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Guide on the Control of Nitrosamines in Active

Pharmaceutical Ingredients and Medicines
Guide No. 50/2021 - version 3

National Health Surveillance Agency - Anvisa

2023
 Guide on the Control of Nitrosamines in Inputs
Active Pharmaceuticals and Medicines
IN FORCE AS OF 06/13/2023

This Guide expresses Anvisa's understanding of best practices with respect to procedures, routines and
methods considered adequate for complying with technical or administrative requirements demanded by the
Agency's legislative and regulatory frameworks.1

This is a non-normative regulatory instrument, of a recommendatory and non-binding nature, therefore, it is

possible to use alternative approaches to the propositions set out here, provided they are compatible with the
requirements related to the concrete case. Failure to comply with the content of this document does not
characterize a health infraction, nor does it constitute grounds for rejecting petitions, provided that the
requirements required by law are met.

The recommendations contained in this Guide take effect from the date of their publication on the Anvisa
Portal.

1Ordinance No. 162, of March 12, 2021 , which deals with guidelines and procedures for improving regulatory quality at the National
Health Surveillance Agency (Anvisa).

Copyright©2021. National Health Surveillance Agency – Anvisa. Partial or total reproduction of this
document by any means is completely free, as long as the source is properly cited. playback stops
any commercial purpose is prohibited.

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SUMMARY

1. SCOPE.................................................................. .................................................................. ............4

2. INTRODUCTION.................................................................. .................................................................. ...4

3. LEGAL BASIS.................................................................. .................................................................. ......5

4. CHEMICAL AND REACTIONAL ASPECTS.................................................................. .................7

a) Formation of nitrosamine as a contaminant in the process of obtaining Active

Pharmaceutical Ingredients.................................................................. .................................. 10

b) Formation of nitrosamine as a contaminant in the drug manufacturing process

.................................................................. .................................................................. ......... 13

5. RISK MANAGEMENT.................................................................. ............................. 14

6. PRIORITIZATION FACTORS FOR RISK ASSESSMENT (STEP 1).................. 19
a) Basic components.................................................................. ......................................19
b) Purge evaluation.................................................................. .................................................... 20

7. CONFIRMATORY TESTS (STEP 2).................................................................. ..............21

a) Analytical method.................................................................. ............................................... 21

b) Batches to be tested.................................................................. .....................................21

8. CALCULATION STRATEGY FOR ASSIGNING ACCEPTANCE LIMITS....22

a) Limits for single nitrosamine.................................................................. ........................ 23

b) More than one nitrosamine.................................................................. ................................24

c) New nitrosamines.................................................................. ......................................................25

d) ApproachLess Than Lifetime.................................................................. ........................27

9. GLOSSARY.................................................................. .................................................................. ..... 28

10. REFERENCES.................................................................. .................................................................. .. 29

Guide on the Control of Nitrosamines in Active Pharmaceutical Ingredients and Medicines

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1. SCOPE

This document presents recommendations regarding the control of nitrosamines in all

Chemically synthesized Active Pharmaceutical Ingredients (APIs) and medicines for human use that contain
them, as well as biological products, when applicable.

The recommendations are equally applicable to post-registration changes that may result
in the formation of nitrosamines, such as changes related to the API, composition and packaging of the
medication, not exclusively restricted to these.

2. INTRODUCTION

N-nitrosamines constitute a class of compounds characterized by the binding of a

nitroso group (-N=O) to an amine functional group (-NRtwo). Among the compounds in this class, there are some
mutagenic, genotoxic and potentially carcinogenic agents in humans and, for this reason, they must be
controlled at levels considered acceptable and safe. These compounds can be found in water, cured meat,
processed fish, beer and other alcoholic and non-alcoholic beverages, cheese, soy sauce, oils, processed
vegetables and human milk and their exposure within safe limits represents a low risk of harm to health. (EFSA
CONTAM Panel, 2023) (EMA 2020). However, exposure above acceptable levels and for a long period may
increase the risk of cancer (Horne et al., 2023). (

In 2018, regulatory agencies around the world became aware of the presence of
nitrosamines above the levels allowed in drugs, after manufacturers of active pharmaceutical
ingredients from the group of drugs commonly called “sartanas” – angiotensin II receptor
antagonists, used to control blood pressure – issued warnings of their possible presence in this class
of drugs .

Since then, these agencies have promoted actions in order to protect the health of patients.
exposure to nitrosamines in medications above levels considered acceptable. In Brazil, the control
actions promoted by Anvisa began with inspections carried out in 30 drug manufacturing
companies, with 111 products having been inspected. As a result, 31 health actions were carried out,
including interdictions, suspensions and recalls. The source of drug contamination was identified as
coming mainly from the presence of solvents under chemical conditions that favor the formation of
nitrosamines.

Faced with the case of “sartanas”, the main regulatory agencies in the world, together with
drug companies began to investigate whether other drugs could also have nitrosamines above
acceptable levels. In 2019, the presence of nitrosamines in other drug classes, such as nizatidine,
ranitidine, and metformin, was reported. Additionally, the possible formation of nitrosamines from
the primary packaging material containing nitrocellulose was evidenced. Since then, formation of
nitrosamines has been observed in several other medicinal products, for example those containing
varenicline, rifampicin, diosmin and sitagliptin.

As part of this investigation process, it was found, for example, that the presence of
nitrosamines in ranitidine presented a different source than previously found in other products. For this drug,
the formation of dimethylnitrosamine (NDMA) originates from an intermolecular degradation that occurs
throughout the storage of the product, and which can be influenced by the crystal morphology of the molecule
(King et al. 2020), in addition to being accelerated by the storage at temperatures above

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room temperature. Such conditions may result in consumer exposure to unacceptable levels of this
impurity (FDA 2020b).

In Europe, in the months following the discovery, several manufacturers of the raw material ranitidine had
their Certificates of Suitability (Certificate of Suitability-CEP) revoked by theEuropean Directorate for the
Quality of Medicines(EDQM). It is also noteworthy that drugs containing ranitidine hydrochloride have
been available to the world's population for over 30 years and have been used for the treatment of ulcers,
esophagitis and gastric reflux, without reports of serious adverse events, which reinforces the need for
evaluation and control of nitrosamines in all drug classes.

It is important to emphasize that although there is a very low risk of nitrosamines being
present in biological products, these cannot be definitively discarded. In the light of current scientific
knowledge, it is known that such risks are concentrated, for example, in products with chemically synthesized
fragments, those packed in blisters containing nitrocellulose, biological products with excipients in their
composition, or in which there is the intentional addition of agents nitrosants in the manufacturing process
(EMA, 2020).

In this sense, although nitrosamines are not expected to be formed during the manufacture of
most APIs and finished products (EMA 2020b), it is strongly recommended that manufacturers, distributors and
fractionators of APIs and drug manufacturing and importing companies evaluate their products for the
possible presence of these contaminants and take the necessary precautionary measures to minimize the risk
and ensure the safety of these products.

This guide presents basic concepts about training, risk management related to
presence, in addition to recommendations on control of nitrosamines in active pharmaceutical ingredients and
medicines, as well as clarifies the responsibility of companies, presents strategies for calculating limits and
addresses other concepts.

It should be noted that this is a document that can be changed as new studies and
information is available regarding this area of knowledge, including acceptable limits of exposure
to nitrosamines, in view of the lack of conclusive studies on long-term intake.

