CuttingEdge Celebrating 13th year of publication

Nitrosamine Impurities & NDSRIs

Dr. BM Rao
- Recent Important Updates
CEO
QDOT Associates
Hyderabad
drbmrao@qdotassociates.com

This is in continuation of the articles published in December 2021, July 2022, February 2023 and
in July 2023 in CuttingEdge. In July and August months there were few very important updates on
Nitrosamine impurities / NDSRIs from key regulatory agencies like FDA and EMA. Comprehensive
summary of these updates were covered in this article.

This article covers the information related to:

A. Update from US FDA: ‘Recommended Acceptable Intake Limits for Nitrosamine Drug Substance
Related Impurities (NDSRIs) Guidance for Industry, Date effective in August 2023’.
B. Update from EMA: 07 July 2023, EMA/409815/2020 Rev.16.
C. Update from EMA: 28 July 2023, EMA/409815/2020 Rev.17.
D. Update from Health Canada: July 24, 2023, Health Canada, Guidance on nitrosamine impurities in
medications.

N
N-Nitroso compounds have been listed as one of the cohorts of concern
as per ICH M7. In recent years, the regulatory focus has shifted
from common nitrosamines to N-nitroso impurities of drug products.
N-Nitrosamine risk assessment of pharmaceuticals has moved from
potential presence of known small-molecule N-nitrosamines (NAs) such
as NDMA, NDEA etc. to NDSRIs in drug substances and drug products.
While N-nitrosation of simple secondary amines is well-understood,
more complex amines can undergo alternative reaction pathways that
can be more challenging to predict. Several such complex amines are known not to undergo
N-nitrosation but are either unreactive or react by alternative pathways such as C-nitrosation
or nitration to generate non-N-nitrosamine products. The detection and quantification of
unacceptable levels of nitrosamine drug substance-related impurities are of great concern for
analytical scientists during drug development. Moreover, risk assessment of nitrosamines is
also an essential part of the regulatory filling.

While there is comprehensive literature about the formation of NAs from nitrite in solution
(e.g., NAP test), the formation of NDSRIs even in solid drug products (DPs) from parts
Spinco Biotech
38 September 2023

