USP’s

Workshop on Nitrosamines Impurities

Analysis, Industry Needs and Regulatory Perspectives

February 16, 2023

Hyderabad
 Potential sources of nitrosamines formation, contamination, and the
need for appropriate GMP controls for APIs

Presentation by:
Dr. Saji Thomas
Vice President & Head-R&D (API)
Jubilant Pharmova Limited
C-26, Sector-59, Noida-201301
[email protected]
9958080487
Contents:
a) Nitrosoamine Drug Substance related impurities
b) Recall of drugs from Market due to Nitrosoamines
c) Classification of Nitrosamines according to their carcinogenic potential
d) Toxicity potential of N-Nitrosoamines
e) Sources of Nitrosoamines - 360° Risk Assessment
f) About Nitrosoamines
g) Potential sources of nitrosamines formation, contamination
h) Control strategy for Nitrosamine formation/contamination in API
manufacturing process
i) Analytical Technologies for Nitrosoamine impurity analysis in Drug
substance and Drug products
j) Importance of Reference standard qualification
k) Nitrosoamines contamination during manufacturing.
l) Static Charge, Air Ionization.
Nitrosoamine Drug Substance related impurities

All the drugs which contain 2 amine group can react with traces of Nitrite/Nitrate (present in solvent/reagent/catalyst)
resulting in formation of NDSRI)
Recall of drugs from Market due to Nitrosoamines
Potentially
API/Product carcinogenic
Manufacturing
process
Product
Drug Detection of RECALL from
product nitrosamine the market
impurities
Degradation No of product recalls due to Nitrosamines
during storage
Metformin 256
e.g. NMDA, NDEA,
NMBA, NDSRI Ranitidine 415
Nizatidine 3
Valsartan 220
Valsartan/HCT 204
Valsartan/Amlodipine 137
Valsartan/Amlodipine/HCT 64
More than 1400 product lots have been
Losartan 324
recalled/withdrawn from the market in the past 2
Losartan/HCT 165
years due to the presence of nitrosamines above their
Irbesartan 38
daily acceptable limit. Irbesartan/HCT 7

0 50 100 150 200 250 300 350 400 450

*J. Med. Chem. 2021, 64, 2923−2936
Classification of Nitrosamines according to their carcinogenic potential

Group 3
Group 1 Group 2B
Group 2 A,
Carcinogenic to Group 1;
humans

Group 2A
Probably
Carcinogenic to
humans
Group 1

Group 2B
Possibly
Carcinogenic to
humans
Group 3
Not classifiable
as to its
carcinogenicity
to humans

*J. Med. Chem. 2021, 64, 2923−2936

Toxicity potential of N-Nitrosoamines
Tobacco The carcinogenicity of N-nitrosamines is based on the fact that they can react with DNA
base pairs after cytochrome P450-mediated metabolic activation to form unstable α-
hydroxymethyl-N-nitrosamines, finally yielding alkyl- or aryl diazonium ions as the
ultimate carcinogens

In-silico testing
Ex. Derek and Sarah

In-vitro OECD 471

AMES test

in-vivo
Rodent carcinogenicity

*J. Med. Chem. 2021, 64, 2923−2936

About Nitrosoamines
❖ Nitrosamines are N-nitroso compounds (NOCs) that contain a common
functional >N–N=O group
❖ Nitrosamine impurities are probable human carcinogens as their long-term
exposure above certain levels may increase the risk of cancer development
❖ 92% of 300+ known nitrosamines are known to show carcinogenicity, thus they Classical mechanism from secondary amines
are part of ICH M7 “Cohort of concern” - very low acceptable amounts,
requiring sensitive analytical methods.

Mechanism: Nitrosonium cation derived from

nitrosating agents attacks on a pair of free electrons on
the amine nitrogen that results in the formation of n-
Carcinogenicity of nitrosamines stems from their biotransformation
nitrosoammonium cation plus nitrite cation
via microsomal liver enzymes, primarily CYP2E1, to their respective
alkyl diazonium ions. These ions react with the DNA and form DNA
adducts, which cause endogenous DNA damage 1EDQM document, Feb 2020
Sources of Nitrosoamines - 360° Risk Assessment

KSM Reagents Excipient

Solvents
Solvents Intermediates

Water
Water and Route of Excipient
atmosphere Synthesis compatibility
DS DP
Degradation Material
recovery Degradation Cross
Contamination
Cross
Cleaning Contamination
validation Packing Lack of
Packing Lack of material control in
material control in process
process Hazard Identification

Dose response evaluation

Effective implementation of 360° risk

assessment and control strategies to Expose
consistently manufacture N-Nitrosoamines free
drug substances in pharmaceutical industry Risk Characterization
 Sources of 2° amines and Nitrite

Sources of Nitrite: Direct sodium Nitrate

Indirect sources: Inorganic bases,
Reagents, KSMs, atmosphere, water etc.
Potential sources of nitrosamines formation, contamination
Before reaction During reaction
1. Solvent & Solvent Contamination 1. Sodium nitrite (NaNO2), or other nitrosating agents in the
Underground Plant Day Tank
Solvent presence of secondary or tertiary amines :
storage Reactor
Tanker Tank in Plant
Tank Use of sodium nitrite (NaNO2), or other nitrosating agents in the
presence of secondary or tertiary amines or quaternary
Solvent flow in a pharma company ammonium salts, or in combination with reagents, solvents and
Solvent contamination can be happened during storage & solvent transfer from catalysts, which are susceptible to degradation to secondary or
one tank to another
tertiary amines.
2. Contaminated Raw material Direct source In Direct source
Raw material contaminated with Nitrosamine impurity is the source of Nitrosamine
Nitric Acid, Sodium Nitrite, Sodium hydroxide, Sodium
contamination in the product
Sodium Azide carbonate, Sodium Bicarbonate,
Potassium Carbonate, Hydroxy
3. Cross contaminations : amine hydrochloride, 4-
Nitrolphenol, Sodium chloride,
Cross-contaminations due to different process run on the same line and due to
Sodium hypochlorite, 2-
operated related error during batch charging.
Nitropropane, Nitromethane
2. Presence of quaternary amines:
Reactions of Tetrabutylammonium bromide in addition to secondary
4. Contaminated water : and tertiary amines (e.g. use of Tetrabutylammonium bromide
Nitrosamines in treated water as a result of the use of chloramine (or chlorine
(TBAB) which could give rise to N-nitrosodibutylamine (NDBA)
which can form chloramines with any amines present) and further reaction to
nitrosamines. Other oxidants (e.g. ozone) can lead to NOx formation which could although the lack of an available Electron lone pair makes this an
then react with amines to Generate Nitrosamines. unlikely mechanism, nevertheless, NDBA has been found in some
instances.
Potential sources of nitrosamines formation, contamination

After reaction
Unit operations & Possibilities for formation/contamination of Nitrosamines wise

❖ Layer separation : There is a risk of nitrosamine formation when a quenching step is performed directly in the main reaction
mixture (i.e., when nitrous acid is added to reaction mixture to decompose residual azide).
❖ Distillation : Possible reactions of volatile low molecular weight amines occurring in the manufacturing
process (solvents, raw materials, in combination with nitrosating agent). Their volatility means they could potentially carry
over during distillation processes
❖ Charcoalization : NDMA can form form DMA in the presence of Active carbon particles (Reference:
Unexpected Role of Activated Carbon in Promoting Transformation of Secondary Amines to N-Nitrosamines
LOKESH PADHYE, PEI WANG,TANJU KARANFIL,AND CHING-HUA HUANG).
❖ Drying :
In FBD, product directly contacts with Hot air. If the Air is contaminated with
NOx, there is a possibility to form Nitrosamine impurity.
❖ Muti Milling, Air jet milling, Sifting :In clean room, product directly contacts with air during Milling, sifting etc.
If the clean room air contaminated with NOx, there is a possibility to form Nitrosamine impurity.
❖ Contaminations from Blister packing materials:
Formation of N-nitrosamines can be caused by reaction of nitrocellulose in the lidding foil with amine containing printing ink
[dimethylamine (DMA) and diethylamine (DEA)] and transferred to the finished product during heat-sealing blistering process
via vaporization and condensation on the finished product
Control strategy for Nitrosamine formation/contamination in API
manufacturing process
Contamination Formation
Control Strategies / Carryover
1. Control of secondary amine & nitrates in specification & periodically cleaning X 
1. Solvent & Solvent contamination of storage tank.

2. Control of secondary amine, nitrates & Nitrosamine in the Raw material X X

2. Contaminated Raw Material specification
Before
reaction 3. As per GMP, two different product can’t be charged at same time in same area.
3. Cross Contamination X X
It can eliminate cross

4. Contaminated water 4. Removed during water treatment by UV irradiation X X

1. Presence of Sodium Nitrite, or Nitrosating

During agents in the reaction 1. Optimized quenching (quenching process outside of main reaction mixture) X 
reaction process can eliminate the Sodium Nitrite, Nitrosating agents.
2. Presence of quaternary amines

1. Layer Separation 1. Accurately layer separation can eliminate the Sodium Nitrite, Nitrosating
X X
agent from process.
2. Distillation
2. Batch to batch water reflux in distillation column can be eliminate batch to X 
batch nitrosamine contamination
3. Charcoalization
After reaction 3. By using Neutral Activated carbon X X

4. Drying & Milling 4. Use of carbon molecular filters during FBD drying: Basic filter (potassium
carbonate impregnated carbon) for control of NOx through air and acidic filter X X
for control of amines.
5. Contamination from Blister packing
material 5. Elimination of Nitrocellulose as packing material X X
Control strategy for Nitrosamine formation/contamination in API
manufacturing process
❖ Supply chain: Regular audit and monitor sourcing activities of raw materials, starting materials, intermediates and solvents.
Complete details of manufacturer, repackers, distributors and transporters should be maintained. Recommended to
establish control strategies to prevent Nitrosoamine contamination.
❖ Recovered solvents: To avoid cross-contamination it is recommended to use recovered material such as solvents, reagents,
and catalysts should be used only in the same step or in an earlier step. Appropriate control strategies for Nitrosoamines
should be in place.
❖ Reaction conditions: Avoiding reaction conditions that may produce nitrosamines whenever possible, demonstrating that
the process is adequately controlled and is capable of consistently reducing nitrosamine impurities through appropriate and
robust fate and purge studies.
❖ Using bases other than secondary, tertiary, or quaternary amines (when possible) if ROS conditions may form
nitrosamines.
❖ Solvents: Using caution when the ROS involves the use of amide solvents (e.g. N,N-dimethylformamide, N,N-
dimethylacetamide, and N-methylpyrrolidone).
❖ Replacing nitrites with other quenching agents for azide decomposition processes.
❖ Quenching steps: Removing quenching steps (when there is a risk of nitrosamine formation, e.g., using nitrous acid to
decompose residual azide) from the main reaction mixture to reduce the risk of nitrosamine formation.
❖ Environmental contamination: Water used in API manufacturing is a major source of nitrite and nitrosoamine and
therefore, there should be an appropriate control strategy and purification process to use nitrosamine free water to be used
in the process.