It is important to register international regulatory cooperation on this topic. In 2018, it was

formed a strategic group composed of regulatory agencies from several countries to deal with the
subject, the NISG, "Nitrosamines International Strategic Group”. In 2020, a spin-off group of the NISG, the
NITWG, or International Technical Working Group on Nitrosamines (Nitrosamines International Technical
Working Group) with the attribution of addressing technical issues related to nitrosamines in more depth.
Anvisa has been part of these groups since 2021.

3. LEGAL BASIS
Law No. 6,360, of September 23, 1976, which provides for the health surveillance to which
subject to medicines, drugs, pharmaceutical and related inputs, cosmetics, sanitizing products and other
products;

RDC Resolution No. 73, of April 7, 2016, which provides for post-registration changes,
cancellation of registration of drugs with synthetic and semi-synthetic active ingredients;

RDC Resolution No. 76, of May 2, 2016, which provides for changes,
inclusion and post-registration cancellation of specific drugs.

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 RDC Resolution No. 166, of July 24, 2017, which provides for the validation of methods
analytics;

RDC Resolution No. 753, of September 28, 2022, which provides for the criteria for the
granting the registration of drugs with synthetic and semi-synthetic active ingredients, classified as new,
innovative, generic and similar;

RDC Resolution No. 359, of March 27, 2020, which establishes the Pharmaceutical Input Dossier
(DIFA) and the Letter of Adequacy of Active Pharmaceutical Ingredient Dossier (CADIFA);

RDC Resolution No. 412, of August 20, 2020, which establishes the requirements and conditions
for carrying out stability studies for the purposes of registration and post-registration changes of biological
products;

Normative Instruction No. 65, of August 20, 2020, regulates the classification of changes
post-registration and conditions and technical documents necessary to instruct requests for post-registration alteration and
cancellation of registration of biological products;

RDC Resolution No. 511, of May 27, 2021, which provides for the admissibility of codes
foreign pharmacists.

RDC Resolution No. 413, of August 20, 2020, which provides for post-registration changes and
cancellation of registration of biological products;

RDC Resolution No. 576, of November 11, 2021, which provides for the notification of
low-risk medications;

RDC Resolution No. 625, of March 9, 2022, which provides for the minimum requirements
relating to the obligation, on the part of companies holding drug registrations, to communicate the
implementation of the drug recall action to the competent health authorities and consumers, in the
event of sufficient evidence or proof of quality deviation that represent a risk, injury or consequence
health, as well as on the occasion of cancellation of registration related to safety and efficacy;

RDC Resolution No. 654, of March 24, 2022, which provides for Good Manufacturing Practices
of Active Pharmaceutical Ingredients;

RDC Resolution No. 658, of March 30, 2022, which provides for the general guidelines of good
drug manufacturing practices;

RDC Resolution No. 677, of April 28, 2022, which provides for risk assessment and risk control
Potentially carcinogenic nitrosamines in Active Pharmaceutical Ingredients (API) and medicines for human
use.

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4. CHEMICAL AND REACTIONAL ASPECTS

N-nitrosamines have the general formula described below:

No

No

Rtwo R1

Figure 1- General structure of N-nitrosamines.

The chemistry of nitrosamines has been studied and documented since the last century, having
this field of study was boosted in the 1950s by the observations of Magee and Barnes (1956) who
related these substances to the onset of cancer. Data obtained in Norway, in the late 1950s and early
1960s, already indicated the presence of nitrosamines as a cause of hepatotoxicity in animals fed
diet treated with nitrite (Ender et al. 1964).

Dimethylnitrosamine (NDMA) is currently classified by the IARC as probably

carcinogenic to humans (group 2A) (IARC 1987), while two tobacco-specific nitrosamines (i.e., 4-(N-
nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine ( NNN)) are categorized as
carcinogenic to humans (group 1) (IARC 2007).

The existence of these compounds is widespread. Fong and Chan (1973) observed that the ability
of theStaphylococcus aureusreduce NO- 3(nitrate) to NO-two(nitrite) from salts present in fish, could
lead to the production of nitrosamines through the reaction of the nitrite formed with trimethylamine, a
substance present in large quantities in decomposing fish. Water and smoked foods are also commonly
cited as sources of nitrosamines.

The genotoxicity of nitrosamines is dependent on alpha-carbon hydroxylation, which occurs

through enzymes of the microsomal P-450 system (CYP 450). Activation involves the production of diazonium
ions, which decompose leading to the formation of carbocations, which are positively charged and electrophilic
species capable of binding to DNA (Rath & Canaes 2009, Carlson et al. 2017). Currently, data from animal
studies of 228 nitrosamines reveal that 82% are considered carcinogens.in vivo(Thresher et al. 2020);
independent of the administered route (Li & Hecht 2022, Preussmann & Stewart 1984).

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 Figure 2- Hydroxylation of nitrosamines mediated by the P-450 system and subsequent generation of diazonium ions. Adapted from EMA (2020a) and
(Rath & Canaes 2009).

Chemically, nitrosamines can be formed from amines and nitrosating agents.

(usually oxidized nitrogen containing NyOx groups) under certain reaction conditions. The nitrosation of secondary amines
(Fig. 3B), tertiary and quaternary ammonium compounds by nitrous acid is a general example of the formation of these
compounds. Nitrous acid is an unstable compound obtained from sodium nitrite in an acid medium leading to the
generation of nitrosonium ion (NO+), which is responsible for the N-nitrosation of amines (Fig. 3A).

H+
NaNOtwo HNOtwo + H+ HtwoO + NO+ (A)

nitrite of acid ion

sodium nitrous nitrosonium

H O
H
AT THE+ No
- HX
No R1 No+ No X- (B)
1
Rtwo No
Rtwo O R1 R two
the mine

ntrosamine

Figure 3 - Formation of nitrosamines by nitrosation with nitrous acid obtained from sodium nitrite in an acid medium.

In general, nitrosamines formed from primary amines are unstable and lead to
direct formation of the corresponding diazonium ion; (Fig. 4C). That is, when one of the ligands R1or Rtwoof the amine
shown in Figure 3 is a hydrogen, tautomeric interconversion occurs in the primary nitrosamines formed (something
analogous to keto-enol equilibrium) (Fig. 4A), which leads to the formation of a corresponding alkyldiazonium.

Diazoic acid tends to dehydrate in the presence of hydrogen ions and form diazonium ion (Fig.
4B). This ion is rapidly decomposed forming a carbocation and releasing nitrogen (Ntwo) (Roberts &
Caserio 1977, Reusch 1999). In summary, there are no appreciable amounts of nitrosamines formed from
primary amines by the decomposition tendency of the nitrosated product.
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 R

No O No oh

H No R No
nitrosamine acid
diazoic (A)
primary

No oh H+
No O+ - HO
two R No+ No (B)
H
No R No
diazonium ion
acid
diazoic

RNoH HONO RNo+ No R++ No No (W)

H+, 0°C
H
diazonium ion decomposition
the mine
primary

Figure 4- Carbocation formation from primary amines.

The structure of the nitrosamine obtained at the end of the nitrosation process will be determined
primarily by the structure of the amine that has been nitrosated. As an example, the formation of
dimethylnitrosamine (NDMA) from the nitrosation of dimethylamine (DMA) is illustrated as follows:

H+
No No O
NaNOtwo
H No
dimethylamine - nitrite of dimethylnitrosamine -
DMA sodium NDMA

Figure 5 - Nitrosation of dimethylamine (DMA) and formation of dimethylnitrosamine (NDMA).

