).

For option 1, the AI limit for total N-nitrosamines should be set in ppm/ppb according to the most
potent N-nitrosamine present at ≥ 10% of its AI. The most potent nitrosamine is the one with the
lowest AI (see table 1). Limits for individual N-nitrosamines can be defined but are not necessarily
needed. However, it should be clearly stated which N-nitrosamines are included in the calculation of
total N-nitrosamines.

For option 2, the limits for N-nitrosamines should ensure an overall risk of not more than 1 in
100,000. Different approaches can be employed to achieve this risk requirement:

Fixed approach: fixed AI limits (in ppm/ppb) are set for individual nitrosamines and no limit for total
N-nitrosamines is needed. The limit for each N-nitrosamine should be set at a percentage of its AI limit
such that the sum of the % AI limits for each specified nitrosamine does not exceed 100%.

Flexible approach: each N-nitrosamine should be specified at its AI limit in ppm/ppb and an
additional limit for total N-nitrosamines is required. The calculation for total N-nitrosamines could be
written as:
𝑛𝑛
𝑋𝑋𝑋𝑋
� 𝑥𝑥 100% ≤ 100%
𝐴𝐴𝐴𝐴𝐴𝐴
𝑖𝑖=2

Where Xi is the amount of each single N-nitrosamine i in ppm and AIi is the AI limit of each N-
nitrosamine i in ppm.

For each batch, to determine whether the limit for total N-nitrosamines is met, the amount of each N-
nitrosamine present (in ppm/ppb) should be converted to a percentage of its respective AI limit. The
sum of % AI limits of specified N-nitrosamines should not exceed 100%.
Example of control options and specifications for multiple nitrosamines in the same finished
product:

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The case of two NAs:

Two NAs both at or above 10% of their respective AI

Example:
NDMA and NDEA are both detected at or above 10% of their respective AI) in a finished product with
maximum daily dose of 300 mg.

AI limit

• NDEA: 26.5 ng/day / 300 mg/day = 0.088 ppm or 88 ppb = most potent N-nitrosamine
• NDMA: 96.0 ng/day / 300 mg/day = 0.32 ppm or 320 ppb
Specification possibilities for different control options:

Nitrosamine Option 1 Option 2 - Fixed Option 2 - Flexible

Example 20:80 ratio2
NDMA Not needed NMT 64 ppb NMT 320 ppb

(320 ppb x 0.2)

NDEA Not needed NMT 70 ppb NMT 88 ppb

(88 ppb x 0.8)

Total Nitrosamines NMT 88 ppb Not needed NMT 100%1

[𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁] 𝑝𝑝𝑝𝑝𝑝𝑝 [𝑁𝑁𝑁𝑁𝑁𝑁𝑁𝑁] 𝑝𝑝𝑝𝑝𝑝𝑝

1 ( + ) 𝑥𝑥 100% ≤ 100%
320 𝑝𝑝𝑝𝑝𝑝𝑝 88 𝑝𝑝𝑝𝑝𝑝𝑝
NMT 100% = 1:100,000 theoretical excess cancer risk.
2 For option 2 fixed approach, a ratio of 20% NDMA to 80% NDEA (20:80) is used as an example only. Different

ratios could be used in different situations dependent on relative amounts present, provided that the sum of the %
AI limits for each specified nitrosamine does not exceed 100%.

Example of presentation of acceptable batch results for each control option:

Model data from 1 batch:

• NDMA found at 38 ppb

• NDEA found at 44 ppb

Option 1 Option 2 - Fixed Option 2 - Flexible

Example 20-80 ratio
Limit Results Limit Results Limit Results
NDMA Not needed - NMT 64 ppb 38 ppb NMT 320 ppb 38 ppb

(12% of AI)
NDEA Not needed - NMT 70 ppb 44 ppb NMT 88 ppb 44 ppb

(50% of AI)
Total NA NMT 88 ppb 82 ppb Not needed - NMT 100% 62%

Control options for Genotoxic APIs

Genotoxicity encompasses mutagenicity, clastogenicity and aneugenicity.

Mutagenic APIs are defined as substances having DNA-reactive properties as described in ICH M7.