Ref: Date of publication: September 01, 2020

Control of Nitrosamine Impurities in Human Drugs
Recommendations for API manufacturers
For drug products with MDD of <880 mg/day;
total NSA of 0.03 ppm is NMT 26.5 ng/day.
For drug products with MDD of >880 mg/day;
Removal of Recovered solvents total NSA should be adjusted as it should not
quenching steps exceed 26.5 ng/day.
It is recommended to
use recovered material

1. Regular audit and monitor sourcing activities

Environmental 2. Complete details of manufacturer, repackers, distributors and
contamination Reaction conditions transporters should be maintained
Water used in API Avoiding reaction
manufacturing is a Reprocessing conditions that may
major source of nitrite API batches can be produce nitrosamines
and nitrosoamine
reprocessed to Nitrosoamine formation
control the • Amide solvents are susceptible to degradation
nitrosoamines
which will be another source of secondary
▪ If the risk assessment determines that there is no amines.
potential for nitrosamine impurities, there is no need to • Recovered materials may pose a risk of
take further action. nitrosamine impurities due to the presence of
Methodology/QL
▪ Manufacturers should conclude a risk assessment of
Preferred QL at or below 0.03 ppm for MDD upto residual amines.
approved or marketed products within 6 months of
<1.2g.
publication of this guidance. • A risk of nitrosamine formation when a
Preferred QL 0.02 ppm for MDD >1.2 g.
▪ Starting materials or outsourced intermediates may be at quenching step is performed directly in the main
risk through cross-contamination. reaction mixture
▪ FDA recommends that applicants conduct a conduct • Inadequate and unvalidated cleaning procedures
confirmatory testing as needed prior to submission of an can also lead to cross-contamination.
original application.
▪ o-xylene and toluene were contaminated during recovery
due to inadequate cleaning and to use of shared storage
equipment between different customers
CONTROL STRATEGY FOR NITROSAMINES

➢ Avoiding use of nitrites or other nitrosating agents along with secondary or tertiary amine or quaternary
ammonium salts
➢ Appropriate process controls for recovered solvents along with spike and purge data.
➢ Proper storage of raw materials, intermediates and reagents to avoid contamination
➢ Equipment should be properly cleaned and checked for contamination
➢ Checking the carryover of Nitrosamine impurities in various intermediate stages
➢ Modify the process to purge out amines, nitrites and Nitrosamine impurities at various stages
➢ Control strategies should be implemented to detect and control Nitrosamine impurities
➢ Use of antioxidants in formulations to minimize chances of nitrosamines formation
➢ Adjusting solid oral dosage form formulations to more neutral pHs

15
Confidential
Analytical Technologies for Nitrosoamine impurity analysis in Drug
substance and Drug products

GC/MS Based Screening and Targeted LC/MS Based Screening and

Quantification Targeted Quantification

GC ‹1469› Nitrosoamine impurities Procedure, Sample Concentration and Limit of Quantification

Chromatography Column Ionization Acquisition Mode Use of Quantitation Impurities Sample LOQ (solution LOQ w.r.t
technique and packing/ internal concentra concentration sample
detection phase standard* tion concentration
Procedure-1 LC -HRMS L-43 Electrospray Multiple and single No Single point NMDA, NDEA, NEIPA, 20 0.001 µg/mL 0.05 µg/g
ion monitoring calibration NDIPA, NMBA, NDBA mg/mL
Procedure-2 GC-HS MS/MS G-16 Electron impact Multiple reaction Yes Single point NDMA, NDEA, NEIPA, 100 0.002 µg/mL 0.02 µg/g
Triple quad monitoring calibration NDIPA mg/mL
Procedure-3 LC-MS/MS L1 Atmospheric Multiple Reaction Yes Calibration NMDA, NDEA, NEIPA, 66.67 0.00066 (NDEA) 0.01 ((other
Triple quad pressure monitoring curve NDIPA, NMBA, NDBA mg/mL 0.0013 (other impurities
chemical ionization impurities µg/mL µg/g
Procedure-4 GC-MS/MS G-16 Electron impact Multiple reaction Yes Calibration NDMA, NDEA, NEIPA, 100 0.0005 0.005
Triple quad monitoring curve NDIPA NDBA mg/mL µg/mL µg/g

16
UV absorbance of the Nitrosoamines

All the Nitrosoamines are UV active, shows absorbance between 230nm-250nm and can be detected on HPLC using UV detector.
Importance of Reference standard qualification
NMR Structure Elucidation Data :
Based on the literature review, the Synthesized molecule’s chemical
general experimental conditions for shifts are quite similar to Irbesartan
except at C-28 which is at 151.98
the preparation of N-Nitroso and a quaternary carbon while in
Irbesartan were employed Irbesartan it is a methylene carbon,
this suggests this carbon modified.
Reaction Conditions employed In the 1HNMR methylene proton
at C-28 is missing and one =N-OH
✓ Solvents/Volume: DMF, DCM,
proton is observed at δ12.01 which
water, DMSO, acetic acid, supported the structure of
methanol, dil. HCl/(2-12 V) synthesized compound is N-oxime
✓ Sodium nitrite: Mole equivalents and not Nitroso Irbesartan
varied from 1.5 to 5.0 w.r.t Conclusion: When Irbesartan was treated with
Irbesartan. sodium nitrite under acidic conditions, it lead to
✓ Acids: Organic, inorganic and formation of Irbesartan Oxime exclusively as
Lewis Acids were employed. The MS/MS spectra of synthesize compound shown in the reaction. And its mechanism is also
✓ Reaction temp/Time: 00C to reflux
displayed the protonated m/z 458.2299 depicted.:
temperature of the solvent/10 min -
24 h (elemental analysis C25H28N7O2+ which is
consistent with N-Nitroso-Irbesartan

Reaction mechanism of synthesis of Irbesartan Oxime

The MS/MS spectra shows common fragment which are originating from biphenyl
tetrazole moiety, which is indicative of the presence of an unmodified tetrazole
moiety. In order to determine the exact structure its NMR structure elucidation was
done.
Nitrosoamines formation during manufacturing

Typical API Production facility Chemical Area Pharma Area

Secondary H.E. Vent Line

storage Tank for workup

Micron filter
CW out CHW out

Solvent from Solvent

Solvent from Solvent
Liquid Raw material

Solid Raw material

from addition tank

Manhole/ Hopper
CHW In

charge through
storage Tank Primary H.E. CW In SS Reactor
Cap : 3 KL
Reflux line To Distil solvent
Collection Tank

Charcoal charging
through Hopper 4. Crystallization

Steam Inlet SS Reactor 5. Filtration

Hot water outlet SS Reactor Cap : 5 KL
Cooling water outlet Cap : 3 KL SS Reactor
Cap : 3 KL
SSCF, 48”
Chilling water outlet
Brine outlet
Hot water outlet Hot Air Outlet
Cooling water Inlet
Steam condensate Chilling water Inlet
Brine Inlet
Hot Air inlet
Unit operations 2. Quenching, Workup, (through Pre-filter followed b
/process 1. Reaction Distillation, Dissolution 3. Charcoalization HEPA filter)
6. Dryer FBD
Possible way of Nitrosamine contamination during batch manufacturing in a API manufacturing plant
A) Sources of Secondary amines as Raw material. F) Nitrosoamine source in ambient air
B) Raw material containing Nitrosamines. Dimethyamine and NO2 reacts &
C) Recovered solvents due to presence of residual amines or nitrites or Nitrosamines. forms NDMA impurity in presence of
D) During Quenching process, when quenching step is directly performed in the main reaction mass. moisture in air.
E) Lack of Process Optimization and Control.
 Types of dryer in Clean room
Utility Utility
1) Hot water
1. Vacuum Tray Dryer 2) Cool water 5. NAUTA Dryer 1) Steam
(VTD) (Indirect contact with product) (Conical Screw Dryer) 2) Hot water
3) Cool water
(Indirect contact with product)

1) Hot Air
2. Fluidized Bed Dryer 6. Rotary Vacuum 1) Steam
(Direct contact with product) 2) Hot water
(FBD) Paddle Dryer (RVPD) 3) Cool water
(Indirect contact with product)

1) Steam 1) Hot Air/ Hot Nitrogen

3. Rotocone Vacuum 2) Hot water 7. Spray Dryer (Direct contact with product)
3) Cooling water
Dryer (RVD)
(Indirect contact with product)

1) Steam
2) Hot water 8. Freeze Dryer 1) Chilled Oil
4. Agitated Nutsche 3) Cooling water (Indirect contact with product)
4) Chilling water
Filter & Dryer (ANFD) 5) Brine
(Indirect contact with product)
 Equipment flow in Powder Processing (PP) Area

Air contact of Product Air contact in

Air contact of Product
during charging head space of blended
during charging

12 kg/cm2
Material Compress Air
from Dryer
Contact time between High
pressure compressed Air
and Product is ~ 4 min.

Air contact of sifted material Air contact of sifted material

loading into drum loading into drum
Vibro sifter machine Milling machine Blending machine Micronization machine (Air jet mill)

Contact of clean room air with product Contact of compressed air with product
❑ Before air jet milling, the nitrosamine impurity was 10 times below the accepted limit. After Air Jet Milling, there was a formation of Nitroso Hydrochlorothiazide upto 3 times
higher than accepted limit.

❑ Static charge plays a role in this impurity formation.

❑ The formation of NNitrosamine in the Thiazidine moiety is due to the reaction between this and Nitrous
NO+
Oxide of the compressed air. R2-NH R2-N-N=O
Nitrosamine formation
❑ This reaction can be considered as an electrophilic attack of the N atom from the Nitrous Oxide on the N
atom of the Thiazidine molecule. Such a reaction can be considered as a Charge-Charge interaction between
the two. (Referance : Brochure of CIR-Q-TechTAKO Technologies, )
Static Charge, Air Ionization, Control strategy for Nitrosamine
formation/contamination in API manufacturing process
Static Charge & Air Ionization

Ions are formed in the Earth’s atmosphere under the action of short-length radiation of a Sun corona. Negative ions may result from attachment
of electrons to oxygen molecules. The rate of ion formation is larger at higher altitudes because both the depth of penetration of X-ray radiation
inside the Earth’s atmosphere and the intensity of X-ray radiation decrease with a decreasing wavelength

❑ Molecular ions are formed in the troposphere as a result of absorption of X-ray radiation X-ray radiation
that is emitted by the solar corona creating an atmospheric current in atmospheric dry
regions that leads to Earth discharging.
❑ The scheme of elementary processes for formation and decay of molecular ions in the Ion formation
troposphere. Decay of molecular ions in a dry atmosphere results from recombination of
positive and negative molecular ions.
❑ Air is a mixture of gases including nitrogen, oxygen, carbon dioxide, water vapor, and other
trace gases, any one or more of which can be ionized. When any one or more of these gas Attachment Recombination
molecules gains or loses an electron, it becomes charged and thus called air ions. to aerosols

Loss of Electrons e
Static charge generation Ion generation (+)
Friction Separation CATION

Neutral
+ + + + + ++ + ++ + + + + + + + Atom

- - - - - - - - - - - - - - - -- (-)
ANION

Gain of Electrons e
Product Contamination due to Static charge
AIR IONIZATION :
Gowning 1. An ion is an atom that is
Entry Particulate electrically charged.
Transfer
2. It is produced when there
is a change in the electrical
balance of a neutral atom.
Gown/Smocks
3. When high voltage is
Contaminated Cleanroom applied on the tip of the ion
Entry emitter, corona discharge is
observed which breaks the
air molecules into + and –
ions.
Charged
Product 4. These ions can be used to
Particulate
safely neutralize the static
Attraction Without Ionization
charge.
Particle Attraction With Ionization
Particle Falls to ground

PRODUCT Photograph of a charged Photograph of an

CONTAMINATION catheter after exposure uncharged catheter
near a cleanroom surface exposed near a cleanroom
with particulate surface for five minutes.
contamination.
Reference : Air Ionization: Theory and Practice for Life Science Manufacturing © 2020 Simco-Ion
Existing static charge neutralization Vs Air Ionization

Human Body Static Dissipater Air Ionization

Limitation of Human Body & Equipment static charge dissipater
❑ Powder itself continues to retain the charge since the power is an insulator.

❑ Conventional practices of inserting a grounding rod into the

powder cannot help as the insulative powder cannot conduct charge into the
grounding rod.

❑ If earthing bonding line not fitted properly, then charge will not be dissipated,
Earthing of Equipments it will accumulated and explosion can be occurred.

Benefits of Air Ionization

❑ Neutralization of Clean room air by Air Ionization.

❑ Charged powder/ Particle will particle will be neutralize immediately after

contact of Ionized air.

❑ No required for eathing bonding except equipment.

Use of Air Ionization
1. Prevent batch rejections occurring due to black particles.
2. Ionized air can be a non-hazardous , economical replacement for nitrogen. It is cheaper than nitrogen in the long term & there are no dangers of ionized air leakage as per OSHA
standards.
3. Prevent powder from forming layers/sheets within process equipment, thus greatly simplifying cleaning during batch changeover.
4. Preventing formation of powder layer/sheets also prevents chances of cross contamination as powder no long sticks in hard to each places, when using ionized air.
5. Achieve more consistent particle size with less iterations & assure QC to get consistent readings across batches.
6. Prevent powder agglomeration which is occurring due to static charge , during storage.
7. Prevent plasticizers from entering the API , which are occurring due to a combination of electro static discharge & sputtering effect.
8. Ionization is also useful in preventing contamination from nitrosamines.
Typical Manufacturing process of Nitrosamines free API

A + B + C + D =
API

Nitrosamines
impurity
free API
Thank you
 All are personal thoughts based on experience.