Likewise, acid nitrosationNo-methyl-4-aminobutanoic acid (MBA) leads to the formation of

carboxypropylmethylnitrosamine (NMBA):

O O
No H+
No O
O H NaNOtwo HO No

N-methyl-4-aminobutanoic acid carboxypropylmethylnitrosamine

- MBA - NMBA

Figure 6 - Nitrosation of N-methyl-4-aminobutanoic acid (MBA) and formation of carboxypropylmethylnitrosamine (NMBA).

Both NDMA and NMBA were detected as impurities arising from the synthesis of APIs
angiotensin II receptor antagonists (EMA 2020, FDA 2020). The reactions above describe what
probably occurred to generate the NDMA and NMBA nitrosamines, i.e. acid formation
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nitrousin situfrom the presence of sodium nitrite, leading to the formation of the nitrosating agent (NO+) and
consequent nitrosation of the amines DMA and MBA, as shown in Figure 3A (EMA 2020).

It is known that other compounds containing NOx groups, such as nitrite salts and alkyl nitrites,
nitrous anhydride (NtwoO3), dinitrogen tetroxide (NtwoO4), nitrosyl halides (NOCl), nitrosylthiocyanate and
nitrosophenol, among others, are capable of nitrosating amines (Figs. 7 to 9). Processes such as curing meat, malting
before fermentation or during chemical reactions can lead to the production of some of these compounds. Nitric
oxide is capable of nitrosing in the presence of metals and organometallic compounds (EMA 2020a).

O
H R H R No
No+ NaNOtwo No+ + HO
Δ R No R two

Cl-
H R H
AT THEtwo
-

Figure 7 - Formation of secondary nitrosamine from ammonium salt and sodium nitrite.

Figure 8- Formation of nitrosamine from tertiary amine and NtwoO3as a nitrosating agent.

NaNOtwo/ H+
No No O
No

triethylamine diethylnitrosamine - NDEA

Figure 9 - Nitrosative dealkylation of triethylamine and formation of nitrosamine diethylnitrosamine (NDEA).

The formation of nitrosamines from tertiary amines is generally a slower process.

than the process of nitrosation of secondary amines, since it involves a dealkylation step, followed by
nitrosation of the dealkylated amine and, regarding the formation of simple nitrosamines, derived from
trialkylamines, a much lower risk is expected when compared to the formation of nitrosamines derived from
secondary amines, although this reduced reactivity may not occur with more complex tertiary amines (López-
Rodríguez et al., 2020 and Ashworth et al., 2023).

a) Formation of nitrosamine as a contaminant in the process of obtaining Active

Pharmaceutical Ingredients

Among the main causes of the formation of nitrosamines as contaminants in API, is the
simultaneous use of secondary or tertiary amines and nitrosating agents, sources of NOx (NaNOtwo, NotwoO3for
example) in the synthesis or obtaining of intermediates or APIs. Not only can the deliberate use of these compounds
in the same step lead to the formation of nitrosamines, but also the carriage of amines or sources of

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NOx from previous steps to subsequent steps can generate a condition conducive to the formation of
nitrosamines. Contaminated reagents, starting materials, solvents and catalysts can also lead to the formation
of nitrosamines during synthetic steps.

Amines can also be generatedin situ. For example, the solvent

dimethylformamide (DMF) which can undergo acidic or basic hydrolysis under favorable conditions and generate
dimethylamine (DMA), which is susceptible to nitrosation (Fig. 10).

H
O CH3

CH3 +
H or OH- + HN
No Δ
H oh
+HtwoO CH3
CH3
dimethylamine - DMA

dimethylformamide - DMF

NaNOtwo/ HX

H3W

No CH3

No

dimethylnitrosamine -
NDMA
Figure 10 - Hydrolysis of dimethylformamide (DMF) and formation of NDMA.

Dimethylamine can also appear as an impurity in the solvent dimethylformamide (DMF),

arising from the DMF synthesis process itself. Similarly, the solvent methylpyrrolidone (NMP) can undergo hydrolysis
and generate substrate amenable to nitrosation (Fig. 11) (Klein 2017, EMA 2020).

O
O

H+or oh- No
No
Δ, HtwoO
HO H
acid
N-methyl-4-aminobutanoic acid

methylpyrrolidone - NMP

NaNOtwo/ HX

No O
HO No

carboxypropylmethylnitrosamine
- NMBA

Figure 11 - Hydrolysis of methylpyrrolidone (NMP) and formation of NMBA.

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 The cases mentioned above are possible causes of the presence of NDMA nitrosamines
and NMBA in some processes for obtaining APIs (EMA 2020), exemplifying how the process conditions
should be evaluated regarding the potential for the formation of amines susceptible to nitrosation,
especially when agents capable of generating nitrosating species are used, even if in different stages of
the process, since the carrying of impurities from one stage to another can lead to the risk of nitrosamine
formation. For example, the nitrite used in a given step can be carried to subsequent steps, going
through processes ofwork up, crystallization or by other purification operations carried out (EMA 2020,
FDA 2020).

In addition to the transformations of inputs used in the process, exemplified with the cases of
hydrolysis of DMF and NMP, direct reactions between ammonium salts and nitrosating agents can also occur.
For example, tetrabutylammonium bromide (TBAB) or triethylamine hydrochloride (TEA.HCl) catalysts undergo
nitrosative dealkylation, a reaction similar to that shown in Figure 9. However, the solvent dimethylacetamide
and other amides can follow the same degradation pattern shown in Figure 9. Figures 10 and 11, generating
amines capable of nitrosation. Worthy of note, in addition to the cases mentioned so far, is the possibility of
oxidation of hydrazines, which can even happen through simple exposure to oxygen in the air ( López-
Rodríguez et al., 2020, Horne et al., 2023).

Due to the wide use of amines in several synthesis processes, it is possible that these
are present as contaminants in various inputs. Additionally, the presence of less substituted amines in reagents
such as tertiary amines or ammonium salts is possible. For example, diisopropylamine and isopropylethylamine
are possible contaminants of diisopropylethylamine (DIPEA), as well as diethylamine is a likely impurity present
in triethylamine (TEA). Furthermore, phase transfer catalysts such as triethylamine hydrochloride (TEA.HCl) have
been identified as a potential source of triethylamine and diethylamine (EMA 2020).

It is of crucial importance to evaluate the process of obtaining an active pharmaceutical ingredient

(IFA) in full, including assessment of procurement of key intermediates and starting materials. The
possibility of carrying a certain substrate from one stage of the process to another, the use of reagents
that have amines in their profile of contaminants or potential nitrosating agents and the process
conditions, such as temperature and pH that may favor the degradation of reagents or solvents, are some
important examples to be observed.

In this sense, it is important to know the degree of purity of the materials introduced and
its potential impurities. Contamination within the same manufacturing unit or resulting from recovery/
reuse of solvents or materials conducted by third parties must always be considered in risk assessments
of synthesis processes. Possible critical combinations that must be observed are situations in which the
general conditions for the formation of nitrosamines are present as in the examples in Table 1.

Table 1 – Examples of critical combinations that can lead to the formation of nitrosamines.