Clastogenic APIs are substances causing structural chromosomal aberrations.

Aneugenic APIs are substances causing numerical chromosomal changes.

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The ICH M7(R1) guideline does not apply to drug substances and drug products intended for advanced
cancer indications as defined in the scope of ICH S9 (Ref. 4). Additionally, there may be some cases
where a drug substance intended for other indications is itself genotoxic at therapeutic concentrations
and may be expected to be associated with an increased cancer risk. Exposure to a mutagenic impurity
in these cases would not significantly add to the cancer risk of the drug substance. Therefore,
impurities could be controlled at acceptable levels for non-mutagenic impurities. Below it is explained
in more detail how this is applied to the control of nitrosamine impurities.

1. Policy for products not within the scope of ICH S9

a. Containing mutagenic or clastogenic APIs:

i. Control nitrosamine at or below ICH Q3A/B qualification threshold1 when genotoxicity of API is
considered to produce a significant risk for mutagenicity/clastogenicity at therapeutic exposures;

ii. The rules established for the control of nitrosamines as explained in the Article 5(3)
referral or elsewhere in the Q&A apply when mutagenicity/clastogenicity of API is considered not
to produce a significant risk for mutagenicity/clastogenicity at therapeutic exposures.

b. Containing aneugenic APIs:

i. The rules established for the control of nitrosamines as explained in the Article 5(3) referral or
elsewhere in the Q&A apply since aneugenicity of API is considered not to produce a significant
risk for carcinogenicity at therapeutic exposures

c. Containing non-genotoxic APIs

i. The rules established for the control of nitrosamines as explained in the Article 5(3) referral or
elsewhere in the Q&A apply

2. Policy for products within the scope of ICH S9

a. Containing genotoxic or non-genotoxic APIs:

i. Control nitrosamine at or below ICH Q3A/B qualification threshold.

Higher limits may be set for nitrosamines in certain cases. However, it is expected that the
Applicant/MAH will ensure that the presence of nitrosamine impurities in their medicinal products is
mitigated as much as possible.
1Wherever it is quoted ”Control nitrosamine at or below ICH Q3A/B qualification threshold”, this
implies that control at the qualification threshold is justified from a safety perspective.

11. What should I do if a nitrosamine is detected in my

medicinal product?
If one or several nitrosamine(s) is detected for the first time in my medicinal product:

The MAH/Applicant should forthwith inform the competent authorities, irrespective of the amount
detected as described in Q&A 3 for medicinal products subject to the call for review.

The levels should be reported in ng and ppm, together with the corresponding calculations used to
describe the potential exposure to the detected nitrosamine based on the maximum daily dosage
recommended in the SmPC. If SmPCs differ between Member States, the calculations should be
provided for each different maximum exposure. Sufficient details should be provided to enable the
calculations to be reviewed and verified.

The calculated exposure(s) should then be compared to the limit defined in Q&A 10:

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• If the limit is not exceeded for the detected nitrosamine or, in case of presence of multiple
nitrosamines, if the total risk remains below a theoretical lifetime excess risk of <1:100,000, the
MAH/Applicant shall control the nitrosamine(s) in the FP at or below this limit (see Q&A 10) and
should take measures to mitigate the risk of nitrosamine formation or contamination in the
medicinal product as much as possible (see Q&A 12).

• Where the limit defined in Q&A 10 for single or multiple nitrosamines is exceeded, the
MAH/Applicant should submit forthwith an (interim) investigation report including (preliminary)
root cause, risk mitigating plan and benefit/risk assessment. The competent authorities will then
assess the impact on the benefit/risk balance and the consequent need for any action to be taken.

Please refer to the Assessment report of the CHMP’s Article 5(3) opinion on nitrosamine impurities in
human medicinal products for further information.

Changes to the marketing authorisation related to measures to prevent or minimise the risk should be
introduced without delay and in accordance with the guideline on classification of variation (please
refer to Q&A 13).

If the presence of specific nitrosamine(s) in a medicinal product has already been reported
to the authorities by the MAH and is below the limit defined in Q&A 10 or a limit approved by
the authorities, there is no need for a further notification to the authorities.