Potential sources of Nitrosamine formation and

challenges associated with “Testing of” drug products
Tushar N. Mehta, Ph. D.
(VP, Amneal Pharmaceuticals)
 What are we going to talk?

Paradigm Shift?
Potential Sources
How do they form?
Why it is Challenging?
Recent Trends? Indicative!
Risk Assessments? Enough!
VUCA Why?
Challenges
Way to Go!
Paradigm Shift
 Potential Sources

Formation during API Processing Carry over from API

Use of Sodium Nitrite and 2/3 Secondary amine with Nitrate/Nitrite

Amine (from excipients) Small & NDSRI

Recycled Solvents, catalyst, Water, Third party recycled Solvents,

Reagents Cross contamination

Third party recycled material Packaging Components

Contaminated Starting Materials False Positives, Degradation

 How are they form? Nitrite itself is not a nitrosating agent and
requires the presence of an acid to become
a nitrosating agent (H2NO2 +, N2O3 , or
other NOx species

• pH and temperature
• Concentrations of nitrosable substance
and nitrosating agents
• pKa of the secondary amine
• Water or moisture in solid products
Reaction with amine • Time (shelf life or process hold time?)

Not all amines are equally vulnerable – Secondary Very strong pH dependence Fastest
>> Tertiary; low pKa > high pKa; primary amines Nitrosation at pH < 3.15 Concentration
not a risk (exceptions! Yet to conclude) dependence
 Why it’s Challenging?
Multiple
Projects

Multiple Projects

Limits?

Number of NAs Toxicity

Protocols

Unknown Ever Changing

Science MA’s Regulations
Recent Recalls/History : What does it indicate?

Regulatory Ranitidine FDA issued Risk

Nitrosamines
requests ,Metformin, the assessment
in Sartans,
from over Varied Losartan guidance completion
Ranitidine
40 Requirements products and
and
countries were recall testing
Pioglitazone

2018 Sep Oct 2019 Nov Dec 2020 2021-22

Year wise segregation numbers of
company
30
0
25
0
20

15
28
10 23
5 0 4
5 3 4
0 1
2018 2019 2020 2021 2022
COC NDSRI
Risk assessment-is it Enough?
Other
Excipient API
parameter
source of source of
Contamination NITROSAMINE
Nitrate & Nitrosable & + IMPURITY/s
Nitrosating
Nitrite agents
Source of
impurity
 Challenges : Let’s Be Aware

Development
• GC- MS/MS & LC
Small Molecule Nitrosamine CoC
MS/MS HRMS
Various References available (General)
GC MS/GC- MS/MS – Suitable with exceptions

Validation
• What Needs to
be Included

NDSRI CT • Samples Matrix

Emerging Science (Challenging)
LC -MS/MS LC/HRMS – Suitable (Limitations)
Limits?
Toxicity?
Controls? Possible? Formulation Changes? BIO? Cost? Viability?
Challenges : Let’s Be Aware
Challenges : Let’s Be Aware

Limits?
 Far Way to Go!

Post approval challenges

Analytical Regulatory Formulation Supply Disruptions
Procurement and right Limits for nitrosamines Designing of formulations
characterization of the specially for complex with less nitrosamines
standards nitrosamines Use of scavengers
Method development Discussion with
challenges FDA/Communications
Limits?

Designs
Protocols
Acceptability
Duration
Challenges associated with Nitrosamines/NDSRIs analysis in API

USP-IPA workshop – 16th Feb 2023

Hyderabad, India

Dr. BM Rao, Ph.D.,

Partner & C.E.O. Qdot Associates
 Disclaimer

The views expressed in this presentation are the

speaker’s personal views and do not represent the views

of their current and past employers.

. 2
 Sources of Nitrosamine Impurities
Potential Source Examples of Identified Risk

Water • Poor quality water or solvents

• Presence of acid, nitrites or other nitrosating agents
• Use of sanitized water (e.g., chloramines)
Solvents • Use or Presence of residual of di- or tri-alkyl amines and amides (e.g., dimethylformamide [DMF], dimethylamine
[DMA], triethylamine [TEA], N-methylpyrrolidone [NMP]) in the presence of nitrites and acid media.
• Limited controls for recycled solvents : Presence of nitrites or other nitrosating agents, Presence of acid.
Degradation of solvents like DMF during its recovery

Raw Material • Use of contaminated raw materials or starting materials

• Raw material related impurities
Manufacturing • Use of sodium nitrite for azide quenching in the synthesis in acid media
process • Need of additional purification steps ? (e.g., crystallization)
Excipients • Presence of nitrites or other nitrosating agents
Degradation • Degradation drug substances or residual raw materials, impurities intermediates in manufacturing processes in
Stability presence of other sample matrix.
Container Closer • Thermal decomposition of nitrocellulose to produce nitrites followed by migration to the drug product
• Biodegradation of nitrocellulose to produce nitrites followed by migration to the drug product
Cross • Different processes run of same equipment at manufacturing sites, transportation or Recovery solvents
Contamination
. 3
 Nitrosamine Drug substance-related impurities (NDSRIs)
o Residual Nitrites from various sources in the manufacturing process react with secondary or tertiary amines functional
groups in drug substance or residual raw-materials intermediate and degradation impurities in the processes and form
corresponding nitrosamines.
o Example : Degradation of Sertraline and Duloxetine to corresponding nitrosamine

Sertraline

Duloxetine

. 4
 Analytical Procedures : USP < 1469 >
Procedure 1 Procedure 2 Procedure 3 Procedure 4
No. of Nitrosamine 7 4 6 6
Impurity names NDMA, NDEA, NDMA, NDEA, NDIPA, NDMA, NDEA, NDIPA, NDMA, NDEA, NDIPA,
NDIPA, NEIPA, and NEIPA NEIPA, NMBA, and NEIPA, NMPA, and
NMBA, NMPA, and NDBA NDBA
NDBA
Sample preparation Extraction by Suspension in Extraction by water Extraction by
Methanol Acetonitrile with 1% formic acid methylene chloride
Separation LC GC LC GC
Sample introduction Direct Injection Headspace Direct Injection Direct injection
Ionization ESI EI APCI EI
Detector HRMS MS (QQQ) MS/MS (QQQ) MS/MS (QQQ)
MS scan mode SIM, PRM (MS) MRM MRM MRM
Use of isotope-labeled No Yes Yes Yes
internal standard
Quantitation Single standard Single standard Calibration curve Calibration curve
. 5
 Analytical Procedures : IPC
In addition to methods published in USP <1469>, Indian Pharmacopeia Commission (IPC) has published additional
two methods as below :
Procedure 5 Procedure 6
No. of Nitrosamine 1 1

Impurity name NDMA in Ranitidine NDMA in Metformin

Sample preparation Extraction by Methanol Extraction by Methanol

Separation LC LC
Sample introduction Injection Injection
Ionization EIC EIC
Detector HRMS HRMS
MS scan mode PRM (MS) PRM (MS)
Use of isotope-labeled internal standard No No

Quantitation Single standard Single standard

. 6
 Analytical methods published (available in public domain)

Method details Published

date
Combined Direct Injection N-Nitrosodimethylamine (NDMA), N-Nitrosodiethylamine (NDEA), N- 04-19-2019
Nitrosoethylisopropylamine (NEIPA), N-Nitrosodiisopropylamine (NDIPA), and N-Nitrosodibutylamine
(NDBA) Impurity Assay by GC-MS/MS .
GC/MS Headspace Method for Detection of NDMA in Valsartan Drug Substance and Drug Products 01-25-2019
Combined Headspace N-Nitrosodimethylamine (NDMA), N-Nitrosodiethylamine (NDEA), N- 04-29-2019
Nitrosoethylisopropylamine (NEIPA), and N-Nitrosodiisopropylamine (NDIPA) Impurity Assay by GC-MS/MS
Combined Direct Injection N-Nitrosodimethylamine (NDMA) and N-Nitrosodiethylamine (NDEA) Impurity 12-11-2018
Assay by GC/MS
Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Method for the Determination of NDMA 10-17-2019
in Ranitidine Drug Substance and Solid Dosage Drug Product.
Liquid Chromatography-Electrospray Ionization-High Resolution Mass Spectrometry (LC-ESI-HRMS) Method 06-03-2020
for the Determination of Nitrosamine Impurities in Metformin Drug Substance and Drug Product
Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS) Method for the Determination of 02-04-2020
NDMA in Metformin Drug Substance and Drug Product
Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS) Method for the Determination of 05-21-2019
Six Nitrosamine Impurities in ARB Drugs
. 7
 Regulatory requirements – Analytical methods

o The analytical methods need to be sufficiently sensitive in order to adequately detect and quantify trace levels of
nitrosamine impurities.
o The following principles apply:
▪ The limit of quantification (LoQ) provides the minimum level at which an analyte can be quantified with acceptable
accuracy and precision and should thus be used for impurity testing and decision making.
▪ LOQ of Method should be less than 0.03 ppm (as per FDA) for specified nitrosamine impurities or 10% of
specification limit (as per EMA) which ever is lower.
▪ Method sensitivity criteria: LOQ should be < 10% of the limit based on AI, If not achieved, LOQ should be < 30% of
the limit based on AI
▪ Exceptions are anticipated for medicinal products used at high daily doses (AI may be below technical feasibility of
the method), or in case more than one nitrosamine is anticipated or identified in each medicinal product.
o Different analytical methods may be used for determination of multiple nitrosamines. If the same analytical method is
used for multiple nitrosamines, the selectivity of the method should be demonstrated for each nitrosamine.

. 8
 Analytical Method : Technical considerations

Technical considerations during analytical method development of Nitrosamine impurities:

o Interference caused by presence of trace amounts of nitrosamines in testing materials utilized : Ex: Water, airborne

sources, plastics products and rubber/elastomeric products)

o Contamination during sample preparation (avoiding cross contaminations from gloves, membranes, solvents etc.) which

could lead to false positive results.

o In situ formation of nitrosamines during analysis.

o Use of accurate mass techniques are required (MS/MS or high-resolution accurate mass systems (HRMS)) in order to

overcome interference in the identification of the specific peak of a certain nitrosamine (Ex: false positives have been

observed from DMF co-eluting with NDMA)

. 9
 Method Development Challenges and suggested solutions for Nitrosamine impurities

Challenges Suggested solutions

▪ Focus on highly selective specific instrument like LC-MS/MS (QqQ), LC-HRMS, GC-MS, GC-
Need highly sensitive methods Triple quadrupole (QqQ).
▪ Short column for achieving good S/N for impurity

▪ Use of Headspace techniques

Matrix interference & separation ▪ Use of UHPLC columns
of other impurities ▪ Solid phase extraction and syringe filters to avoid sample matrix interference
▪ In case of matrix interference use HRMS

Solubility variation between ▪ More focus on GC- Headspace techniques

analytes and impurities ▪ Optimization of sample preparation techniques

Reproducibility of detector at ▪ Anticipating feature needs method sensitive should be set high during development stage.
quantitation limits & Method ▪ Instrument maintenance i.e. regular source cleaning and use of valco to avoid detector
transfer challenges contamination is paramount importance.

▪ Most of impurities (NDSRIs) exists as E/Z isomers. Need to Optimize of concentration of

Peak shape of the Nitrosamine organic modifier.
varies ▪ Screening various column chemistries for improved peak shape and separation between
analyte and product peak.

. 10
 Method Development Challenges and suggested solutions for Nitrosamine impurities
Challenges Suggested solutions
➢ Dedicated place for standard and sample preparation.
Environment contamination
➢ Avoid exposure of impurity standard in instrumentation lab and with samples and
Absorption of impurities on
diluent.
exposure
➢ Adequate glassware’s cleaning is important aspect.
LOQ recovery in drug product is a ➢ Use of extraction technique for sample preparation.
challenge ➢ Adequate S/N ratio for impurity
➢ For long sustainability and early phase method harmonization is paramount important.
Vast product range & Regular
➢ Analyzing different label claim drug products by using single method/same standard
monitoring at QC
concentration
Single product
➢ Where possible method should be impurity specific. Focus on use of same method for
Multiple methods, Release time &
multiple product.
Cost-effective approach
➢ Working standard evaluation of impurity standard is by different technique is upmost
important.
➢ Interference from sample matrix or other impurities having similar structure likely cause
NDSRIs false positive result.
➢ Lack of information on stability data
➢ Poor resolution between API and Nitrosamine impurities due to structural similarity.