per million levels of nitrite was
surprising. It was recently shown
that up to 40% of common APIs
and 30% of API impurities are
potential NAs precursors, as they
contain vulnerable amine moieties.
If only the more reactive secondary
amines are considered, still
13−15% of APIs are potentially
at risk. Not surprisingly, NDSRIs
have become the focus from
both an industry and regulatory
perspective. It is a challenging
situation for both industry and
regulators, that NDSRIs may be
present in hundreds if not thousands
of medicinal products, some of
them affecting whole essential
drug classes such as β-blockers.
A survey conducted by Medicines
for Europe among pharmaceutical
manufacturers and presented at
the fourth meeting of the EMA
Nitrosamine Implementation
Oversight Group (NIOG)
with Industry Associations on
November 30, 2022, revealed that
so far 90% of potential NDSRIs
identified in NAs risk assessments
were later confirmed by analytical
testing.
In connection to the above stated
information on NDSRIs a few
recent published guidance to
industry by USFDA, EMA and
other key regulatory agencies are
summarized below.
Summary of recent
regulatory updates on
Nitrosamine impurities/
NDSRIs
A: USFDA guidance update in
August 2023
FDA has issued a Guidance
and a webpage in August 2023
to help industry through the
NDSRI questions we all have.
Specifically, FDA intends to
include on this website updated
September 2023
Nitrosamine Impurities & NDSRIs
information on: (1) recommended
AI limits for certain NDSRIs based
on their predicted carcinogenic
potency categorization listed
by APIs that hypothetically
could be at risk of forming such
NDSRIs (2) recommended AI
limits for certain NDSRIs based
on compound-specific data or
read-across analysis from a
surrogate; (3) recommended
interim AI limits for certain
NDSRIs; (4) recommended
testing methods for confirmatory
testing of certain NDSRIs; and
(5) recommended safety testing
methods for NDSRIs.
Let’s first discuss the
Guidance for Industry (GFI),
'Recommended Acceptable Intake
Limits for Nitrosamine Drug
Substance-Related Impurities
(NDSRIs)'. As they state in
the preface, this GFI is being
implemented immediately
without a comment period (so
it is FINAL not DRAFT). A
high-level overview of what FDA
is providing in this GFI follows:
• This GFI applies to APIs, OTC,
and prescription drug products
(including biological products
regulated by CDER, either
as standalone or biologic-led
combination products), as well
as drug products not marketed
under a drug application. It
includes drugs in clinical
development, those that have
pending applications, and those
that are approved. However,
it does not apply to NDSRIs
that are detected in products
indicated for use in patients
with advanced cancers (refer
the Scope of the GFI for
additional details).
• The GFI contains a ng/day to 1500 ng/day. This
recommended methodology
for Acceptable Intake (AI)
limit determination to generate
predicted carcinogenic po-
tency categorization and
corresponding AI limits that can
be applied by manufacturers
and applicants when there
are no FDA published
recommended AI limits. The
guidance complements the
approach recommended in
ICH M7 (R2) that recommends
the use of structure-activity
relationship (SAR) concepts.
• FDA recognize that not
all NDSRIs have the same
carcinogenic potency, therefore
'it is currently unknown if
all or some NDSRIs are in
fact high potency mutagenic
carcinogens'. Therefore, section
IV of ‘Recommendations for
AI limits based on predicted
carcinogenic potency categ-
orization’. The approach
assigns a recommended AI
limit to an NDSRI based on
its activating and deactivating
structural features alone.
• The predicted carcinogenic
potency categorization and
resulting recommended AI
limit approach described in this
guidance should not be applied
to NDSRIs in circumstances
in which FDA otherwise
recommends an AI limit (e.g.,
based on compound-specific
assessments or read-across
analysis from a surrogate).
• Table 1 (FDA Recommended AI
Limits for Certain Hypothetical
NDSRIs) in the GFI provides
five (5) Potency Categories
and provided with associated
recommended AI limits for
NDSRIs that range from 26.5
is followed by a flowchart
Spinco Biotech
39
Nitrosamine Impurities & NDSRIs
(Figure 1) which can be used
to predict the Carcinogenic
Potency Category of an
NDSRI. Appendix A further
gives information on how to
determine a potency score
based on selected structural
features present.
• Section V of the GFI discussed
expectations from FDA for
timing, implementations, etc.,
specifically when a product
is marketed versus one in
development or under review.
• For better understanding on the
timelines, please refer to the
below statements:
a) Recommended Timeline for
Approved or Marketed Drug
Products
- FDA recommends that if
NDSRIs were not considered
in previous risk assessments,
manufacturers and applicants
re-evaluate the risk within
3 months of publication
of this guidance, with a
recommended completion
date by November 1,
2023, as part of overall
risk management. FDA
recommends conclusion
of NDSRI confirmatory
testing of drug products
and submission of required
changes in drug applications
by August 1, 2025.
b) Recommended Timeline
for Drug Products in
Development and Under
FDA Review
- Applications pending with
Agency: Applicants with
pending applications should
conduct the risk assessment
expeditiously and inform
FDA if confirmatory testing
Spinco Biotech
40
finds NDSRI levels above
the AI limits recommended
in this guidance. If an
NDSRI is detected above the
recommended AI limit, the
applicant should amend the
application as appropriate.
The Agency will work with
the applicant in an effort to
resolve issues during the
review cycle.
- Pre-submission stage:
FDA recommends that an
applicant conduct a risk
assessment for NDSRIs
and conduct confirmatory
testing as appropriate prior
to submission of an original
application. However,
the risk assessment and
submission of confirmatory
testing, if appropriate, and
changes to the drug master
file or application may be
submitted in an amendment
if these are not available
at the time of the original
application submission.
Such an amendment should
be submitted as quickly as
possible after the original
application submission to
minimize any potential
adverse impact on the
application assessment
timeline.
FDA has also provided approaches
on how to justify or qualify a
proposed alternative AI.
In addition to this helpful new GFI,
FDA has created an accompanying
webpage (here) where they plan to
provide more real-time updates to
NDSRI-specific information as it
arises. According to the webpage,
FDA intends to provide updated
information and recommendations
on the following topics:
- AI limits for certain NDSRIs
based on their predicted
carcinogenic potency
categorization listed by APIs
that could hypothetically be at
risk of forming such NDSRIs.
- AI limits for certain NDSRIs
based on compound-specific
or read across analysis from a
surrogate.
- Interim AI limits for certain
NDSRIs.
- Testing methods to be used for
confirmatory testing of certain
NDSRIs.
- Safety testing methods for
NDSRIs.
- FDA has started out strong,
providing (in Table 1) Recom-
mended AI limits for 247 certain
hypothetical NDSRIs, including
the API source, Potency
Category, and recommended AI
Limit. In Table 2, there are 3
out of 4 (except Ciprofloxacin)
NDSRI AI limits provided
based on compound-specific
or read-across analysis from a
surrogate.
B: EMA guidance updates
from July 2023
On 7 July 2023, the European
Medicines Agency (EMA) updated
its guidance on nitrosamine
impurities. The regulatory
body has amended Q&A 10 to
include the Carcinogenic Potency
Categorization Approach (CPCA)
and the enhanced Ames test (EAT)
for establishing acceptable intakes
(AIs) for N-nitrosamines. The
assessment report of the Committee
for Medicinal Products for Human
Use (CHMP)’s Article 5(3) of
Regulation (EC) No 726/2004
opinion on nitrosamine impurities
September 2023
 Nitrosamine Impurities & NDSRIs