Batch records are subject to inspection by competent authorities.

12. Which are the measures to mitigate the risk of presence

of nitrosamines?
The presence of N-nitrosamines in the FP shall be mitigated as much as possible and shall be at or
below a limit defined in Q&A 10.

MAHs shall design or adapt the manufacturing process of their medicinal products to prevent formation
of and contamination with nitrosamines whenever possible.

MAHs should implement a control strategy regarding N-nitrosamines, which should include current and
prospective measures to minimise the risk of generation of/contamination with nitrosamines (e.g.
change of manufacturing process, change of raw material quality, introduction of appropriate
specifications and development of appropriate methods, and measures on the premise and equipment
such as cleaning procedures and environmental monitoring). MAHs should control nitrosamine levels in
accordance with the limits defined in Q&A 10 and any future changes that may impact on the risk (e.g.
change of supplier, change of manufacturing process and change of packaging).

MAHs shall also ensure that active substances and excipients used in their FPs are manufactured in
compliance with good manufacturing practices in line with Article 46(f) of Directive 2001/83/EC.

Please refer to the Assessment report of the CHMP’s Article 5(3) opinion on nitrosamine impurities in
human medicinal products for further information.

13. Which changes would be required for Marketing

Authorisations?
MAHs should introduce changes to their API and/or FP (e.g. manufacturing process, controls and
specification, product formulation, raw materials and packaging), through the timely submission of
appropriate variation(s) in accordance with the guideline on classification of variations.

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When nitrosamine(s) is (are) identified, the corresponding limit(s) as defined in Q&A 10 should be
introduced in the specifications of the FP. Please refer to Q&A 15 for information on the test modalities.

The application for a variation should contain information on amendments to the marketing
authorisation – i.e. in module 3 (3.2.S and 3.2.P), the active substance master files (ASMF) or the
Certificate of Suitability to the monographs of the European Pharmacopoeia (CEP) that is necessary to
control nitrosamine impurities in the active substance and/or FP. Variations should be submitted
according to the existing variations classification guideline: EUR-Lex - 52013XC0802(04) - EN - EUR-
Lex (europa.eu)

Depending on the root cause identified and extent of changes to be made, grouping of variations or
use of work-sharing procedures might be applicable: https://www.ema.europa.eu/en/human-
regulatory/post-authorisation/variations/worksharing-questions-answers.

14. What is the approach for new and ongoing marketing

authorisation applications (MAA)?
Applicants shall design their manufacturing processes and controls to prevent if possible or mitigate as
much as possible the presence of N-nitrosamines in their API and FPs (please refer to Q&A 12).

The potential presence of nitrosamines must be evaluated as part of the MAA as follows:

• At the submission stage:

− For the risk evaluation, Applicants are required to follow the principles for step 1 as per Q&A 2.
The risk evaluation should be submitted as an attachment to Module 1 with a corresponding
reference in Module 3.2 of the marketing authorisation dossier. To supplement the detailed risk
evaluation, the template located on the CMDh nitrosamine website (section “For additional
specific information related to nationally authorised products (including MRP/DCP)”) could also
be submitted: https://www.hma.eu/human-medicines/cmdh/advice-from-cmdh/nitrosamine-
impurities.html . The template is optional for CAPs. For NAPs, and DCPs, the template is
mandatory and the CMDh practical guidance located in the same section of the same website
should be followed.

− If a risk of presence of nitrosamines in the medicinal product is identified, applicants are

required to provide the risk assessment outlining the impact on the benefit-risk balance of the
product and a risk mitigation strategy. Applicants should also submit confirmatory testing plans
or confirmatory testing data as mentioned in step 2 (see Q&A 2).

− In case applicants have not submitted a risk evaluation and, if applicable, confirmatory testing
plans with their MAA, these should be submitted during the marketing authorisation review
procedure.

• During the Marketing Authorisation (MA) evaluation procedure:

− If the risk evaluation was not submitted as part of the MAA, it will be requested during the MA
review process. Risk evaluation will have to be adequately documented and, if applicable,
supported by confirmatory testing in case a possible risk of presence of nitrosamines has been
identified. This information should be submitted as part of the responses to the list of
questions.