. 11
Method Development key challenges - NDSRIs

Challenge 1 : Sensitivity Challenge 2 : Stability of solution

a) ESI (+) Polarity preferred mode for NDSRIs a) Impurity response was increased on storage
b) Inclusion of additives i.e. Ammonium acetate, b) Hydrolytic and pH dependent degradation
Ammonium format etc. to improve the sensitivity
c) Diluent (basified as per need)
c) Optimizing ratio of Organic modifier
d) Auto sampler temperature
(combination of acetonitrile and methanol..)

Challenge 3 : Recovery Challenge 4: Carry Over

a) Not meeting at lower level (LOQ and/or a) Mostly associated with column stationary phase,
Specification) shall be minimized by optimizing the LC Gradient
b) Higher recovery due to stability concern
c) Interfering of other peaks

12
.
Case study-1 : NDBA content in API by GC-MS

o Problem Statement : Observed %RSD failure during GC-MS analysis.

• During execution of NDBA by GC-MS in one of the API Product, observed standard peak area increasing significantly

form injection to injection.

• The diluent used is a combination of dichloromethane (DCM) and methanol.

• After multiple experiments, optimized needle wash diluent to only methanol.

• After diluent optimization, the obtained %RSD results were well within the acceptable range.

• The DCM present in diluent hampering the free movement of needle during the needle wash leads to area variability

from injection to injection, which was resolved by use of only methanol.

. 13
 Case Study-2
o N-Nitroso Dabigatran Etexilate Mesylate impurity in Drug Substance:

Area Observed in API control Samples 46225.9

Area Count Observed for Standard Samples Area Count Observed for Spiked API Samples
(0.011 ng/mL) (0.113 ng/mL) (0.503 ng/mL) (0.011 ng/mL) (0.113 ng/mL) (0.503 ng/mL)
(0.002 PPM) (0.023 PPM) (0.101 PPM) (0.002 PPM) (0.023 PPM) (0.101 PPM)
Average 2358.5 21453.5 95979 46589.0 66872.5 139894.2
STDEV (%) 83.08 219.48 NAV 484.85 1042.46 2451.67
% RSD 3.52 1.02 NAV 1.04 1.56 1.75
Area observed in API control + Standard samples 48584.4 67679.4 142204.9
% Recovery 95.89 98.81 98.38
. 14
 Case Study- 2 (continue…)
o N-Nitroso Dabigatran Etexilate Mesylate impurity in Drug Substance:

• LC-MS/MS Method (QTRAP 4500) Flow Gradient

‒ Column: Time Flow A.Conc B.Conc B.Curve
• Phenomenex Luna Omega PS C18, 3µ 0 0.600 90 10 0
‒ Mobile Phase: 3 0.600 90 10 0
• Pump A: 6 0.600 90 10 0
‒ Acidified Ammonium trifluoroacetate acetate
• Pump B: 18 0.600 30 70 0
‒ 50:50 Methanol : Acetonitrile
20 0.600 30 70 0
‒ Flow Rate: 0.600 mL/min
21 0.600 5 95 0
‒ Injection Volume: 10 µL
24 0.600 5 95 0
‒ Needle Wash:
24.1 0.600 30 70 0
• 80:20, Methanol: Water with 0.1% Formic Acid
26 0.600 30 70 0
Challenges and suggested solutions: 26.1 0.600 90 10 0

1) Method recovery is on higher side 29 0.600 90 10 0

2) Mesylate salt gives acidic environment in the diluent and leading to

formation of NDSRI impurity Q1 Mass Q3 Mass Dwell Time DP EP CE CXP
(Da) (Da) (msec)
3) Blank Interference
657.2 364.2 200 85 10 26 20
Solution: Hydrolytic degradation of API taking place leading to failure in
recovery as well as pH dependent stability improved (basic diluent pH 657.2 433.2 100 85 10 34 20
supported in optimum stability along with removal of water from
657.2 627.1 100 85 10 19 20
diluent/reconstitution solution). Blank Interference (Carry Over). Resolved by
optimizing the Gradient conditions.

. 15
 Case Study-3
Time A% B%
o N-Nitroso Propranolol in Drug Substance (min)
• Method 0.00 98 2
3.00 95 5
‒ Column:
6.00 90 10
• Phenomenex Biphenyl; 150 X 3 mm; 2.6 micron
8.00 63 37
• Mobile Phase:
15.50 63 37
• Pump A: 1 mM Ammonium formate with 0.1% Formic
17.00 35 65
acid in water
20.00 25 75
• Pump B: 0.1% formic acid in Acetonitrile
22.00 25 75
‒ Flow Rate: 0.400 mL/min
23.00 98 2
‒ Injection Volume: 25 µL 26.00 98 2

Challenges and suggested solutions:

API Load: 1 mg/mL Limits Achieved 1) NDSRI level increased over period under Autosampler Condition (Propranolol
HCl acidic pH of API solution leading to favored condition). Diluent pH was
Limit of Detection (ng/mL) [(ppm)] 0.005 optimized to provide extended stability.

Limit of Quantitation (ng/mL) [(ppm)] 0.010 2) False Positive results in Control Substance because of Co-eluting other drug
substance related impurities (N-Formyl Impurity of Propranolol)
Specification Limit (ng/mL) [(ppm)] 1.000 3) Chromatographic resolution was improved by optimizing the Gradient.

Range (ng/mL) [(ppm)] 0.010 – 10 4) Issues with method recovery was successfully resolved

. 16
 Case Study-4
o N-Nitroso Meglumine in Tafamidis Meglumine Drug Substance
‒ Column: Time
A (%) B (%)
(min)
• Luna® Phenyl-Hexyl Column 250×4.6 mm; 5 µ 0 100 0
• Mobile Phase: 13 100 0
• Pump A: 0.1% Acetic acid in water 15 5 95
• Pump B: 0.1% Acetic acid in Acetonitrile 21 5 95
‒ Flow Rate: 0.400 mL/min 23 100 0
‒ Injection Volume: 5 µL 35 100 0

‒ Needle Wash:
• 80:20 v/v Acetonitrile: Water

Challenges and suggested solutions:

1) Meglumine is a sugar alcohol derived from glucose
that contains an amino group modification. It is
often used as an excipient in pharmaceuticals.
2) Highly Polar NDSRI Impurity. Selection of Column
was very critical.
3) Solubility Challenges: Lower API load to overcome

.
17
 Thank You

Thank you for your attention !!

Any questions ??

Email : [email protected]
[email protected]
. 18
Analytic-al – OSD

Nitrosamine in Drug Products –

Analytical Challenges

L. Kalyanaraman Ph.D
Head – AR&D (OSD & Advanced Characterisation)
Dr Reddy’s

Dr. Reddy’s Laboratories Ltd. 1

DISCLAIMER
Analytic-al – OSD

The opinions expressed in this presentation are solely those of the

presenter and not necessarily those of Dr. Reddy’s.

Dr. Reddy’s Laboratories Ltd. 2

CHEMISTRY
o NDSRI - Active Ingredient with secondary amine or degradation
product/residual API Intermediate

o Three thing are required – Amine substrate, Nitrite source and

chemical environment
o Weaker bases – nitrosamine chances are high
o Most ideal pH to form nitrosamine 3-4 – at basic pH nitrosating
agent will be weaker – amine protonation at very low pH
Dr. Reddy’s Laboratories Ltd. 3
CARCINOGENICITY
o ICH M7 – Cohort of Concern – more potent than TTC
o Alpha hydrogen is required for carcinogenicity
o Diazonium ion leading to formation of carbocation responsible for
DNA alkylation

o TD50 of the structurally closest N-nitrosamine with robust data

used for calculating limits
So it is essential to understand the Limits & Sensitivity of Method
required before jumping on the development
Dr. Reddy’s Laboratories Ltd. 4
LIMITS
• Limits as per EMA (21st Dec 2022, Rev.14) based on SAR & Read across approach

Analytic-al – OSD

• EMA - t-AI limit of 178 ng/day EMA - 12 months for commercial products
• CAPA implementation of 3 years – 13.3xAI (upto 12 M treatment) & 6.7xAI (>12 M
upto 10 years treatment)
• Any new filings/approval – 18 ng/day (EMA) & 26.5 ng/day (FDA) of unknown
nitrosamine
• For multiple nitrosamine the total should meet 26.5 ng/day.
ANALYTICAL METHOD PLAYS A VITAL ROLE IN MAKING A STABLE PRODUCT
Dr. Reddy’s Laboratories Ltd. 5
REFERENCE STANDARD
o General
Analytic-alNitrosamine
– OSD standards – Widely available – No major issue

o NDSRI – Standards need careful characterisation

o Cooking API with Nitrite may not always lead to NDSRI

o Mass fragmentation & NMR study (N15 in some cases)

o Ensure the purity by mass balance method

Dr. Reddy’s Laboratories Ltd. 6

MASS TECHNIQUE
o GC-MS Triple quadrupole - Direct Injection or Head Space
Suitable –forOSD
o Analytic-al Simple Nitrosamines – Except non volatiles (Eg. NMBA)
o Solvent extraction – High Sensitivity & Low Matrix Interferences
o Thermal Degradation – Eg. Ranitidine
o May require a deuterated internal standard

o LCMS/MS
o ESI – Matrix impact/Ion Suppression – APCI is preferred for Simple Nitrosamines
o Positive mode is preferred (except NMBA – negative mode)
o Deuterated Internal standard – avoids matrix interferences & Ion suppression - Difficult to
get NDSRI deuterated standard
o Quantifier & Qualifier MRM fragments – helps during development for interference check

o LCHRMS
o Specificity through accurate mass measurement of ions – BEST TO USE IF AVAILABLE
Dr. Reddy’s Laboratories Ltd. 7
METHODOLOGY
o Mass is selective
Analytic-al – OSD – but chromatographic separation of interfering
compounds is ideal
o Ionisation suppression/enhancement with excipient co-elution
o Isobaric interference – 15N DMF (m/z 75.0569) & NDMA (m/z 75.0553)
o Choose the MRM transition specific to structural feature
o Rotamers show as two peaks – unsymmetrical substitution

Dr. Reddy’s Laboratories Ltd. 8

SAMPLE PREPARATION
o In-situ – OSD
formation
Analytic-al during analysis - Trace level Nitrites
o pH of the Diluent
o Extraction process – Sonication temperature is critical
o Photolytic degradation during preparation
o Cross contamination – Gloves, Filters, Solvents
o Plastic Centrifuge tubes & Nilon filters – Potential NDBA source
o Control experiments & Orthogonal method to be used to cross
check

SCAVENGER ADDTION TO ELIMINATE TRACE NITRITES WILL ENHANCE ROBUSTNESS

Dr. Reddy’s Laboratories Ltd. 9

FORMULATION ASPECTS
Analytic-al – OSD
o Trace level nitrite Eg. SSG, CCS, Pregel Starch, Povidone,
Crospovidone & Lactose
o Nitrocellulose in the lidding foil with amine in printing ink with Heat-
sealing blister
o Oxidative degradation on storage can enhance – Eg. Metformin
o Aerial oxidation during formulation process – Eg. FBG
o Formulation microenvironment pH
o Temperature enhance the kinetics – higher temp drying
o Packaging configuration

Dr. Reddy’s Laboratories Ltd. 10

ACKNOWLEDGEMENTS
Analytic-al – OSD
o Advanced Characterisation Team – ACT-SEL
o OSD-AR&D Team

Dr. Reddy’s Laboratories Ltd. 11

Analytic-al – OSD

Thank You

[email protected]
Dr. Reddy’s Laboratories Ltd. 12
USP Approaches to Impurities

Edwin L. Gump, Ph.D.