Potency Recommended 83 known nitrosamine impurities

Comments
Category AI Limit (ng/day) present (In previous version AI
1 18
N-nitrosamines assigned to Category 1 are predicted to values for 27 known nitrosamine
have high carcinogenic potency. impurities were published).
The recommended AI limit of 100 ng/day is representative
of two potent, robustly tested N-nitrosamines, NDMA and Annex 2: Carcinogenic Potency
NNK (4-(methylnitrosamino)-1-(3-pyridyl)-1-(butanone)), Categorization Approach (CPCA)
2 100 which have recommended AI limits of 96 ng/day and 100
ng/day, respectively.
for N-Nitrosamines
-N-nitrosamines assigned to Category 2 are predicted to EMA Rev.16 document describes
have carcinogenic potency no higher than NDMA and NNK.
an approach for assigning an
N-nitrosamines in this category have lower carcinogenic
potency than Potency Category 2, due to the presence of a
N-nitrosamine impurity/NDSRIs to
3 400 weakly deactivating structural feature. a predicted carcinogenic potency
The recommended AI limit was set to reflect a 4-fold category, with a corresponding
decrease in carcinogenic potency from Category 2. acceptable intake (AI) limit, based
-N-Nitrosamines assigned to Category 4 may be
on an assessment of activating or
metabolically activated through an α-hydroxylation pathway
4 1500 but are predicted to be of low carcinogenic potency. deactivating structural features
Eg: the pathway is disfavoured due to steric or electronic present in the molecule.
influences, or because clearance pathways are favoured.
-N-Nitrosamines assigned to Category 5 are not predicted In the context of this document,
to be metabolically activated via an α-hydroxylation activating or deactivating
5 1500 pathway due to steric hindrance or the absence of α-
hydrogens or are predicted to form unstable species that
features are defined as molecular
will not react with DNA. substructures that are associated
The Five Predicted Potency Categories and Associated AI Limits for N-Nitrosamines
The Five Predicted Potency Categories and Associated AI Limits for N-Nitrosamines with an increase or decrease
in carcinogenic potency.
The Carcinogenic Potency
in human
Annex medicinal
3: Enhanced Amesproducts offers
Test Conditions conducted by groups such as the
for N-nitrosamines Categorization Approach is based
guidance and enhanced
For N-nitrosamines, recommendations UStheFood
testing conditions for Ames and
assayDrug Administration
are recommended due to the on structure-activity relationship
reported reduced sensitivity
on mitigation and prevention of of the assay under standard
(FDA)’s National Center forsuch
conditions for some N-nitrosamines
as NDMA.
(SAR) concepts.
nitrosamine-contaminated
If a standard Ames assay is conducted human Toxicological
and produces a positive result,Research (NCTR)
there is no need to conduct
medicinal
an additionalproducts. and The
assay using enhanced testing conditions. have beenAmes
enhanced evaluated
assay test for a
conditions The approach assumes that the
are described in the new EMA Q&A’s guidance. variety of N-nitrosamines including α-hydroxylation mechanism of
New inclusions in the EMA’s metabolic activation is responsible
NDSRIs. Evaluation of Ames assay
revised guidance on nitrosamine for the mutagenic and highly potent
test conditions for N-nitrosamines
impurities carcinogenic response observed for
EMA/409815/2020 Rev.17 and Effective from 28 July is2023
ongoing, with a goal to identify
Included in the revision is the the most robust Ames testing many N-nitrosamines. Structural
addition of Appendix 1. It lists conditions, EMA reported in the features that directly increase or
the nitrosamines for which AI revision document. Deviations from decrease the favorability of the
have been established by the the recommended conditions should activation mechanism. Therefore, a
Non-clinical Working Party be justified. prediction of the mutagenic potential
(NcWP), including new AIs for and carcinogenic potency of an
N-nitrosamines determined using
EMA/409815/2020 Rev.16 and N-nitrosamine can be generated
the CPCA.
Effective from 7 July 2023 based on its structural features.
In the recent revision of EMA Annex 3: Enhanced Ames Test
Also added Annex 2, which Q&A’s guidance, Annexure-2 Conditions for N-nitrosamines
describes the CPCA for and Annexure-3 which details
N-nitrosamines. The guidance on ‘Carcinogenic Potency Categ- For N-nitrosamines, enhanced
nitrosamine impurities also has orization Approach (CPCA)’ and testing conditions for the Ames
addition to Annex 3. It describes the ‘Enhanced Ames Test Conditions assay are recommended due to the
Enhanced Ames Test Conditions for for N-nitrosamines’. reported reduced sensitivity of the
N-nitrosamines. EMA highlighted assay under standard conditions
that the enhanced Ames assay test At present as per the latest EMA for some N-nitrosamines such as
conditions are informed by work Q&A’s guidance (Rev. 16) there are NDMA.