− If the applicant is not able to provide satisfactory information and justification of a favourable
benefit-risk profile of the product at this stage, a request to further assess the risk of presence

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 of nitrosamine will be part of the further list of questions / outstanding issues depending on the
stage of the MA procedure.

− Any outstanding issues related to the quality requirements of the product would have to be
addressed before the final opinion on the granting of the MA.

For new and on-going marketing authorisation applications, the number of batches to be tested as part
of any confirmatory testing should be commensurate with the risk in line with ICH M7(R1) guideline.
The source of risk has to be well understood (e.g. by spike and purge studies) such that impurity levels
are expected to be consistent from batch to batch. Test results from a minimum of 6 pilot scale
batches or 3 production scale batches may be sufficient. Depending on the risk factors for nitrosamine
presence, e.g. with risk factors being closer to the FP, more batches may need to be tested. If multiple
manufacturers, manufacturing processes and/or sources of at-risk raw materials are used, (or were
used historically during development), then testing of additional batches would be necessary to cover
these risk factors.

If a product is available in multiple strengths of the same dosage form with the same risk factors
applicable to each, then testing could be rationalised by testing only the worst-case scenario strength.
The worst-case approach should be justified by the MAH on a case-by-case basis.

During development of an analytical method, a reference standard of the relevant nitrosamine impurity
is generally needed. If, despite extensive efforts, it becomes apparent that the relevant nitrosamine
impurity cannot be synthesised, then this could be an indication that the nitrosamine either does not
exist 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. The justification could include relevant literature, information on
structural/stereo-electronic features and reactivity of the parent amine, stability of the nitrosamine and
experimental data to illustrate the efforts made to synthesise and to analyse the impurity. The
justification should be included in the submitted risk assessment.

15. When should a test for nitrosamines be included in the

MA dossier?
When a nitrosamine is identified after Step 2 confirmatory testing, a limit will usually need to be
included in the specifications of the finished product and the product must comply if tested. If the root
cause has been identified in the finished product manufacturing process or storage, or nitrosamines
have been detected in the finished product, but the actual source of contamination remains unclear,
routine testing of the finished product is required by default.

The control point (finished product, API or an intermediate) for nitrosamines should be selected in such
a way that it will give assurance of presence of the impurity below the acceptable limit based on
acceptable intake (AI) in the finished product. Testing is usually expected to be carried out in the
finished product, however if the source of a nitrosamine impurity is identified in the active substance
manufacturing process, control options 1 to 3 as stated in ICH M7(R1) guideline could be used to
demonstrate that the nitrosamine will not be present above the acceptable limit based on AI in the
finished product. Testing of raw materials (e.g. excipients) should also be considered if these are
potential sources of nitrosamine impurities. Exceptions from routine testing may be possible, if the root
cause of contamination is demonstrated to be well-understood:

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• Only if the amount of nitrosamine present is consistently below 10% of the acceptable limit based
on AI in the API or in the finished product, then a test for the nitrosamine could be omitted from
the specification.

• Only if levels of a single nitrosamine are consistently below 30% of the acceptable limit based on
AI in the API or the finished product, skip-testing according to the ICH Q6A definition could be
acceptable.

16. What are the responsibilities of MAHs for APIs with CEPs
or ASMFs?
MAHs/Applicants, manufacturing authorisation holders and API manufacturers should work together
and take precautionary measures to mitigate the risk of presence of nitrosamines during the
manufacture and storage of all medicinal products containing chemically synthesised APIs.

MAHs/Applicants must ensure that appropriate and robust risk evaluations are carried out by the
relevant manufacturing authorisation holders and API manufacturers (including ASMF or CEP holders)
in accordance with Article 46 of Directive 2001/83/EC.

17. How does the lessons learnt exercise from presence of

nitrosamines in sartans relate to the Article 5(3) Referral
Outcome?
The lessons learnt exercise was conducted by experts from the EU Regulatory Network to determine
which lessons can be learnt from the handling of the cases of sartans with nitrosamine impurities. The
objective is to make recommendations on how to reduce the risk of such impurities in medicines and to
ensure that regulators are better prepared to manage cases of unexpected impurities in the future.
Although the exercise focussed on lessons learnt from the assessment conducted for the sartans with a
tetrazole ring, the recommendations apply to all human medicines.