Vice President, Small Molecules

16 February 2023
New Monograph OI Format

2
© 2021 USP
Reporting Thresholds

3
© 2021 USP
Impurities in OTC products

4
© 2021 USP
Mutagenic Impurities

5
© 2021 USP
Nitrosamine impurities: Current
Approaches and Future Strategy - USP
Perspective

Mrunal A Jaywant, Ph.D.

U.S. Pharmacopeia, India
([email protected])

February 16, 2023

Summary of content

 Simple to Complex Nitrosamines

– The journey so far…
– USP’s Nitrosamine Program
– USP’s Tools and Solutions

 USP’s Current Strategy

– Non-compendial solutions
– Pharmaceutical Analytical Impurities
– Strategy for Excipients

 Future Roadmap

2 2
© 2019 ©
USP
2019 USP
Simple to Complex Nitrosamines
The journey so far..
EDQM was informed
EMA alerted the EDQM EDQM was informed about the presence of
and suspended the about the presence of traces of NDMA in
valsartan CEP for low levels of NDMA in metformin HCl. EDQM
Zhejiang Huahai ranitidine HCl and the requested holders of
Pharmaceuticals (ZHP) CEPs for ranitidine HCl metformin CEPs to NDMA
were suspended. address this issue.

EDQM EDQM EDQM

July-2018 Sep-2019 Dec-2019 Apr-2020 Jul-2020

FDA FDA FDA FDA FDA

NDMA identified in one valsartan FDA found NDMA impurity The FDA is aware that The agency determined that FDA recalled several
API producer (ZHP-China) by in ranitidine and alerted some metformin diabetes the impurity in some ranitidine extended release (ER)
voluntary reporting healthcare professionals. medicines in other metformin tablets and
products increases over time
Valsartan recall initiated due to the FDA initiated recall of countries were reported and when stored at higher published the list including
presence of NDMA NDMA containing ranitidine to have low levels of than room temperatures and details about metformin
FDA announcement for the recall products. NDMA may result in unacceptable products.
of several medicines containing levels of this impurity.
valsartan

3
© 2019 USP
Nitrosamines in Rifampin and Rifapentine
• Rifampin and Rifapentine are antibacterial drugs
used to treat tuberculosis; rifampin is also used to
treat or prevent other serious infections
• The acceptable intake limits (in terms of
concentration in ppm) are 0.16 ppm for MNP in 1-methyl-4-nitrosopiperazine
rifampin and 0.1 ppm for CPNP in rifapentine. (MNP)

• [[8/26/2020] The agency will not object to certain

manufacturers temporarily distributing rifampin
containing MNP below 5 parts per million (ppm). Rifampin
The agency also will not object to certain
manufacturers temporarily distributing rifapentine
containing CPNP below 14 ppm..

• Update [10/29/2020] To continue to mitigate or avoid 1-Cyclopentyl-4-nitrosopiperazine

a shortage and to help ensure patients have access (CPNP)
to rifapentine, FDA will not object to certain
manufacturers temporarily distributing the medicine
containing 1-cyclopentyl-4-nitrosopiperazine
(CPNP) above the acceptable intake limit of 0.1
parts per million (ppm) and at or below 20 ppm
until they can reduce or eliminate the impurity. Rifapentine
4
Reference: FDA Updates and Press Announcements on Nitrosamines in Rifampin and Rifapentine
https://www.fda.gov/drugs/drug-safety-and-availability/fda-updates-and-press-announcements-nitrosamines-rifampin-and-rifapentine#:~:text=8%2F26%2F2020%3A%20FDA,or%20prevent%20other%20serious%20infections. © 2019 USP
 Simple to Complex Nitrosamines
The journey so far.. Varenicline
FDA did not object to certain
manufacturers temporarily distributing
Varenicline Varenicline varenicline tablets containing N-nitroso-
HC became aware of N- FDA announced Pfizer’s varenicline above the FDA’s acceptable
nitrosovarenicline being voluntary recall of varenicline intake limit of 37 ng per day but below the
detected in Teva- (Chantix) drug products due to interim acceptable intake limit of 185 ng
varenicline drug potentially unacceptable levels per day. In May 2022, FDA announced
N-nitroso varenicline the new acceptable intake limit is 37 ng
products of nitrosamine impurity
per day.

October 2020 June 2021 July 2021 October 2021

April 2021 July 2021

Irbesartan
Varenicline Varenicline
In Sep. 2020, Health Canada (HC) Voluntary recall of all
Health Canada requested that Pfizer
became aware of N-nitrosovarenicline Irbesartan tablets and
Canada ULC recall the 5 impacted
being detected in an API. Hence, HC Irbesartan-
lots of CHAMPIX (varenicline) with
requested all companies marketing hydrochlorothiazide tablets
varenicline to evaluate the risk of the levels of a nitrosamine impurity, N-
due to potential presence
presence of nitrosamine impurities in nitrosovarenicline, above the
of N-nitrosoirbesartan
their drug product and to conduct testing acceptable intake limit established by
impurity
as necessary Health Canada.
N-nitroso irbesartan

5
© 2019 USP
 Simple to Complex Nitrosamines
The journey so far..
Sitagliptin
FDA recently became aware of a
Propranolol hydrochloride nitrosamine impurity, Nitroso-STG-19
Voluntary recall of Inderal-LA (known as NTTP), in certain samples of
(propranolol hydrochloride) capsules in sitagliptin. To avoid a shortage, FDA
Canada due to the presence of a allowing temporary distribution of
N-nitroso-propranolol
nitrosamine impurity (N-nitroso- Sitagliptin products with NTTP impurity
propranolol) above the acceptable level. above the acceptable intake limit of 37 ng
Nitroso-STG-19
per day, and up to 246.7 ng per day.

March 2022 March 2022 Dec. 2022

March 2022 Aug 2022

Orphenadrine Citrate Quinapril and Hydrochlorothiazide Quinapril Tablets

Voluntary recall of Orphenadrine Tablets
Citrate 100 mg Extended Voluntary recall of Quinapril and Voluntary recall of
Release (ER) Tablets due to Hydrochlorothiazide Tablets due to the Quinapril Tablets due
potentially unacceptable levels of presence of a nitrosamine, N-nitroso- to the presence of a
NMOA or Nitroso-orphenadrine quinapril, above the Acceptable Daily nitrosamine, N-
impurity
Nitroso-orphenadrine Intake (ADI) level. N-nitroso-quinapril nitroso-quinapril,
above the Acceptable
Reference:
Daily Intake (ADI)
https://www.fda.gov/drugs/drug-safety-and-availability/fda-updates-and-press-announcements-nitrosamine-varenicline-chantix#60f8a171a4486 level.
https://www.fda.gov/safety/recalls-market-withdrawals-safety-alerts
https://www.fda.gov/drugs/drug-safety-and-availability/
6
https://recalls-rappels.canada.ca/en/alert-recall/pfizer-recalls-accupril-blood-pressure-tablets-due-nitrosamine-impurity © 2019 USP
 Recent Recalls Due to NDSRIs

 The October announcement of a recall of two lots of the hypertension

medication Quinapril and Hydrochlorothiazide from the U.S. market is putting
nitrosamine drug substance related impurities (NDSRI) in the spotlight once
again.
 In December 2022 the FDA announced a voluntary recall of four lots of Quinapril
Tablets due to the presence of a nitrosamine impurity observed in testing above
FDA’s proposed interim limit, adding to the growing number of recalls due to
nitrosamine impurities in recent years.
 Amidst the latest recall related to NDSRIs, USP continues to lead the charge by
providing quality standards-based solutions, organizing workshops and training
courses and hosting a forum for the exchange of crucial knowledge to help keep
our medicine supply chain strong and protect patient health.
Reference:
https://www.fda.gov/safety/recalls-market-withdrawals-safety-alerts/aurobindo-pharma-usa-inc-initiates-voluntary-nationwide-recall-two-2-lots-quinapril-and 7
https://www.fda.gov/safety/recalls-market-withdrawals-safety-alerts/lupin-pharmaceuticals-inc-issues-voluntary-nationwide-recall-four-lots-quinapril-tablets-due
© 2019 USP
USP’s Nitrosamine Program:
Accomplishments so far…

1
Documentary Standard
To address the
nitrosamine impurities
safety concern from a
pharmacopeial
2 Reference Standard
Eight USP Reference
Standards have been
perspective, a USP Joint established to support
Expert Subcommittee
(JSC) was convened in USP’s General Chapter <1469>
Nitrosamine Impurities
February 2020 to
develop General Nitrosamine
Chapter <1469>
Nitrosamine Impurities. Program
3
Advocacy and capability
building
USP Education course
Webinar, Round Table
Discussion, Workshop, User
Forums
Trainings to Regulators
8
© 2019 USP
GC <1469> Nitrosamines Impurities
Timeline

1 2 3 4 5 6

01 Sep 2020 30 Nov 2020

GC <1469> End JSC addressed Standard is Published in GC <1469>

publication commentary comments and balloted USP-NF 2021 Issue 3 became
in the PF reviewed proposal 1st Jun ‘21 official
Comments period Sub-committee
1 Dec ‘21
st
<1469> GC is balloted for
published in end (ALL addressed public approval by Chemical
Pharmacopeial stakeholder are comments and Analysis General
Forum 46 Issue encouraged to revise chapter if Chapter Expert
5, available on- participate) necessary committee
line

9
© 2019 USP
GC <1469> Nitrosamines Impurities

Content
1. INTRODUCTION
2. NITROSAMINE IMPURITIES
3. SOURCES OF NITROSAMINES
4. NITROSAMINE RISK ASSESSMENTS – DEVELOPMENT OF A CONTROL STRATEGY
5. LIMITS OF NITROSAMINE
6. TESTING FOR THE PRESENCE OF NITROSAMINES
7. TEST METHOD PERFORMANCE CHARACTERISTICS OF NITROSAMINE METHODS
8. ANALYTICAL PROCEDURES
9. ADDITIONAL SOURCES OF INFORMATION

10
© 2019 USP
USP Nitrosamine Reference Standards
 USP developed initially six USP Nitrosamine Reference Standards for
use with General Chapter <1469> Nitrosamine Impurities
Catalog # Structure Catalog # Structure Catalog # Structure
Name Name Name
Label value Label value Label value
1466674 1466663 1466607
N-Nitroso dimethylamine N-Nitroso N-Nitrosomethyl
(NMDA) diisopropylamine (NDIPA) phenylamine (NMPA)
1.00 mg/mL in Methanol 1.00 mg/mL in Methanol 1.00 mg/mL in Methanol

1466652 1466641 1175800

N-Nitroso diethylamine N-Nitroso dibutylamine Deutero N-
(NDEA) (NDBA) Nitrosodimethylamine D3C N
1.00 mg/mL in Methanol 1.00 mg/mL in Methanol (NDMA-d6) N O
1.00 mg/mL in Methanol
CD3

1466685 1466696
N-Nitroso N-Nitroso methtylamino
ethylisopropylamine butyric acid (NMBA)
(NEIPA) 1.00 mg/mL in Acetonitrile
1.00 mg/mL in Methanol

11
© 2019 USP
Nitrosamine Training Materials

Introduction to Proposed USP General Chapter

<1469> and Handling of Nitrosamine Impurities
Reference Standards: Posted on YouTube in
Nov. 2020
12
© 2019 USP
Nitrosamine Exchange – Online Community

Nitrosamine Exchange
Knowledge Community

Join http://nitrosamines.usp.org 13
© 2019 USP
Overview of USP Nitrosamine activities

Nitrosamine USP
Nitrosamine Training
Documentary Workshops / Global Public
USP Reference material/
Standards Webinars / Health
Standards Education
course Conferences
Scientific Training and
NDIPA Webinars/
Developed a guidance for
NDMA Workshops
tutorial and global
education regulators
<1469>- NDBA Round table
course on
Nitrosamine discussions/
NDEA Nitrosamine stakeholder Nitrosamine
Impurities
impurities to forums test methods
NMBA train industry for essential
stakeholders tuberculosis
Industry
NEIPA connect forums drugs

14
© 2019 USP
 Nitrosamine Impurities Survey 1.0
Research Goal
Understanding current challenges and Key findings:
practices for controlling & testing
Nitrosamines’ impurities and what else is • Nitrosamines is the topmost impurity of
required in this space. concern for Drug products and Drug
What? substances, whereas Elemental impurities and
Residual solvents top the list in Excipients
Online survey distributed via Qualtrics and category.
emails from regional USP teams. • Uncertainty in observing and controlling
When? nitrates and nitrites levels is noted for each
Fieldwork date: August 9 – August 31, 2021 product category.
• This uncertainty level goes even higher for
Who? Excipients.
Survey targeted USP stakeholders and
customers.
Total sample for analysis and reporting
=242 (incl. 18 partials)
15 15
© 2019 USP
USP’s Current Approaches
 Non-compendial solutions:
– Publications
– Analytical Hub
• Analytical Procedures:
 Solvent Method
 Ranitidine
 Rifampin and Rifapentine
 Universal Method
– Pharmaceutical Analytical Impurities (PAI)

 Strategy for Excipients:

– Nitrite and Nitrate in Excipients

 Advocacy and Capability Building:

– Pharmacopeial Education
– Nitrosamine Workshop/ User Forums
16 16
– Nitrosamine Tutorial © 2019 ©
USP
2019 USP
Non-compendial solutions: Publication

• Solvents are widely used as a reaction media

and other steps in the production of drug
substances and products in pharmaceutical
industries.