Spinco Biotech
September 2023 41
 21. What is the approach to control the presence of nitrosamines until a substance specific AI is
established? (Updated)
Considering the new approaches for setting nitrosamines limits using the carcinogenic potency
categorisation approach (CPCA) and the enhanced AMES test (EAT) protocol (see Q&A 10
above), the approach for a universal temporary AI (t-AI) while a formal AI is established is no longer
Nitrosamine Impurities & NDSRIs
considered necessary, as such the contents of this question has been deleted in July 2023.
22. What is the approach to control presence of N-nitrosamine exceeding the AI during CAPA
implementation? (Updated)
EMA/409815/2020 Rev.16
Effective from 7 July 2023
The less-than lifetime (LTL) concept or the
use of interim limitƐapproach is applicable to
all authorised products that have:
• a duration of treatment not exceeding
10 years;
The less-than lifetime (LTL) concept or the
use of interim limitƐapproach is not
applicable to the below instances:
• Authorised medicines taken for a
lifetime (>10 years);
*In any case the limit should not exceed 1.5
μg/day unless the established AI (Table 1,
Q10) is > 1.5 μg/day.
If a standard Ames assay is
C: Health Canada guidance update from July 24, 2023
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 are
described in the new EMA Q&A’s
guidance.
EMA/409815/2020 Rev.17 and
Effective from 28 July 2023
20. What are the regulatory steps
taken by authorities following the
identification of an N-nitrosamine
exceeding the AI? (Updated)
The use of the temporary AI (t-AI:
178 ng/day) while a formal AI is
established, as described in Q&A
21 concept was removed from
the guidance. The AI limit to
be calculated as defined in EMA
Q&A’s guidance Rev-16.
21. What is the approach to control
the presence of nitrosamines
until a substance specific AI is
established? (Updated)
Spinco Biotech
42
EMA/409815/2020 Rev.17
Effective from 28 July 2023
This point updated as, less-than lifetime
(LTL) concept or the use of interim limitƐ
approach is applicable to all authorised
products that have:
• CAPA implementation timeline of up
to 3 years from the establishment and
publication of the AI
(nevertheless, MAHs are expected to expedite
CAPAs implementation).
This point is removed in current revision.
*In any case the limit should not exceed 1.5
μg/day unless the established AI (Table 1,
Q&A10) is > 1.5 μg/day or the nitrosamine
concerns a category 5 according to CPCA or
the nitrosamine is shown to be negative in
an enhanced Ames test (EAT).
Considering the new approaches
for setting nitrosamines limits
using the carcinogenic potency
categorisation approach (CPCA)
and the enhanced AMES test
(EAT) protocol (see Q&A 10), the
approach for a universal temporary
AI (t-AI) while a formal AI is
established is no longer considered
necessary, as such the contents of
this question has been deleted in
July 2023.
22. What is the approach to
control presence of N-nitrosamine
exceeding the AI during CAPA
implementation? (Updated) (refer
above table).
C: Health Canada guidance
update from July 24, 2023
The updates include two significant
approaches recently finalized by
the Nitrosamines International
Technical Working Group
(NITWG) that will assist industry
and regulators in setting Acceptable
Intake (AI) limits for nitrosamines
in drug products, namely the
Enhanced Ames Test (EAT)
conditions and the Carcinogenic
Potency Categorization Approach
(CPCA).
The updates to the guidance
document include the addition of
AI limits for several nitrosamines
that are considered acceptable to
Health Canada. The application
of the CPCA has revised already
established AIs for various
nitrosamines, these include
N-nitroso-dabigatran, N-nitroso
-tamsulosin, N-nitroso-rasagiline
and 1-methyl-4-nitrosopiperazine
(MNP).
Reference: https://www.
canada.ca/en/health-canada/
services/drugs-health-products/
compliance-enforcement/
information-health-product/
drugs/nitrosamine-impurities/
medications-guidance.html
Conclusions
Nitrosamine impurities focus has
been extended to over the past two
years, from highly potent small
molecule N-nitrosamine impurities
formed during manufacture of
drug substances, to NDSRIs
predominantly formed during
formulation/storage of the drug
product. All three (US FDA, EMA,
HC) are continuously providing
the guidance to the industry on
Nitrosamine impurities/NDRSIs.
So far published AI limits for 250
impurities by FDA, 83 impurities
by EMA and 101 impurities by
HC. Now it is the responsibility
of the manufacturer to evaluate
these impurities presence through
risk assessment and submit the
outcome to the agencies against
the target timelines. Effective
strategies to avoid or mitigate
the presence of N-nitrosamine
September 2023
 Nitrosamine Impurities & NDSRIs