The recommendations set forward include new or additional guidance on areas such as the control of
impurities (including cohort of concern compounds), Good Manufacturing Practice, the roles and
responsibilities of manufacturers and MAHs/Applicants but also proposals for improvement of
communication with patients and healthcare professionals and cooperation with international partners.
The full recommendations are available on EMA’s website. The European medicines regulatory network
will develop an implementation plan and then work with the parties that will implement each action.

It should be noted that the lessons learnt exercise outcome has been taken into account in the Article
5(3) procedure. The implementation of recommendations of the lessons learnt exercise will strengthen
the regulatory framework and complement the outcome of this Article 5(3) procedure which provides
the scientific opinion on the presence of nitrosamine impurities in human medicines.

18. What about regulatory requirements in other regions?

Regulatory authorities in the EU have been cooperating with international partners in the United
States, Canada, Japan, Singapore, Switzerland, Australia and other countries to mitigate presence of
nitrosamines in medicinal products and to align requirements. For questions about regulatory
requirements outside the EU, please contact the relevant authorities.

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19. What is the approach for line extensions and variations
applications not linked to changes required as part of article
5(3) recommendation?
No risk evaluation is generally necessary when submitting line extension or variation application. The
risk evaluation is only required to be submitted for products in scope of the call for review as reported
in Q&A 3.

Nevertheless, in some exceptional cases questions on the presence of nitrosamines in the product may
be raised if a potential risk is identified during the assessment.

20. What are the regulatory steps taken by authorities

following the identification of an N-nitrosamine exceeding
the AI?
The regulatory process dealing with the outcomes of the call for review is outlined in European
Medicines Regulatory Network approach for the implementation of the CHMP Opinion pursuant to
Article 5(3) of Regulation (EC) No 726/2004 for nitrosamine impurities in human medicines.

Chapter 3.2 provides a description on how regulators will approach the outcome from the call for
review in accordance with the different scenarios reported by MAHs.

In case of identification of one or more N-nitrosamine exceeding the AI in the finished product, or in
case that the sum of all detected N-nitrosamines exceeds the 1 in a 100,000 lifetime risk (scenario A),
the following steps are taken in order to protect public health and ensure availability of critical
medicines:

• A lead authority is identified as responsible for reviewing the information available and for
providing the (preliminary) assessment of the case. The lead authority is selected as outlined in
chapter 5.1.

• The Rapid Alert Network (RAN) and the availability Single Point Of Contacts (SPOCs) are informed
in order to determine the criticality of the product (in accordance with Criteria for classification of
critical medicinal products for human and veterinary use).

• The feedback from RAN and availability SPOCs is taken into account by the lead authority when
providing the preliminary recommendations on any interim or eventual required market actions
and on the acceptability of corrective and preventive actions proposed by the MAH.

• The Incident Review Network (IRN) is consulted in order to facilitate the exchange of information
and to evaluate whether additional measures are needed or whether a different regulatory pathway
is warranted.

• If market actions are recommended, each National Competent Authority (NCA) will follow up in
accordance with their national procedures and depending on the criticality of the product for their
markets.

• The use of the temporary AI (t-AI) while a formal AI is established, as described in Q&A 21, or of
an interim limit based on the LTL approach during CAPA implementation, as described Q&A 22,
may be considered, as applicable, by the lead authority and NCAs on a temporary basis for market
action purposes. Please refer to chapter 3.2.1.1 of the regulatory process dealing with the
outcomes of the call for review referenced above.

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21. What is the approach to control the presence of
nitrosamines until a substance specific AI is established?
Q&A 10 provides guidance on the calculation of the limit when a new nitrosamine is identified. If N-
nitrosamines are identified without sufficient carcinogenicity data to derive a substance-specific limit
for lifetime exposure as recommended in ICH M7(R1) guideline, and the class specific TTC for
nitrosamines of 18 ng/day is not used for controlling the levels of the nitrosamine in the finished
product, an AI agreed by the Non-clinical Working Party (NcWP) and adopted by the CHMP is required
to decide on control options for the nitrosamine in the finished product.