• Nitrosamine contamination can occur when fresh

solvents (ortho-xylene, toluene, and methylene
DOI:https://doi.org/10.1016/j.xphs.2022.11.024 chloride) get contaminated during shipment from
vendors (e.g., during transfer between storage
vessels).

• The determination of nitrosamines in solvents

plays an important control in the manufacturing
of quality drug substances and drug products.

• The current study provides a highly sensitive

procedure for the determination of six
nitrosamines in widely used solvents:
dichloromethane, ethyl acetate, toluene, and o-
xylene.
17
https://www.fda.gov/media/141720/download
© 2019 USP
Non-compendial solutions: Publication

• An in-silico analysis of more than 12,000

small molecule drugs and drug impurities.

• In total, 41.4 % of the APIs and 30.2 % of

the API impurities listed in the GSRS
database are potential nitrosamine
precursors.

• Most structures identified through this

workflow could form complex API-related
nitrosamines (NDSRIs).

• Analytical standards that would allow for

quantification in the pharmaceuticals
concerned are currently only available for
less than 5 % of all potential NDSRIs.

18
© 2019 USP
 Non-compendial solutions: USP Analytical
Hub
• Launched in December 2022  Public online repository containing non-compendial
analytical procedures (analytical notes) for the testing
of nitrosamine impurities and related substances.

 USP’s scientists curate these analytical procedures

through internal development/validation or through
scientific review of non-compendial donations. They
are NOT compendial standards.

 The procedures contained in the analytical notes

should be validated by the user. USP is not and will
not be responsible for the use or implementation of the
procedures.

 Hosted in The Nitrosamine Exchange. The Analytical

Hub allows keyword searches and the view of key
USP App Note - Nitrosamines analysis in analytical procedure parameters and chromatograms.
Solvents by GC-MS-MS V2.pdf
19
© 2019 USP
Non-compendial solutions: Common Method
Approach
Sample preparation Mass spectrometric
Different class of drugs
optimization detection

Angiotensin II Receptor Blockers

(Sartans)
Total Matrix
Histamine-2 Receptor Antagonists Dissolution precipitation
(Ranitidine and Nizatidine)

Antidiabetic Agents Solid phase Selective

(Metformin Hydrochloride) extraction extraction

Antimicrobial Agents NDMA, NMBA, NDEA, NEIPA,

(Rifampin and Rifapentine) NDIPA, NDPA, NMPA, NDBA, and additional nitrosamines

Optimization in-process 20
© 2019 USP
Pharmaceutical Analytical Impurities (PAI)
Available in April 2023 and later
Impurity name or Chemical Molecular
RFI CAS API
formula Formula
621-64-7 N-Nitrosodipropylamine (NDPA) − C6H14N2O

61379-66-6 1-Cyclopentyl-4-nitrosopiperazine Rifapentine C9H17N3O

16339-07-4 1-Methyl-4-nitrosopiperazine Rifampin C5H11N3O

930-55-2 N-Nitrosopyrrolidine -- C4H8N2O

138768-62-4 N-Nitroso Metoprolol Metoprolol C15H24N2O4

2248746-67-8 N-Nitroso Carvedilol Carvedilol C24H25N3O5

84418-35-9 N-Nitroso Propranolol Propranolol C16H20N2O3

2820170-74-7 N-Nitroso Labetalol Labetalol C19H23N3O4

134720-04-0 N-Nitroso Atenlol Atenolol C14H21N3O4

2820170-76-9 N-Nitroso Bisoprolol Bisoprolol C18H30N2O5 21

© 2019 USP
Additional PAIs being prepared and coming
soon!
 Includes a mix of both simple nitrosamine impurities and Nitrosamine Drug
Substance Related Impurities (NDSRI)
 Therapeutic categories of medicines with the potential to be affected by these
impurities include:
– Antidotes, Deterrents, and Toxicologic Agents
– Central Nervous System Agents
– Cardiovascular Agents
– Genitourinary Agents
– Blood Products/Modifiers/Volume Expanders
– Antidepressants
– Antiparkinson Agents
22
© 2019 USP
 Strategy for Nitrosamines in Excipients
Scope: Formation of NDMA from Nitrite

To develop a strategy for the control of Nitrosamines in

Excipients in collaboration with the Excipients Test Method
Expert Committee

Work plan:
Nitrite in Excipients
Determination of Nitrates and Nitrites in at risk excipients
 As part of risk assessment, the
Preliminary Findings: level of Nitrites and Nitrates in
 Challenging sample preparation excipients needs to be evaluated
 Interferences from other ions and a control strategy needs to be
 Inconsistent recoveries established by the drug product
manufacturers.
Status:
 Work in-progress to establish a sensitive and robust method
23
© 2019 USP
Advocacy and Capability Building:
Nitrosamine Tutorial
Scope:
To design and create video tutorials on Nitrosamine methods highlighting critical troubleshooting involved in
nitrosamine methods (LC-MS/MS & GC-MS/MS) covered under the nitrosamine education course.

Work Plan:

Content development Technical and legal

Tutorial design Video Creation Launch
review

Status:

Content development Technical and legal Launch

Tutorial design Video Creation
review

Scheduled Scheduled in 24
in Feb 2023 March 2023 © 2019 USP
Future Roadmap

 Method for NDSRIs

 Risk Assessment Tool

 Additional Nitrosamine RS (PAI)

 Training to Regulators: Lab Demonstration

 Strengthening collaboration with FDA

 Launch of Nitrosamine Survey 2.0 questionnaire

25
© 2019 USP
Method For NDSRIs
Scope:
N-Nitrosoatenolol
To develop analytical procedure for
determination of NDSRIs. N-Nitrosobisoprolol
Work Plan:
Identify a specific class of drug products. N-Nitrosocarvedilol
Synthesis and characterization of reference
materials. N-Nitrosolabetalol
Develop sensitive and robust analytical
procedures (LC-MS/MS). N-Nitrosometoprolol

Status:
 Synthesis and characterization completed N-Nitrosopropranolol
 Method development is in progress
26
© 2019 USP
Risk Assessment Tool
Scope:

To develop a ‘practical’ guidance document for

conducting Risk Assessment [What   How]
Work Plan:

Development through a collaborative process

with Nitrosamine Exchange community members

Inputs from Expert Committee and FDA liaisons

Publication of final guidance document (White

Paper, Stimuli Article, Peer-review article)
Status:
27
 Kicked off Jan ‘23 © 2019 USP
Future projects under consideration…
Harmonization
(Convergence) with
other pharmacopeias for
analytical procedures

Collaboration with WHO

Discussion with
EC for Packaging
component
Analytical services
standards
??? Method
development,
training, etc.

28
© 2019 USP
THE EUROPEAN
DIRECTORATE FOR THE
QUALITY OF MEDICINES
& HEALTHCARE
(EDQM)
 The EDQM response
to nitrosamines
USP/IPA Workshop on Nitrosamines Impurities
Analysis, Industry Needs and Regulatory Perspectives

February 16, 2023

Bruno Spieldenner
European Pharmacopoeia Department
EDQM, Council of Europe

2 © EDQM, Council of Europe, 2022. All rights reserved.

The EDQM
• The European Directorate for the Quality of
Medicines & HealthCare (EDQM):
• Is part of the Council of Europe, an
international organisation promoting
human rights, democracy and the rules
of law
• Located in Strasbourg, FR
• Is in charge of the European
Pharmacopoeia (Ph. Eur.),
Certification/CEP procedure, Official
Medicines Control Laboratories
(OMCL) network, etc
• Is not the European Medicines Agency
(EMA)
3 © EDQM, Council of Europe, 2022. All rights reserved.
Nitrosamines - The road in Europe
EMA webpage: https://www.ema.europa.eu/en/human-regulatory/post-authorisation/referral-procedures/nitrosamine-impurities

2018 2019 2020

June: EU notified that an API February: CHMP opinion on nitrosamine impurities in July: CHMP opinion pursuant to Article
manufacturer detected presence of sartans containing a tetrazole group made public in an 5(3) of Regulation (EC) No 726/2004 for
NDMA in valsartan assessment report. nitrosamine impurities in human medicinal
products
July:EC triggered a review in March: Adoption 5 revised sartan monographs including CHMP opinion of 25 June 2020 ≠
accordance with Article 31 of temporary limits for NDMA/NDEA European Commission’s Decision of 2
Directive 2001/83/EC to be carried Request for revision of 2 general monographs on Substances April 2019
out by EMA’s Committee for for pharmaceutical use (2034) and Pharmaceutical
Medicinal Products for Human Use preparations (2619) July: Outcome of a Sartans Lessons Learned
(CHMP) exercise (also triggered by the Art. 31 referral)
April: EC’s Implementing Decision concerning, in the
by the European medicines regulatory network
framework of Article 31 of Directive 2001/83/EC of the
September: scope of the referral
European Parliament and of the Council, the MA of medicinal
widened to include all sartans with November: CHMP Opinion concluding that
products for human use which contain the active substances
a tetrazole moiety in their Article 31 outcome on sartans should be
“candesartan”, “irbesartan”, “losartan”, “olmesartan”,
molecular structure. aligned with Art. 5(3) outcome.
“valsartan” => CHMP opinion rendered legally binding in EU
Adoption Ph. Eur. general chapter 2.5.42. N-
Nov: Request for minor revision: & EEA member states
nitrosamines in active substances
5 sartan monographs
Addition on Ph. Eur. work prog. of Sept: start of Art. 5(3) of Regulation (EC) No 726/2004,
general chapter 2.5.42 whereby EMA’s Executive Director requested a CHMP opinion
– to provide guidance to MAHs and manufacturers of
medicines containing chemically synthesised APIs

4 © EDQM, Council of Europe, 2022. All rights reserved. Light blue: Ph. Eur. specific items
Nitrosamines - The road in Europe
2021 2022
February: implementation plan agreed on how the December: publication by the
European medicines regulatory network, together with the EMA of revision 14 of Questions
EDQM, will implement the outcome of the CHMP's review. and Answers for marketing
authorisation holders/applicants
February: The Nitrosamine Implementation Oversight on the CHMP Opinion for the
Group (NIOG) was set up to oversee the harmonised Article 5(3) of Regulation (EC)
implementation of the CHMP's Article 5(3) opinion on No 726/2004 referral on
nitrosamines. It contains representatives from the CHMP, nitrosamine impurities in human
CMDh, EMA and its working parties and EDQM medicinal products
(EMA/409815/2020).
April: implementation date of the five monographs on
sartans with a tetrazole ring, namely Valsartan (2423),
Losartan potassium (2232), Irbesartan (2465),
Candesartan cilexetil (2573) and Olmesartan medoxomil
(2600) which have been revised to align them with the
latest regulatory recommendations issued by the CHMP
published on November 2020.

5 © EDQM, Council of Europe, 2022. All rights reserved.

EDQM response to nitrosamines
https://www.edqm.eu/en/n-nitrosamine-contamination-in-brief

Ph. Eur strategy

Sampling & testing by OMCL network

CEP procedure

6 © EDQM, Council of Europe, 2022. All rights reserved.