Nitrosamine formed Source amine source nitrosated amine

in
NOx
HN
No O NaNOtwo No O
No
NDMA DMF DMA

No NaNOtwo No No

No O
N,N-DMA N,N-DMA
NMPA

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 H
No O
NaNOtwo No No
No
NDEA
TEA DEA
H
No
No+
Cl–
TEA
TEA.HCl

NaNOtwo No
No O No
No

DIPEA
DIPNA DIPEA

NaNOtwo
No O No No
No

EIPNA DIPEA DIPEA

O O
NaNOtwo O
No O No H No
HO No MPN O H
NMBA MBA
O
NaNOtwo No+
NN No

Cl– TBAB
NDBA TBA

NH

DBA

In addition to nitrosation of amines, other factors must also be considered during the evaluation.
less classic conditions that allow the formation of nitrosamines mentioned in the literature (López-Rodríguez et
al. 2020), such as the reduction of nitramines, oxidation of hydrazines and formation from organometallic
compounds.

b) Formation of nitrosamine as a contaminant in the drug

manufacturing process

The formation of nitrosamines during drug production is less predictable in

comparison with the API production. However, the considerations made in the previous section about the
possible ways of formation of nitrosamines in the API may remain valid for the drug, as long as the
reaction conditions are present.

The formation of nitrosamines in drugs can occur due to some factors

linked to the production process of the drug and its unit operations, the interaction between the API and the
excipient during the production process or during storage, the degradation of the API and other materials, the
interaction of the formulation with the packaging material and, also, by cross-contamination between processes.

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 In the context of nitrosamine formation in the finished product, agent availability
nitrosant can come from the raw materials used, it is known that excipients such as crospovidone,
hypromellose, magnesium stearate, among others, may contain nitrite as impurities (Boetzel et al. 2022). The
nitrite interaction of the excipients must be evaluated against the presence of amines, mainly secondary,
arising from other excipients, API or API-related substances.

Some unit operations present potential risks for the formation of nitrosamines,
such as processing in fluidized beds, due to the possibility of oxidation of disubstituted hydrazines or
exposure of secondary amines to oxidized nitrogen species (López-Rodríguez et., 2020) (Horne et al.,
2023). Wet granulation can facilitate contact between the API, or any substances related to amine
function, and the nitrosating agents, especially if this process step occurs in an ideal pH range
(Horne et al., 2023). Although the risks associated with water quality are generally considered to be
low, it is essential to properly control the water quality and estimate the amount of nitrite, or the
very presence of nitrosamines in the water. Depending on the conditions, the formation of
nitrosamines can become significant at lower pH values (<

The degradation of ranitidine, which occurs in the finished product and which can be influenced by
crystal morphology of the molecule, is an example of nitrosamine formation that involves API degradation and
is influenced by factors other than those classically attributed to nitrosamine formation.

Packaging has already been associated with the presence of NDMA and NDEA, due to reactions between the
amines present in printing inks and nitrocellulose present in packaging sealing sheets. During the
high temperature sealing process, nitrosamines can evaporate and contaminate other
pharmaceuticals in the package.

5. RISK MANAGEMENT

It is suggested that companies use the risk management principles described in the Guide
ICH Q9 -Quality Risk Management(ICH 2005) as support in carrying out risk analyses. In this guide, we
propose carrying out this analysis in three stages: Risk Assessment (1), Confirmatory Tests (2) and
Nitrosamine Control for Regularized Products (3).

Step 1 called Risk Assessment consists of identifying and assessing the risk of
formation and presence of nitrosamines. It is suggested that this step be initiated by analyzing the API synthesis route,
which may be performed by the API manufacturer, distributor or fractionator, holder of the API registration (holder) or even
by the drug manufacturer, if it holds the complete synthesis route of the active. The knowledge of the complete API
synthesis encompasses the knowledge of the synthesis routes of the starting materials, the control of the materials and the
knowledge of the synthesis route in detail. This information is commonly found in the restricted parts of the Active
Pharmaceutical Ingredient Dossier (DIFA). After this evaluation, the company will proceed with the evaluation of the
production process of the finished product and the potential for the formation of nitrosamines throughout the shelf life.

If a risk of the presence of nitrosamines is identified as a result of Step 1, the company

must proceed to Step 2 and carry out the confirmatory tests in order to confirm or refute the presence of
nitrosamines in the API or finished product, depending on the origin of the risk. At this stage, the company
must verify that the identified and quantified nitrosamine has an acceptable intake limit defined and published
by Anvisa as per Table 3 of this guide.

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 If the company disagrees with the limits of nitrosamines established in Table 3 (Limits of
acceptance for nitrosamines) or the nitrosamine impurity identified does not yet have a harmonized and
published acceptable intake limit, it must notify Anvisa and propose new limits based on scientific
rationale through a protocol in the system Requests to GESEF (Security Assessment Management and
Efficacy) using subject code 12194 - Nitrosamine Safety Limit Assessment – Company.

To carry out the tests, the company must use appropriate analytical procedures
sensitive for the quantification of these impurities. The absence of nitrosamines is considered when they are
below 10% of the acceptable intake limit, but other approaches may be justified, not exceeding the limit of 30%.
Once the presence of nitrosamines has been confirmed based on the tests carried out in Step 2, the company
must move on to Step 3, where the controls for nitrosamines in its products are defined.

In situations where there is identification of the presence of nitrosamines above acceptable limits,
the company must adopt the necessary risk mitigation actions and notify Anvisa (Inspection and Surveillance
Management - GGFIS) through the protocol in the Solicita system using the following subject codes: 70788 -
ACTIVE PHARMACEUTICAL INGREDIENTS - Notification for Nitrosamine Assessment above of Acceptable Limits
(manufacturers, distributors and fractionators) (company petition) and/or 70789 - MEDICINES - Notification for
Evaluation of Nitrosamines above Acceptable Limits (manufacturers and importers).

It is important to highlight that in all cases the company is responsible for guaranteeing the quality
and safety of its products with the assessment of the risk of ingestion by patients and adoption of appropriate
actions to avoid or minimize the exposure of individuals to nitrosamines, including in situations of
disagreement with the limits described in Table 3 and in the period until the limit is defined by Anvisa.

Risk control actions must include changes related to the root cause(s)
identified in the investigation, as well as assessment of the need to adopt measures that restrict the
availability of the product on the market (for example: suspension of distribution and marketing,
recall) taking into account actions adopted for markets in other countries and aspects such as risk of
market shortages and availability of therapeutic alternatives.

For known impurities above the limit stipulated in Table 3 - Acceptance Limits
described in section 8 of this Guide, companies must immediately suspend the distribution and
marketing of the drugs or APIs involved, in addition to assessing the need for collection, which must
be treated as provided in Resolution RDC nº 677/2022 and nº 625/2022 and their updates .