impurities originating during drug substance manufacturing

processes are well-known and have been documented, but
the control of NDSRIs in drug products is presently a
significant challenge.

MAHs and API manufacturers should bear in mind

not only whether the structure of the API bears a risk
of formation of NDSRIs, but additionally whether
nitrosatable impurities or degradants may pose a risk of
N-nitrosamine formation during drug product manufacture.
The mitigating measures proposed by FDA should be
considered where an NDSRI is identified in a medicinal
product above the acceptable intake. MAHs, drug product
manufacturers, drug substance manufacturers, as well as
excipient and packaging manufacturers are encouraged
to collaborate further to expand the understanding of risk
factors for N-nitrosamine formation in medicines. Recent
EMA and FDA guidance offers some hope for those of us
in the pharma industry who have been waiting for specific
guidance from regulators, so that the industry can continue
to provide safe and effective drug products to the patients.
Hopefully this is the light at the end of the tunnel we have
been waiting for.
Acknowledgements
BNV Ganapati Rao, Head - CQC & ASAT, Brahmaji Mavuri, Lead –
QC from Dr. Reddy's Laboratories Ltd and Ramesh Nethala, Senior
Manager – CQC from Eugia Pharma Specialities Ltd., for their immense
contributions to this article.

References
1. Recommended Acceptable Intake Limits for Nitrosamine Drug Substance Related Impurities
(NDSRIs) Guidance for Industry, Date effective in August 2023. (https://www.fda.gov/drugs/
guidance-compliance-regulatory-information/guidances-drugs)
2. FDA’s National Centre for Toxicological Research (NCTR) (Li et. al., 2023).
3. OECD Test Guideline No. 471 “Bacterial Reverse Mutation Test (Ames Test, OECD 471)”.
(https://www.creative-bioarray.com/Services/bacterial-reverse-mutation-test-ames-test-
oecd-471.htm)
4. New guidance for Industry - Acceptable Intake limits for NDSRIs and a new web page from
FDA, Lachman Consultant Services, Inc. / Blog / Analytical Methods / New Guidance for
Industry
5. Revisiting the mutagenicity and genotoxicity of N-nitroso propranolol in bacterial and
human in vitro assays. Regulatory Pharmacology and Toxicology. 2023
6. EMA, 07 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.
7. EMA, 28 July 2023, EMA/409815/2020 Rev.17, 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.
8. Health Canada updated Guidance on nitrosamine impurities in medications, Date adopted:
July 24, 2023, Effective date: July 24, 2023.
9. Recommended Acceptable Intake Limits for Nitrosamine Drug Substance Related
Impurities; Guidance for Industry; Department of Health and Human Services Food and
Drug Administration (FDA) [Docket No. FDA–2020–D–1530], (Federal Register / Vol. 88,
No. 150 / Monday, August 7, 2023 / Notices).
10. Formation of N Nitrosamine Drug Substance Related Impurities in Medicines: A Regulatory
Perspective on Risk Factors and Mitigation Strategies by Răzvan C. Cioc, Ciarán Joyce,
Monika Mayr, and Robert N. Bream, Organic Process Research & Development (OPR&D)
Article (https://doi.org/10.1021/acs.oprd.3c00153).
11. Spinco Biotech, CuttingEdge, July 2023, Dr BM Rao ‘Nitrosamine impurities and NDSRIs
– Current regulatory updates’.
C E

Spinco Biotech
September 2023 43