Ph. Eur analytical procedures
• Development of General Chapter on Control of N-nitrosamines in active substances
(2.5.42)
• Based on analytical procedures developed by the OMCLs
• Analytical toolbox, 3 procedures relying on
different techniques (LC-MS/MS, GC-MS and GC-MS/MS)
• Focuses on the detection of 7 nitrosamines in sartans active substances (NDMA, NDEA,
NDBA, NMBA, NDIPA, NEIPA, NDPA)
• Validated as limit tests at 0.03 ppm (proc. A and B) and quantitative test (proc. C)
• Text available in Supplement 10.6
• Availability of official nitrosamines reference standards (CRS) for testing
• May be used to detect nitrosamines in other active substances or in medicinal products
 require appropriate validation

7 © EDQM, Council of Europe, 2022. All rights reserved.

 Ph. Eur. Strategy – on-going work 1/3
Aiming at a moving target
Revision of General monograph 2034 “Substances for pharmaceutical use”
2020

• Draft published in Pharmeuropa 32.1 (January 2020)

• To trigger reactions and suggestions on the best way forward
• Outcome of the CHMP Article 5(3)  focus is on Finished Product and no longer on the API
 Adapt approach
• (2nd) revision of General Monograph 2034 “Substances for pharmaceutical use”
2021

• Revision of General Monograph 2619 “Pharmaceutical preparations”

Both published for comments in Pharmeuropa 33.2
Tabled initially at the November 2021 Commission => postponed to Nov. 2022
2022

Evolution of the scope / discussion on the setting of limits

Adopted in November 2022 to be implemented Ph. Eur. Suppl. 11.3 (Jan 2024)

8 © EDQM, Council of Europe, 2022. All rights reserved.

Ph. Eur. Strategy – on-going work 2/3
Statement in General Monograph 2034 Substances for Pharmaceutical Use
“N-Nitrosamines. As many N-nitrosamines are classified as probable human carcinogens, manufacturers of active substances for
human use are expected to evaluate the potential risk of N-nitrosamine formation and contamination occurring throughout their
manufacturing process and during storage. If the risk is confirmed, manufacturers should mitigate as much as possible the
presence of N-nitrosamines – for example by modifying the manufacturing process – and a control strategy should be
& 2619 (Production)
implemented to detect and control these impurities. General chapter 2.5.42 N-Nitrosamines in active substances is available to
assist manufacturers.”

Statement in General Monograph 2619 Pharmaceutical Preparations

“N-Nitrosamines. As many N-nitrosamines are classified as probable human carcinogens, manufacturers of medicinal products,
except products for veterinary use only and unlicensed pharmaceutical preparations are expected to evaluate the potential risk
of N-nitrosamine formation and contamination occurring throughout their manufacturing process and throughout their shelf-life,
according to the requirements of the relevant competent authorities. If the risk is confirmed, manufacturers should mitigate as
much as possible the presence of N-nitrosamines – for example by modifying the manufacturing process – and a control strategy
must be implemented to detect and control these impurities. General chapter 2.5.42 N-Nitrosamines in active substances is
available to assist manufacturers.”

Both texts to appear in the Production Section  Applicable to manufacturers

9 © EDQM, Council of Europe, 2022. All rights reserved.
Ph. Eur. Strategy – on-going work 3/3
Strategy for genotoxic impurities in individual monographs
Since 2016: implementation of ICH M7 (DNA-reactive) for active substances: 2 options
1 Statement in Production section: when no suitable (selective or sensitive) test is known
or it requires less common equipment.
 Manufacturer has to ensure the compliance of production with defined requirements.

2 Tests section: specific test when suitable analytical procedure available and limit known.

Nitrosamines already mentioned in some monographs / missing in others KEEP ? ADD ? DELETE ? REVISE

With further development and gain of knowledge :additional questions

Secondary amines
Nitrosamine drug substance-related impurities (NDSRIs)
Nitrites in excipients

10 © EDQM, Council of Europe, 2022. All rights reserved.

EDQM response to nitrosamines
https://www.edqm.eu/en/n-nitrosamine-contamination-in-brief

Ph. Eur strategy

Sampling & testing by OMCL network

CEP procedure

11 © EDQM, Council of Europe, 2022. All rights reserved.

Sampling and testing by OMCLs
• Official Medicines Control Laboratories:
• public institutions which test quality of medicines independently from manufacturers (no conflicts of interest, guarantee of impartiality,
respecting confidentiality)
• The network comprises OMCLs from countries that are members or observers of Ph.Eur. Convention (“full” and “associated” members).

• For nitrosamines, a network of 13 labs collaborating for development of methods and testing
• A number of in-house methods established, published and used for the Ph. Eur. General Chapter 2.5.42
• Sampling and testing of medicinal products containing sartans:
• NDMA: 2000 DP and 249 API samples
• NDEA: 1007 DP and 637 API samples
• Work further extended to other nitrosamines, other APIs and associated medicinal products
• Ranitidine  870 DP and 215 API samples
• Metformin  476 DP and 214 API samples
• Rifampicin  44 DP and 38 API samples
• Some OMCLs developed methods for the identification and quantification of selected NDSRIs

12 © EDQM, Council of Europe, 2022. All rights reserved.

EDQM response to nitrosamines
https://www.edqm.eu/en/n-nitrosamine-contamination-in-brief

Ph. Eur strategy

Sampling & testing by OMCL network

CEP procedure

13 © EDQM, Council of Europe, 2022. All rights reserved.

Nitrosamines in (all) active substances covered by CEPs
• EDQM has aligned with EU call for review in October 2019:
 https://www.edqm.eu/en/news/announcement-all-cep-holders-synthesised-apis-regarding-presence-nitrosamines

• Stepwise approach for CEP holders:

• STEP 1: RISK EVALUATION:
o conduct a risk assessment to identify any risk of nitrosamine formation
o EDQM to be informed with a testing plan and timelines if risks identified
• STEP 2: CONFIRMATORY TESTING:
o provide test results to EDQM, and if needed a corrective actions plan with timelines (deadline 26
September 2022)
• STEP 3: UPDATE OF CEP APPLICATION
o implement additional controls or process changes
o send revision application to EDQM as needed
o completion by 1 October 2023 at the latest (extended from September 2022)

CEP holders should be supportive to MAHs and provide them with relevant information

14 © EDQM, Council of Europe, 2022. All rights reserved.

Nitrosamines in (all) active substances (2)
• Assessment of CEP applications since January 2019:
• Routine assessment by EDQM of risks for nitrosamines in the context of
new dossiers, renewals, and in case of changes to routes of synthesis/
changes of suppliers
• Implementation of controls to prevent presence of nitrosamines when
needed

• CEP applicants should include risk assessments in new CEP

dossiers, renewals, and revisions where a risk of nitrosamine
formation may be introduced (i.e. changes to the manufacturing
process, change of suppliers of starting materials or
intermediates, etc.) since 1 October 2020

15 © EDQM, Council of Europe, 2022. All rights reserved.

Nitrosamines in active substances – CEP reviews
For sartan active substances:
 All CEPs are in compliance with the Ph. Eur monographs in force in April 2021
 Limits and test methods applied by manufacturers are mentioned on CEPs

Other substances:
 A couple of specific reviews following information received from CEP holders or from
authorities
 Current focus is on NDSRIs, formed during manufacture or during storage of the API
 EDQM uses the principles described in the latest version of the EMA Q&A (including
the Acceptable Intakes)
 When needed, a limit and a test method for nitrosamines are mentioned on the CEP

16 © EDQM, Council of Europe, 2022. All rights reserved.

GMP inspections of API manufacturers
• Triggered a number of EDQM on-site inspections in 2018-2019
• Carried out jointly with EU, USFDA, Swissmedic
• A couple of non-compliances  Actions on CEPs and marketing
applications
• Samples taken, tested by OMCLs

17 © EDQM, Council of Europe, 2022. All rights reserved.

Authorities : information sharing & communication
• Close cooperation with EMA and the EU network
• Close cooperation with other authorities worldwide – via the
Nitrosamines International Strategic Group (NISG – chair Health
Canada) and its Technical Group (NITWG)
• Sharing information with international partners under confidentiality
agreements:
• presence of nitrosamines in sources of APIs & in medicinal products
• information on analytical methods and test results
• To trigger alignment of decisions

18 © EDQM, Council of Europe, 2022. All rights reserved.

Conclusion
• Evolving environment
• The Ph. Eur. text provides a frame for addressing the risk of
nitrosamines impurities
• The EDQM monitors closely, shares information and implements the
latest regulatory requirements on nitrosamine impurities, in
collaboration with authorities worldwide

Watch the space !

EDQM webpage

19 © EDQM, Council of Europe, 2022. All rights reserved.

 Thank you for your attention

Stay connected with the EDQM

EDQM Newsletter: https://go.edqm.eu/Newsletter

LinkedIn: https://www.linkedin.com/company/edqm/
Twitter: @edqm_news
Facebook: @EDQMCouncilofEurope

20 ©©EDQM,
EDQM,Council
CouncilofofEurope,
Europe,2021.
2022.AllAllrights
rightsreserved.
reserved.
 BACKUP SLIDES

21 © EDQM, Council of Europe, 2022. All rights reserved.

EU Article 5(3) call for review to MAHs
• STEP 1: RISK EVALUATION
• Conduct a risk evaluation to identify active substances and finished products at risk of N-
nitrosamine formation or (cross-)contamination and report the outcome by:
• Chemical medicines: before 31/03/2021
• Biological medicines: before 01/07/2021
• If a risk is identified for an active substance, MAH should submit the step 1 response template
and proceed with step 2 confirmatory testing of the finished product.
• If no risk is identified for an active substance, MAH should conduct a risk evaluation of the
finished product and submit the outcome of step 1 only when they reach a final conclusion on
the active substance and finished product.

• STEP 2: CONFIRMATORY TESTING

• Perform confirmatory testing on the products identified to be at risk of N-nitrosamine formation
or (cross-)contamination and report confirmed presence of nitrosamines in a timely manner

• STEP 3: UPDATE OF MARKETING AUTHORISATIONS

• Chemical medicines: before 26/09/2022 01/10/2023 (deadline for submission of any variations)
• Biological medicines: before 01/07/2023 (deadline for submission of any variations)

22 © EDQM, Council of Europe, 2022. All rights reserved.

EU CHMP Article 5(3) opinion
• Applies to all human medicines, including biologicals
• Presence of nitrosamines should be mitigated and their levels should be within ICH
M7 limits – consider lifetime daily exposure
• Guidance on what to consider for risk evaluations
• Guidance on setting limits (e.g. giving acceptable intakes for known nitrosamines,
for “new” nitrosamines, in case there is more than 1 nitrosamine, etc)
• Limits by default should be in the finished product specification
• Considerations for analytical methods
• Q&As updated regularly with new knowledge

! Decision was taken in November 2020 to apply these principles to medicinal

products containing sartans, so to change the initial EU commission decision for
these products

23 © EDQM, Council of Europe, 2022. All rights reserved.

Nitrosamine Workshop 2023 2/28/2023

Nitrosamine Exchange
A knowledge community for
all-things Nitrosamines

Naiffer Romero, MSc

Scientific Affairs
Principal Scientist
2023

Information + more information

2
© 2019 USP

For personal reference only 1

Nitrosamine Workshop 2023 2/28/2023

Knowledge Hub

 Unleashing the power of online communities

 Increase and accelerate early scientific knowledge
exchange in select topics
 Strong sense of community and belonging, despite not
operating in physical space
 Democratization and Inclusion of Knowledge
 Hosted by USP, BUT defined by the members
 A new tool in USP’s ecosystem of engagement
approaches

3
© 2019 USP

Nitrosamine Exchange Community

Nitrosamine Exchange
Knowledge Community

Join http://nitrosamines.usp.org 4
© 2019 USP

For personal reference only 2

Nitrosamine Workshop 2023 2/28/2023

Learnings Nitrosamines Exchange – Can we

do it?
Apr’21 Oct’21 Feb’22 Today
Launch Analytical Redesign & Collaboration Hub
expansion Multi-language

 2000+ members, 90 countries

 70% new to USP; 86% outside U.S.
– new in 2022: ability to translate text between 22 languages
 198k+ page views
 60% give 4 or 5 on usefulness (scale 1-5)

5
© 2019 USP

Examples

6
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For personal reference only 3

Nitrosamine Workshop 2023 2/28/2023

Examples
Increasing diversity by breaking down language barriers

7
© 2019 USP

Read out from Nitrosamine Exchange

Nitrosamine Chemistry – Risk vs NO Risk?