Finally, if the presence of nitrosamines is confirmed and, as long as it is within the limits
acceptable, the company must file with the Agency, if applicable, the respective post-registration petitions
in accordance with the regulations applicable to each case. Test additions are immediate implementation
or immediate notification and performed via subject-specific code. For petitions applicable to synthetic
and semi-synthetic drugs and APIs, the following subject codes should be used:

• 12195 - GENERIC - Inclusion of the critical control test for nitrosamines in the medication - RDC
677/2022
• 12198 GENERIC - Inclusion of the control test for nitrosamines in the API with CADIFA - RDC 677/2022
• 12201 GENERIC - Inclusion of the control test for nitrosamines in the API without CADIFA - RDC 677/2022
• 12197 NEW - Inclusion of the critical test for control of nitrosamines in the drug - RDC 677/2022 NEW -
• 12200 Inclusion of the control test for nitrosamines in the API with CADIFA - RDC 677/2022 SIMILAR -
• 12196 Inclusion of the critical test for control of nitrosamines in the medication - RDC 677/2022 SIMILAR -
• 12199 Inclusion of the control test for nitrosamines in the API with CADIFA - RDC 677/2022 SIMILAR -
• 12202 Inclusion of the nitrosamine control test in the API without CADIFA - RDC 677/2022

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 It should be noted that in addition to the inclusion of tests, other post-registration changes may apply
in order to mitigate the risk of the presence of nitrosamines. The cases below illustrate some examples of necessary
post-registration alterations according to risk assessment notes:

Example 1: After identifying and assessing the risk of a certain product X, the company
performed confirmatory tests and verified the presence of NDMA above the acceptable limit.
According to the company's risk assessment, the root cause of the presence of nitrosamine was the
reaction of nitrocellulose, a primary packaging component, with an amine present in the product's
excipients during the process of closing the blister by heating. In order to adapt the product, the
company proposes, as a post-registration change, a packaging change in order to remove
nitrocellulose as a component of the blister. For this, a protocol of a type 7.c alteration (major change
in the composition of the primary packaging) is carried out under the terms of RDC nº 73/2016,
which requires an individual protocol and must await a manifestation from Anvisa. Considering the
correct risk assessment carried out,

Example 2: After detecting and confirming the presence of more than one nitrosamine reaching
unacceptable limits, the registration holder, through its investigation, detects that the root cause involves the
API itself and probably the presence of nitrosamine from solvents that were used in the synthesis route.
Assuming that the approved API manufacturer chose to discontinue the API in question, the applicant decides
to change the API manufacturer to adapt the product. For this case, the condition of the presence or absence of
CADIFA under the terms of RDC No. 359/2020 must be considered. If the proposed manufacturer already has
CADIFA and the conditions described in amendment 1.g - Replacement of CADIFA holder (immediate
implementation) of Annex I of RDC 361/2020 are also met, this change may be implemented immediately. If
such conditions are not met or the proposed manufacturer does not have CADIFA, the change must await
approval. In this case, considering that changing the manufacturer mitigates the risk of nitrosamines that
existed due to the approved manufacturer, the previous risk assessment may justify the non-need for
additional parallel post-registrations, and it may be sufficient to change the manufacturer of the IFA.

Example 3: As per the example above, the root cause was identified as a nitrosamine
from the API manufacturing process, but in this case the company will make a change to the API process that will
eliminate this risk. For this case, the condition of the presence or absence of CADIFA under the terms of RDC No.
359/2020 and whether the change involves an impact on the impurity profile must be considered. Considering the
understanding of Questions and Answers of the RDC 73 Edition 4.2 (January 2021), that the removal of an impurity
does not fit into the impact on the impurity profile, this can be considered of immediate implementation under the
terms of RDC nº 361/2020 since that the change does not otherwise impact the impurity profile (eg generating
another impurity) or the API specifications (eg changing its crystalline form). For example, in the case of changing
DIFA without CADIFA, meeting the requirements of the standard, the change can be classified as a type 1.d change
(Change from DIFA without CADIFA (immediate implementation)). In this case, as in the previous example, there
would be no need for additional parallel post-registrations, it being sufficient to just change the IFA process.

Post-registration changes must be filed in accordance with current regulations, and containing
all relevant documentation. Thus, for example, in cases where the nitrosamine control test is included in an API
or synthetic medication, the petition will be for an individual protocol and immediate implementation, pursuant
to RDC 73/2016. In cases where there is a change in the synthetic API synthesis route to mitigate the risk of
nitrosamine formation, the change can be classified as a prior analysis (depending on the conditions in the
specific case), and the fact that it has the objective of mitigation of the risk of nitrosamine formation does not
mean that it can be implemented before analysis.

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 As for nitrosamines that do not yet have a defined acceptance limit, it is not expected that
a post-registration test inclusion change is filed until the limits are approved by ANVISA. However, it
is emphasized that the company holding the registration must adopt the necessary control
measures and carry out the test as soon as possible after identifying its need.

It is important to mention that all documentation related to the risk management of

nitrosamines must be filed at the company and at any time may be required by the Agency or verified
during Good Manufacturing Practices Certification (CBPF) inspections, investigative inspections or
registration audits.

Figure 12 presents a summary of the steps related to risk assessment and the necessary actions
arising from the identified risk.

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Figure 12- Steps related to risk assessment and necessary actions arising from the identified risk

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6. PRIORITIZATION FACTORS FOR RISK ASSESSMENT (STEP 1)
It is known that the number of drugs registered by a company can be significant
and, for this reason, it is suggested that the product evaluation sequence be established based on
prior knowledge about them. Companies may consider factors such as recommended daily intake,
duration of treatment, therapeutic indication, number of patients treated or others that they deem
relevant according to the portfolio or the situation of the product. For example, the lack of
commercialization of a certain product or condition that is approved (such as an alternative API
manufacturer that is approved, but not used by the company) may justify this assessment not being
prioritized at first. However, in cases where an approved condition has not been evaluated for the
risk of containing nitrosamines because it was not being marketed,

Table 2 presents an example of prioritization, using the criteria “Duration of treatment

versus Maximum Daily Dose”, which was proposed by the entities representing the Brazilian
Pharmaceutical Sector in response to the “Discussion Panel on the control of Nitrosamines in medicines”,
held on February 5, 2020 at the Anvisa Auditorium.

Table 2 - Prioritization by Duration of treatment X Maximum Daily Dose.

duration of treatment
Maximum Daily Dose > 1 year 1 to 12 months ≤ 1 month

> 1000mg Very high High Average

100mg to 1000mg High Average Low

<100mg Average Low Very low

To carry out the evaluation of drugs with identified risk, companies can also
use tools such as Failure Mode and Effects Analysis (FMEA) and Failure Mode, Effects and Criticality
Analysis (FMECA), as described in the ICH Guideline guideline Q9 -Quality Risk Management(ICH
2005).

a) Basic components
It is expected that manufacturers, distributors and fractionators of APIs and manufacturing companies and
drug importers to work together and carry out risk assessments using quality management
principles. In addition, the result of the work carried out must be based on scientific knowledge,
always linking the protection of the patient and observing that the level of effort, formality and detail
of the documentation is proportional to the level of risk.

In addition, the principles described in the ICH Guide M7(R1) -Assessment and Control of Reactive DNA
(Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk(ICH 2017) regarding
mutagenicity assessment, control strategies and changes in manufacturing processes of active
substances can be applied.

It is relevant to point out that if the risk of nitrosamine formation has been assessed during
the development phase of the API/finished product manufacturing processes, this information can
be used to support the assessment referred to in this guide.