To protect public health, to inform decisions on required market actions while ensuring at the same
time availability of medicines while a formal AI is established, a temporary AI (t-AI) of 178 ng/day
(total nitrosamines) can be adopted by the relevant authorities for marketed medicines identified to
contain one or more nitrosamines exceeding the TTC of 18ng/day. This t-AI has been derived using
TD50 values calculated in the Lhasa carcinogenic potency database and is based on a probabilistic
approach that there is a 33% risk that the “true” AI is below the t-AI. It is expected that the t-AI
would be used for a period of less than 12 months, as an exposure over this period of time is not
expected to increase the theoretical overall lifetime risk above 1:100,000.

In practice, this means that when competent authorities are notified about a product containing a new
N-nitrosamine exceeding the TTC limit of 18 ng/day, no market actions may be required for batches
with N-nitrosamine levels ≤178 ng/day at the MDD pending the agreement of the AI. The adoption of
the t-AI is not automatic and is evaluated by the relevant authorities at the time of notification. Use of
the t-AI beyond 12 months will require additional consultation with competent authorities.

In terms of application of this approach on cases for some products where interim limits higher than
178 ng/day were evaluated and agreed by the lead authority as part of the assessment, no changes to
these limits are expected in order to avoid potential shortages. If the previously established limits are
lower than 178 ng/day, the MAHs can request these limits to be changed to the t-AI of 178 ng/day.

The t-AI should not be used as a target for development of validated analytical methods to quantify
new nitrosamines since the long-term limits adopted by CHMP might ultimately be lower than the t-AI.

22. What is the approach to control presence of N-

nitrosamine exceeding the AI during CAPA implementation?
In accordance with the regulatory steps taken by authorities following the identification of an N-
nitrosamine exceeding the AI and outlined in Q&A20, the less-than lifetime (LTL) concept or the use of
interim limits may be considered by the lead authority and NCAs on a temporary basis in order to
inform market actions and at the same time ensure availability of medicines. MAHs are expected to
establish and implement corrective and preventive actions (CAPAs) in authorised medicines without
any delays in order to ensure patients safety and product quality. Nevertheless, it is recognised that
implementation of CAPAs may require some time before the MAH is able to mitigate the presence of
the identified N-nitrosamine below the established AI. Therefore, in order to avoid unnecessary risk of
supply disruptions, a harmonised approach promoting the establishment of interim limits in a
streamlined way is agreed. The approach is applicable to all authorised products that have:

• a duration of treatment not exceeding 10 years;

and

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• CAPA implementation timeline of up to 3 years from the establishment and publication of the AI
(nevertheless MAHs are expected to expedite CAPAs implementation).

Treatment duration Up to 12 months >12 months up to 10years

Interim limit 13.3 x AI* 6.7xAI*

*In any case the limit should not exceed 1.5 µg/day unless the established AI (Table 1, Q10) is > 1.5
µg/day.

The approach is not applicable to the below instances where other approaches may be considered on a
case-by-case basis in consultation with the appropriate regulatory authority:

• Authorised medicines taken for a lifetime (>10 years);

• CAPA implementation exceeding 3 years from the establishment and publication of the AI;
• New/ongoing regulatory applications.

The above interim limits are based on the LTL approach outlined in the ICH M7 guideline, using the two
most conservative adjustment factors (6.7 and 13.3 x AI). The application of these adjustment factors
would not be expected to exceed a theoretical excess cancer risk of 1 in 100,000 during the period of
CAPA implementation.

The approach is intended to be evaluated by the lead authority during the assessment of the case and
is expected to be communicated by the lead authority to the concerned MAH as part of assessment
conclusions. In terms of retrospective application, where more restrictive interim limits were previously
agreed for some products as part of case assessment, upon request from the MAH, the lead authority
can re-assess interim limits taking into consideration this approach to control presence of N-
nitrosamine exceeding the AI during CAPA implementation.