Finally, at the end of Step 1, companies should have sufficient arguments to

answer the following questions:

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 • What is the risk of nitrosamine formation in the API synthetic process, taking into account the
combination of reagents, solvents, catalysts and starting materials used, intermediates formed,
impurities and degradants?
• What is the risk of nitrosamine contamination (eg from recovered materials such as solvents,
reagents and catalysts, equipment, starting materials or intermediates)?
• What is the risk of nitrosamine formation during manufacture of the finished product or
during storage throughout its shelf life (eg possible degradation or interaction with
excipients and packaging material)?

b) Purge evaluation

If the presence of potential risk of formation of nitrosamines in the process is verified

synthetic API, taking into account the reagents, solvents, catalysts, starting materials, formed
intermediates and other impurities, a detailed evaluation of the entire process of obtaining the drug
can be carried out with regard to the possibilities of elimination/purge of the(s) ) potentially present
nitrosamine(s).

In this analysis, if mastery of the process for obtaining the API, and its associated parameters,
is such that it is possible to determine that the risk of the presence of nitrosamine(s) above the maximum
permitted limit(s) is negligible, the control strategy may be based solely on process control , with no need
for analytical tests. This strategy is analogous to option 4 of the ICH M7 Guide.

For such a control strategy to be acceptable, a formal risk analysis is required,

considering the physicochemical properties of the nitrosamine(s) in question and the factors of the
process for obtaining the API that impact on the fate and elimination/purge of the nitrosamine(s),
including chemical reactivity, solubility, volatility, ionizability and processes specifically designed to
remove the nitrosamine(s) in question. It is crucial that all factors considered by the company and which
supported the elimination estimate at each stage of the process are clearly recorded and readily available
to Anvisa. This type of analysis must necessarily be conducted by qualified personnel who have
knowledge of the API obtaining process, in order to avoid erroneous purge estimates due to lack of
knowledge of the specific process or erroneous application of elimination factors and other applicable
concepts described in the literature. The outcome of the risk analysis should estimate an elimination/
purge factor for the contaminating nitrosamine(s) in question.

Contamination/elimination studies may be used as evidence of the

ability of the process to eliminate/purge the nitrosamine(s) in question. When it is not possible to
determine the absence, or the presence at negligible levels, from the risk analysis/estimated purge factor,
the control strategy must include analytical tests on the API or on isolated intermediates.

Some scientific publications are available and can be consulted to guide how much
to the best practices for calculating the purge factor, establishing necessary requirements to justify the
elimination of the impurity, including for cases involving nitrosamines, and can be used as a reference in this
step, for example: Teasdale et al. 2010, Teasdale et al. 2013, Barber et al. 2017, Burns et al. 2019, Burns et al.
2020.

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7. CONFIRMATORY TESTS (STEP 2)

a) Analytical method

As with other impurities, laboratory evaluation of the presence of nitrosamines requires

the application of sensitive and selective analytical procedures, often requiring the association of different
analytical techniques such as chromatographic separation associated with identification and quantification by
mass spectrometry, for example.

Although the Brazilian Pharmacopoeia still does not have a method for analyzing nitrosamines,
several procedures have been developed and made public including by regulatory authorities such
as the European Medicines Agency – EMA (EDQM 2020), American Food and Drug Agency
- FDA (FDA 2019) and Canadian Medicines Agency - Health Canada (Health Canada 2019).

It is also necessary to consider the foreign pharmacopoeias admitted in Brazil, according to the
RDC Resolution No. 511, of May 27, 2021 or its updates. These methods, if available, can be used by
companies as a basis for analyzing their products.

Considering the aspects related to the formation of these contaminants and the catalog of
company's products, a strategy to be considered is the company's development of a general and
comprehensive method that may be suitable both for the analysis of active inputs, excipients, and
finished products in their different stages (production, stability studies and others). However, considering
the specificity of each product and the different types of matrix, a single method for all cases may not be
possible.

It should be noted that, regardless of the strategy adopted and the adjustments made, the
method of use must meet the criteria established in current legislation, Resolution RDC No. 166, of July 24,
2017, which provides for the validation of analytical methods, or their updates, or the ICH Guide Q2(R1)
Validation of Analytical Procedures . In this sense, it is highlighted, but not restricted, the need to observe
the limits of detection or quantification and selectivity, especially when this is applicable to different
analysis matrices. It is important to highlight that the analytical validation must be planned considering
the data available for the product. With this, it can be concluded that it is possible to use partial validation,
limit testing, or even the need to perform additional tests, among other situations.

There are reports of the formation of nitrosamines even in the steps of sample preparation well
and interference in the analysis by solvents commonly used in laboratory routine. Therefore, proper
sample preparation is a critical step in the evaluation of these analytes, either due to the loss of impurities
or their generation during this step (EMA 2020, King et al. 2020).

b) Batches to be tested

The step at which nitrosamine will be tested (intermediate, API or finished product) depends on
the origin of the impurity. For example, when confirmatory testing is required for impurities from the
API manufacturing process, these can be tested in the API or in a process intermediate, if their origin is
prior to this intermediate. If the impurity is degradation, it is recommended that the tests be carried out
on samples that represent the product during its validity, for example, batches submitted to stability
studies, samples close to expiration, among others. In this case, the test is applicable both to the API and
to the finished product.
The quantification assays of the API batches made by the API manufacturer can be used
by the drug registration holder provided that there is a critical evaluation of the results, and that the
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manufacturer has been qualified according to current Good Manufacturing Practices legislation. However, this does
not exempt the need for risk assessment in the finished product, since the formation of the contaminant can occur
in several stages of the production process.

Regarding the number of batches to be tested, this must be defined by evaluating

company risk, as recommended below:
• For registered drugs, a minimum of 10% of annual batches, or 3 batches per year, whichever is
greater, must be properly sampled and tested.
O If less than 3 batches are produced in the year, all manufactured batches must be tested.
O If more than one manufacturer, manufacturing process and/or sources of risk-related
materials are used, more batches must be tested in order to cover all risk factors.

O When nitrosamine(s) are degradation impurity(s), at least 3 representative batches of

the product throughout its shelf life must be tested.

OOther technically justified approaches may be accepted.

• For new registration or post-registration petition, the number of batches tested must be consistent with the
quantity required by the current RDC.

O In addition, the number of lots required in the previous paragraph must also be
observed. For example, for petitions requesting less than three batches, the
implementation will be conditional on the company's commitment to test the
implementation batches also later, in order to complete the required 3 batches. These
data must be available for presentation to Anvisa, when requested or during inspection.

• When the drug manufacturer needs to perform the API analysis, the number of batches must be
defined by the company's risk assessment.

8. CALCULATION STRATEGY FOR ASSIGNING ACCEPTANCE LIMITS

The compoundsNo-nitrosos are carcinogenic genotoxic agents of the group called “Group
of concern” orCohort of Concern.These are more potent impurities than most other mutagenic
compounds, hence the use of the threshold based on the concept of “Threshold of Toxicological Concern”
or TTC (Threshold of Toxicological Concern)established in the ICH Guide M7(R1) of 1.5µg/day is not
applicable.

As described in the ICH Guide M7(R1), for such compounds it is expected that the acceptable intake
is significantly lower than for the other potentially mutagenic impurities, so that the establishment of
the limit should ideally be done on a case-by-case basis, using, for example, carcinogenicity data of
structurally similar compounds.

The risk assessment approaches described in this Guide apply to all avenues of
administration, and threshold corrections for different routes of administration are not applicable. Cases
in which scientific data justify particularities of a specific route of administration must be evaluated
individually.

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

a) Limits for single nitrosamine

The determination of acceptable limits or AI (Acceptable Intake) of the nitrosamines listed in this
guide was based on the specific calculation guidance for each compound set out in the ICH Guide M7(R1)
and harmonized with the limits already accepted by other regulatory authorities (EMA 2020b, EMA 2023,
FDA 2020), which, in turn, were obtained from from carcinogenicity studies in animals or rationale
supported by structural similarity. Limits for some known nitrosamines are described in Table 3.

Table 3 - Acceptance limits for nitrosamines

Nitrosamine acceptable intake
Nomenclature CAS
(acronym) (ng/day)
NDMA1 dimethylnitrosamine 62-75-9 96.0
NDEA1 diethylnitrosamine 55-18-5 26.5
EIPNAtwo ethylisopropylnitrosamine 16339-04-1 26.5
DIPNAtwo diisopropylnitrosamine 601-77-4 26.5
NMBAtwo carboxypropylmethylnitrosamine 61445-55-4 96.0
MeNPtwo methylnitrosopiperazine 16339-07-4 26.5
NDBAtwo dibutylnitrosamine 924-16-3 26.5
NMPA1 phenylmethylnitrosamine 614-00-6 34.3
NMOR3 nitrosomorpholine 59-89-2 127.0
NNV4 nitrosovarenicline - 37.0
NDPAtwo dipropylnitrosamine 621-64-7 26.5
NMPH5 methylphenidatonitrosamine 55557-03-4 1,300
- nitrosamineparoxetine5 - 1,300
NPIP1 nitrosaminapiperidine 100-75-4 1,300
NDLX6 duloxetinanitrosamine 2680527-91-5 100
- fluoxetinenitrosamine6 - 100
NTTP4 trifluoromethyltetrahydrotriazolnitrosopyrazine - 37
NTHP1 nitrosaminetetrahydropyridine 55556-92-8 37
NMPEA1 methylphenylethylnitrosamine 13256-11-6 8
NNORT7 nitrosaminanortriptyline 55855-42-0 8
NNK3 butanonapyridinylmethylnitrosamine 64091-91-4 100
NDAB8 dabigatrananitrosamine - 18
- nitrosaminarasagiline8 2470278-90-9 18
- nitrosaminatamsulosin8 - 18
NDELA3 diethanolaminenitrosamine 1116-54-7 1,900
NPYR3 nitrosaminepyrrolidine 930-55-2 1,700
NDPh9 diphenylaminenitrosamine 86-30-6 78,000
- mephenamiconitrosamine acid10 78,000
1Limit calculated from the TD50obtained by the harmonic mean of the carcinogenicity studies listed in the database
Carcinogenicity Potency Database(CPDB) available athttps://files.toxplanet.com/cpdb/
index.html

twoThreshold established based on structure-activity relationship (Q)SAR strategy with NMDA or NDEA.

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3Limit based on TD value50more sensitive derived from the TD study50most robust available in the CPDB database

4Threshold derived using SAR and approachread-across having the value of TD50of N-nitroso-1,2,3,6-tetrahydropyride as a starting
point

5Threshold derived using SAR and approachread-across having the value of TD50NPIP as a starting point

6Threshold derived using SAR and approachread-across having the value of TD50of NNK as a starting point

7Threshold derived using SAR and approachread-across having the value of TD50of NPEA as a starting point

8TTC-derived limit for nitrosamines of 18 ng/day

9Limit based on TD value50more sensitive derived from the TD study50most robust available in the CPDB database with application of the
lower confidence interval (((% CI) of the estimated TD50 (TD50L01)

10 Threshold derived using SAR and approachread-across having the value of TD50of NDPh as a starting point

These acceptable intake values apply to a finished product containing only one
nitrosamine. The limit determined for a specific product in ppm can be calculated through the ratio of the acceptable
intake (in ng) to the Maximum Daily Intake (DMD) of the product (in mg). For example, considering the maximum
daily dose of metformin of 2550 mg and the limit of 96 ng for NDMA, we have 0.038 ppm (96/2550) as an acceptable
daily limit.

This calculation can also consider the factor referring to the duration of treatment, when the
treatment lasts less than 10 years, according to the equation presented in item "Less than Lifetime Approach”,
when applicable.

The acceptable intake values predicted in this guide, especially those calculated based on
structure-activity relationship, are interim limits, considering the best evidence available at the time of
publication of this guide. These limits can be changed in case of availability of new scientific evidence for
these compounds.

The company must notify Anvisa and present technical justification in cases of definition
of limits higher than those presented in table 3, through a protocol in the Request system using subject
code 12194 - Evaluation of safety limits for nitrosamines – Company.

b) More than one nitrosamine

Considering the case of detection of more than one nitrosamine in the same product, two
alternatives can be used to define the limit of the sum of nitrosamines:

Option 1:the sum of all nitrosamines present in the product must not exceed the
acceptance limit for the most potent nitrosamine among those present.
Option 2:individual limits for each nitrosamine are adjusted to ensure that the total risk
from exposure to them does not exceed the negligible risk.

Option 2 considers a risk-based approach to accepting the presence of multiple

nitrosamines, provided that it is demonstrated that the final risk does not exceed the ratio of 1:100,000, defined in the
ICH Guide M7(R1) as an acceptable risk of cancer. Thus, if more than one nitrosamine needs to be controlled in the
specification of the API or the finished product, a limit must be established for the sum of these nitrosamines, which
must ensure that the risk remains negligible. A calculation example considering option 2 is described in Figure 14.

These approaches are only applicable if more than one nitrosamine is actually present.
and needs to be controlled in the specification of the API or finished product. If there is a theoretical possibility of
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presence of nitrosamines, but whose absence has been demonstrated and which do not need to be controlled in the
specification, the limit for total nitrosamines must not consider such impurities.

Example:

For an API that contains only NDEA, the acceptable limit of 26.5 ng/day
corresponds to a risk of 1:100,000. On the other hand, if it only contains NDMA,
the acceptable limit of 96 ng/day corresponds to the same risk of 1:100,000.

In another scenario, for an API that contains both nitrosamines, if the

original limits were maintained, the risk would correspond to the sum of both,
therefore twice the initial risk - greater than the negligible risk. Thus, in order
for the risk to be maintained at 1:100,000, the nitrosamine limits must be
reduced in proportions that ensure a negligible total final risk, as described
below:
If the limit of each one is reduced to 50% of the original value (13.25 ng/
day for NDEA and 48 ng/day for NDMA), each one will represent 50% of the
initial risk, and the sum of both will be equivalent to the negligible risk
(1:100,000). Alternatively, variable proportions between the individual limits
established for each nitrosamine can be used, for example, 30% of the NDEA
limit (7.95ng/day) and 70% of the established limit for NDMA (67.2 ng/day), as
represented in Figure 14.

Figure 13 - Example of setting limits for more than one nitrosamine

c) New nitrosamines

During the risk assessment there is still the possibility of finding other nitrosamines,
potential or real ones, in addition to those listed in Table 3. In view of the absence of specific limits, an
approach similar to that previously performed for nitrosamines with limits listed in this guide is
recommended, with the determination of a specific limit based on carcinogenicity studies, when
available . In cases where carcinogenicity studies are not available, it is recommended to derive a
threshold from structure-activity relationship comparison (Structure Activity Relationship-SAR) with known
nitrosamines or the application of a specific TTC for the class of nitrosamines.