MAHs are expected to ensure that the implementation of adequate controls for the detected
nitrosamines is done as a matter of priority. During the use of the interim limit, monitoring measures
may be evaluated by the lead authority as required. However, it is not the expectation that MAHs
include these interim limits in specifications via variation.

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Annex 1: Decision tree with control options for products
containing multiple N-nitrosamines:

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Annex 2: Carcinogenic Potency Categorization Approach 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 compounds 13 and
also used a set of 84 N-nitrosamines with either rat TD50 values from the Carcinogenic Potency
Database (CPDB) and/or the Lhasa Carcinogenicity Database (LCDB) 14, relative potency
classifications as defined by Rao et al. (1979) 15, and/or AI limits based on previously-conducted
surrogate analyses. 16 The approach assumes that the α-hydroxylation mechanism of metabolic
activation 17 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 recognised 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.

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 (i.e., 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 (e.g., nitrosated indole). For N-
nitrosamines containing two N-nitroso groups, the group with the highest predicted carcinogenic
potency (i.e., the group with the lowest numerical potency category) defines the AI for the entire

13 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.
14 See Lhasa Carcinogenicity Database at https://carcdb.lhasalimited.org/.
15 Rao TK, Young JA, Lijinsky W and Epler JL, 1979. Mutagenicity of Aliphatic Nitrosamines in Salmonella typhimurium,

Mutat Res, 66:1-7.

16 Questions and answers for marketing authorisation holders / applicants on the CHMP opinion for the Article 5(3) referral .
17 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.

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molecule. 18 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.

18 For N-nitrosamines containing more than two N-nitroso groups, the applicant or manufacturer should contact the

applicable drug regulatory authority for further guidance.

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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.

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

Potency Recommended AI Limit

Comments
Category (ng/day)
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
1 18
sufficiently protective to patients.

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,
2 100
respectively. N-nitrosamines assigned to Category 2 are predicted to have carcinogenic potency
no higher than NDMA and NNK.

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
3 400
recommended AI limit was set to reflect a 4-fold decrease in carcinogenic potency from
Category 2.
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
4 1500
pathways are favored. The recommended AI limit of 1500 ng/day is set at the TTC per ICH
M7.**

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
5 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 M7Assessment 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.

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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.

Count of Hydrogen Atoms on Each α-Hydrogen

Example
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.

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

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the N-nitroso group is within more than one ring, the feature score for only the smallest matching
ring should be applied. Examples are intended to be illustrative only and are not intended to be
exhaustive.

Individual
Deactivating
Deactivating Feature Example
Feature
Score

Carboxylic acid group anywhere on

+3
molecule

N-nitroso group in a pyrrolidine ring +3

N-nitroso group in a 6-membered ring

+3
containing at least one sulfur atom

N-nitroso group in a 5- or 6-membered ring* +2

N-nitroso group in a morpholine ring +1

N-nitroso group in a 7-membered ring +1

Chains of ≥5 consecutive non-hydrogen

atoms (cyclic or acyclic) on both side of
+1
acyclic N-nitroso group. 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-nitroso group +1
(cyclic or acyclic)

Electron-withdrawing groups** bonded to α-

carbons on both sides of N-nitroso group +2
(cyclic or acyclic)

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

Hydroxyl group bonded to β-carbons*** on

both sides of N-nitroso group (cyclic or +2
acyclic)
*Excludes examples where N-nitroso group is in a pyrrolidine ring, a 6-membered ring containing at least one sulfur
atom or a morpholine ring (all counted separately).
**Excludes carboxylic acid and aryl (counted separately), and ketone (conflicting data). Additional electron
withdrawing group examples are limited to those described in Cross KP and Ponting DJ, 2021, Developing
Structure-Activity Relationships for N-Nitrosamine Activity, Comput Toxicol, 20:100186, where they are referred to
as “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 on N-nitroso -1
group)

Methyl group bonded to β-carbon (cyclic or

-1
acyclic)

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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.

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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.

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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.

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Example 4 – N-Nitroso-l-nebivolol

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

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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.

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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.

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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.

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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.

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Annex 3: Enhanced Ames Test Conditions for N-nitrosamines

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 (e.g., 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

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 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 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

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