For limit determination based on TD50carcinogenicity studies, these should

meet criteria for quality and robustness as described in the ICH Guide M7(R1), eg studies with multiple
doses (at least 3 groups) and 50 animals per dose per sex. The studies used for the purpose of
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determination of the limit will be evaluated on a case-by-case basis, even if the harmonic mean of the DT50be presented in
Carcinogenicity Potency Database (CPDB).

In the absence of robust studies that can support this limit, the SAR approach is
recommended, as long as it is used as a comparator to TD50of a nitrosamine whose threshold has been derived
from a robust carcinogenicity study with the most similar structure to the compound under test. The use of the
SAR approach must be scientifically justified and adequately documented.

Finally, as an alternative to deriving a limit based on SAR, the specific TTC for the
class of nitrosamines can be applied. Based on scientific knowledge and data available to date, the
TTC for the nitrosamine class corresponds to 18 ng/day. This value corresponds to the 5th percentile
of the TD values50for 45 nitrosamines available in the LCDB Carcinogenicity Database.Lhasa Limited
Carcinogenicity Database) (LhasaLimited 2020), whose methodology for deriving the DT50was
published by Thresher et al. (2019).

Although there are carcinogenicity data for a higher number of nitrosamines in the base
of dataCarcinogenicity Potency Database-CPDB, from which the data used for the determination of the
general TTC for genotoxic carcinogens (1.5 µg/day) were extracted, the TD values50of LCDB were
calculated by selecting only studies that met additional quality criteria. Among these criteria are the
removal of data for which there is no dose-response or the dose-response curves are not linear, exclusion
of studies with a single group and TD values50above 1,000,000 mg/kg (Thresher et al. 2019).

More recently, Thomas et al. (2021) corroborated the applicability of using the database
LCBD data considering only more robust studies and statistically more conservative 5th percentile
derivation. The authors argue that the limit of 18 ng/day allows considering a confidence limit that
estimates the uncertainties and the different potencies of the nitrosamine class.

Options for deriving thresholds for new nitrosamines are illustrated in Figure 15.

Figure 14- Decision tree for establishing limits for new nitrosamines

The company must notify Anvisa in cases of identification of a new nitrosamine (not
contemplated in table 3) and present technical justification for defining its limit through a protocol in
the Solicita system using subject code 12194 - Evaluation of safety limit for nitrosamines – Company.

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 d) ApproachLess Than Lifetime

The term "Less Than Lifetime” is loosely translated to “less than a lifetime”. That
This approach corresponds to the possibility of establishing higher limits for potentially mutagenic
impurities when the duration of treatment is shorter than a lifetime.

This means that limits vary depending on the duration of treatment, based on a concept
foundation of toxicology established by Haber's Law:

Concentration x Time = Constant

Therefore, the carcinogenic effect is based on both dose and duration of exposure. That
concept is set out in Note 6 of ICH M7 (R1) (ICH 2017). Based on this concept, TTC values were
established in ICH M7 (R1) (ICH 2017) that vary according to the duration of treatment, with limits
greater than the limits defined for a lifetime (lifetimeor over 10 years) safety factors were also
incorporated to mitigate the risk of acute effects that could occur, such as a possible saturation of
DNA repair enzymes.

The factors are presented in Table 4 and the rationale for their derivation is illustrated in Figure
16.
Table 4 - Factors for the duration of treatment in the calculation of the acceptable limit (Bercu et al, 2021).

duration of
< 1 month 1 to 12 months 1 to 10 years > 10 years
treatment
factor to be
applied in 80 13.3 6.7 1.0
limit calculation

Figure 15- Correlation between duration of exposure and acceptable daily intake for mutagenic impurities and nitrosamines.
Adapted from Guide ICH M7(R1)

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 In the case of nitrosamines, the TTC of 1.5 µg/day is not applicable due to their greater potency,
placing them in the concern group (cohort-of-concern). However, the ICH Guide M7(R1) does not state
that its concepts, such as the LTL approach, are not applied to compounds included in this group.

Considering the proposed factors described in Table 4, the acceptance limits for
nitrosamines would result in higher limits than applied for lifetime exposure listed in Table 3. A case
study for the NDEA was recently published suggesting its applicability (Bercu et al, 2021), considering
that nitrosamines have similar toxicodynamics to other impurities potentially mutagenic, despite
being more potent.

The applicability of the LTL concept for nitrosamines has been discussed internationally,
however, there is still no consensus on the safety of its use as a starting point for calculating
acceptable limits. Thus, analytical methods must be developed and validated so that their sensitivity
(quantification limit) is adequate for the concentrations presented in situations (a), (b) and (c) of item
8 of this guide and the absence of nitrosamines during the Confirmatory tests, discussed in item 5 of
this guide, should not be based on the limits obtained through the LTL approach. Given the lack of
robust scientific evidence at this time, the possibility of overriding DNA repair mechanisms is
questioned, especially considering other possible sources of nitrosamines such as polypharmacy,
dietary and environmental exposure,

Thus, this Anvisa recommends that the LTL approach not be applied as a point of
starting point for establishing the safety limit for nitrosamines. Anvisa recognizes that, when the safety
limit for a lifetime is lower than the values of a single nitrosamine or more than one present in the
product, considering the technical impossibility of adapting the product to the limit for a lifetime, the LTL
approach may have its justified use, accompanied by a technical-scientific rationale, considering, for
example, indication, dosage or risk of shortages, to be evaluated on a case-by-case basis by Anvisa. For
such cases, the company must present the information through a protocol in the Solicita system using
subject code 12194 - Evaluation of safety limit for nitrosamines – Company.

9. GLOSSARY
Risk analysis: process consisting of three components, namely (1) risk assessment, (2) risk
management and (3) risk communication.

ApproachLess than Lifetime: established assessments for cancer risk based on exposures when
these are less than lifetime (70 years).

cohort-of-concern: group of highly potent mutagenic carcinogens comprising aflatoxin-like, N-

nitroso- and alkyl-azolic compounds.

Acceptable Intake: An intake level that poses a negligible risk of cancer, or for serious/life-
threatening indications, where risk and benefit are adequately balanced.

Threshold of toxicological concern, from EnglishThreshold of Toxicological Concern – TTC: concept

developed to define an acceptable intake for any unstudied chemical that poses a negligible risk of
carcinogenicity or other toxic effects.

Acceptable limit: maximum acceptable concentration of an impurity in a drug substance or drug,

derived from the acceptable intake and daily dose of the drug.

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 Material: term used to denote raw materials (starting materials, reagents, solvents), auxiliary materials,
intermediates, active pharmaceutical ingredients, and packaging and labeling materials.

NDSRIs: From EnglishNitrosamine Drug Substance Related Impurities–refer to nitrosamines complex or

structurally related to API.

Regularized products/products: drugs and active pharmaceutical ingredients (API) registered, notified or
registered.

(Q)SAR and SAR: refers to the relationship between the molecular (sub)structure of a compound and its mutagenic
activity using (Quantitative) Structure-Activity Relationships derived from experimental data.

Negligible risk: risk corresponding to a cancer incidence of 1 in 100,000.

TD50: Chronic dose rate in mg/kg body weight/day that would cause tumors in half of the animals at the end
of a standard lifespan for the species, taking into account the frequency of this type of tumor in the control
animals.

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