Genotoxic Nature of Nitrosamine – Are all N-nitrosamine impurities a

concern?

In-silico Risk assessment (SAR) How / When / Why?

When Nitrosamine RISK is identified – Remove or Control? (Packaging)

Ranitidine – What’s the real root cause?

Less-than-lifetime VS Not Less-than-lifetime

Analytical Challenges – we are still learning

Innovative and in-silico tools to approach novel Nitrosamines?

Science and best practices sharing

8
© 2019 USP

For personal reference only 4

Nitrosamine Workshop 2023 2/28/2023

Nitrosamine Timeline
Timeline Legend:
ZH: Zhejiang Huahai Pharmaceuticals
Release of USP
ZT: Zhejiang Tianyu nitrsoamine
NDMA in HL: Hetero Labs standards NDMA, FDA issues
EMA ZT Valsartan
EMA’s human
medicines NDEA, NDIPA guidance
Release of USP
committee (CHMP) NDBA, NEIPA, NMBA “Control of N- nitrsoamine
NDEA in NDIPA in Nitrosamine
ZH Valsartan valsartan has recommended Impurities in standards
suspension of Huma Drugs” NMPA
NDMA in NDEA in NDMA in ranitidine medicines DNMA-d6
ZH Valsartan HL Losartan Pioglitazone in the EU
Taiwan FDA

Rapid Alert

informs EU network
NDEA in NMBA in
Health Canada
Network NMPA in
OMLC in Germany

Aurobindo HL Losartan Divi Valsartan

Irbesartan USP-NF
USP PF(46) <1469>

Article 31 Article 31

Jul
review Extend the Lessons

1 Sep
of valsartan scope to review Sartans Ranitidine Learnt
20 Sep

24 Jun
12 Sep

1 Sep
26 Jun

16 Jul

2 May
all sartans with Nitrosamines

Jun
medicines; End Article 31
Dec

Jan

July

Apr
recalls tetrazole ring Article 31 triggered in Sartans

2018 2019 2020 2021

16 Apr
Oct

Voluntary recall

Sept
Feb
Jul

Nov
Recall of valsartan

Oct19
of ranitidine

26Aug
irbesartan, losartan in the US
in the US

1 Apr
Nitrosamine Voluntary MNP/CPNP
Voluntary recalls recall of Rifampin/
of valsartan Recall expanded Posting - Laboratory found in
NDMA Nizatidine Nizatidine Rifapentine
to other sartans test results: NDEA
Found in Ranitidine NDMA
levels in recalled
Ranitidine FDA warns Torrent Market in metformin 9
FDA valsartan
medicines for cGMP violations Withdrawal products
© 2019 USP

Nitrosamine Timeline
Timeline
Updates on
EMA possible mitigation
strategies to reduce
the risk of
nitrosamine drug N-Nitroso-irbesartan
N-nitroso- substance-related
impurities in drug
Veracline products N-Nitroso-Desloratadine

N-Nitroso-Folic Acid

N-Nitroso-Quinapril

N-Nitroso-Orphenadrine
Oct

N-Nitroso-Propranolol

2021 2022 Beyond

nitrosamine drug
substance-related
impurities (NDSRIs)

10
© 2019 USP

10

For personal reference only 5

Nitrosamine Workshop 2023 2/28/2023

Next Challenge…

12,000 USP DB

4,848 APIs
(40.4%)

3,552 Impurities
(29.6%)

The Landscape of Potential Small and Drug Substance Related Nitrosamines in Pharmaceutical. Journal of Pharmaceutical Science Nov’23 - https://doi.org/10.1016/j.xphs.2022.11.013 11
© 2019 USP

11

How-To?

12
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Nitrosamine Workshop 2023 2/28/2023

Nitrosamine Exchange Community

Nitrosamine Exchange
Knowledge Community

Join
http://nitrosamines.usp.org 13
© 2019 USP

13

Risk Assessment

 Is there a risk that secondary or tertiary

amine contaminants are present in any
primary amines used in your
manufacturing process?

 Are any components

containing/potentially containing nitrites
and amines present together in solution
or in suspension during processing (e.g.,
during granulation, coating)?

 Are nitrites (NO2-), nitrous acid, nitrates

(NO3-), nitric acid, or azides (N3-) or their
sources present in chemically
synthesized APIs?

14
© 2019 USP

14

For personal reference only 7

Nitrosamine Workshop 2023 2/28/2023

http://nitrosamines.usp.org

Naiffer Romero
[email protected]

15

For personal reference only 8

 Setting Limits for Complex
Nitrosamines

Raphael Nudelman, PhD, Registered Toxicologist (ERT)

Senior Director Impurity Expert
Teva Pharmaceutical Industries Ltd., Israel

USP’s (USP + IPA) Workshop on Nitrosamines Impurities

February 16, 2023
 Disclaimer

The opinions expressed in this presentation are those of the

presenter and not necessarily those of Teva Pharmaceutical
Industries Ltd. or its affiliates (collectively “Teva”). This presentation
has been prepared for discussion purposes only. Neither Teva nor
any of its employees or representatives make any representation or
warranty, express or implied, as to the accuracy or completeness of
any information contained herein. The information and examples
presented originate from individual experience and may not
represent the full scope and/or examples of Teva.
Nothing contained within the presentation is, or should be relied
upon as, a promise or representation as to the future and Teva
expressly disclaims any obligation to update the information if it
should change.
2 NOT FOR DISTRIBUTION
 2019 – General request for nitrosamines risk assessments
• Companies to address risks of nitrosamines in all products, via a 3 step process

3
 Challenges
Endogenous
or
exogenous Very low
exposures acceptable
not intakes
Major safety considered
in the limits
interpretation
differences Uncertainty
around
Less than
lifetime not Purge factor

between ICH M7 acceptance (fully) not accepted

of Ames accepted

and nitrosamine
test
Unclear
guidances Method for
which in-
vivo tests
are relevant
read-across and how
not clear their results
can be
used

4
 Read-Across
The read-across approach is introduced in ICH M7: Surrogate
identification
…a case-by-case approach using e.g., carcinogenicity data
from closely related structures, if available, should usually be
developed to justify acceptable intakes…
SAR with
justification
FDA Guideline for Nitrosamines:
Where carcinogenicity study data for an impurity are of lesser
quality as described in ICH M7, a surrogate compound with
carcinogenicity data may be used to derive an acceptable intake AI for
NDSRI
but should be scientifically justified

EMA Article 5(3)

an approach based on SAR considerations to derive an
acceptable intake limit is acceptable, if appropriately justified
5 NOT FOR DISTRIBUTION
 Read-Across
Surrogate
identification
Read-across considerations
• Computational analysis
SAR with
• Structural alert environment and electronic influences justification

• Degree of substitution and steric hindrance/bulk

• Potential for metabolic activation AI for
NDSRI
• Molecular weight and physicochemical parameters

6 NOT FOR DISTRIBUTION

 Read-Across

7 NOT FOR DISTRIBUTION

8 NOT FOR DISTRIBUTION
 Comparison of Surrogate Selection
N N

N N

Activated α-carbon N
N O
N
N O

N O N O N O
N N
Read across N

N-Nitroso-1,2,3,6- N-Nitroso- N-Nitroso-

surrogate
tetrahydropyridine hexamethylenimine piperidine
(NTHP) (NHEX) (NPIP)
Proposed AI for NNV = 1300 ng/day
TD50 37 µg/kg/day 313 µg/kg/day 1300 µg/kg/day

9 NOT FOR DISTRIBUTION Proposed AI = 313 ng/day

 In vivo Genotoxicity

10 NOT FOR DISTRIBUTION

 In vivo Genotoxicity

?
?
11 NOT FOR DISTRIBUTION
 In vivo Genotoxicity

ICH M7 Q&A # 7.2

Q: If an Ames positive impurity is subsequently tested in an appropriate in vivo

assay and the results are positive, does that support setting compound-specific
impurity limits?

A: No. In vivo gene mutation assays are currently not validated to directly assess
cancer risk because the endpoint is mutation and not carcinogenicity (i.e., they are
used for hazard identification). Results from these tests could identify mode of
action and/or direct further testing strategy to complement the available data for a
weight of evidence approach.

12 NOT FOR DISTRIBUTION

 In vivo Genotoxicity

Negative Ames • (currently) not good enough to de-risk NDSRIs

tests • Can be used as part of WoE approach

Negative Ames test • Should be good enough to consider as Class 5

+ Negative TGR

Positive TGR • Cannot be used to set compound specific AI (PDE)

Positive TGR + • Cannot be used to set compound specific AI (PDE)

• Determine mutagenic potency by comparing
BMDL BMDL to nitrosamines with known carci data

Most NDSRIs are not potent mutagens (non-Cohort of Concern)

13
 In vivo Genotoxicity
• Measures mutagenicity (mutation
frequencies)
TGR • Long, expensive, low availability

• Detects DNA damage in

relevant organs (eg liver) • Measures mutagenicity
• Wild type animals (mutation frequencies)
Mutagenicity
• Short term admin (rapid results) • Wild type animals
• High correlation with TGR • detects down to 1 in 10
million mutations (narrow
• measures DNA strand Error confidence interval)
breaks in individual cells Corrected
Comet assay • Rapid results
(not a measurement of Duplex
mutagenicity) • No OECD guideline
Sequencing

Most NDSRIs are not potent mutagens (non-Cohort of Concern)

14
Control Options / Testing for Nitrosamines
Risk of Limited
robust carci Read- Bioassays WoE approach
formation
data across
Interim
Can AI be AI=1500 Non-CoC
Is there a risk Is there
Yes No derived by No Ames ng/day AI=1500
of formation of robust
SAR/read- test ng/day
a nitrosamine? carci data? -ive
across? -/+ive
Low potency
Comparative
Yes Yes In vivo
No comet/Duplex Relative
studies Sequencing potency
Calculate AI Is read-across No
No action assay analysis
by linear AI accepted by
needed +ive
extrapolatio HA? Exposure -/+ive
Yes
n from TD50 Yes in plasma +ive Calculate
TGR
or liver (If available) BMDL
Control at read- -ive
(PK/TK)
across AI
Class 5
No

Control
No exposure =
at ICH
15 NOT FOR DISTRIBUTION No tox risk (?)
Q3A/B
Questions?
Application of in silico tools
(QSAR) to predict toxicity of
nitrosamines
Dr David J. Ponting
Principal Scientist
[email protected]
 What are structure-activity relationships?
• SAR is the association of chemical structure with behaviour
• (Sub)structural features impact reactivity
• Metabolic activation
• Detoxification
• Phase II metabolism
• Reaction with water
• Reactions with DNA and proteins
• Shape of structure affects active site/receptor docking
• To metabolically active enzymes
• Non-covalent toxicity
• Properties of the entire structure affect ADME
• Does it penetrate a cell membrane?
• Does it bind to plasma protein?
• Does it cross the blood-brain barrier?
• Which organs are more exposed?
• Can it be excreted unchanged or is transformation needed?
• Quantitative and statistical (QSAR) or based on expert assessment (SAR)

Manso et al (2008), J. Phys. Org. Chem., 21, 932–938

 Principal SAR features
• Pharmacokinetics
• Carboxylic acids weak or negative
•
Capable of forming Can be DNA Alkylation
Already polar, and ionised in vivo persistent DNA adduct metabolised
• Skip phase I metabolism?
• Change preferred P450 isoform?
• Steric hindrance
• Anything bulkier than -CH2- results in significant diminution in activity
• Electronic interactions
• Carbonyls potent carcinogens
• Acidity of enol-like α-hydrogen promotes metabolism
• Benzylic/allylic also potent
• Conjugation stabilises radical and ionic intermediates
• C-H dissociation energy significantly reduced
• Strong EWGs reduce potency
• Electron withdrawal strengthens C-H bond
• Reduces rate of metabolic activation
Cross and Ponting (2021), Comput. Toxicol., 20, 100186; Thomas et al (2022), Chem. Res. Toxicol., 35, 1997-2013; Ponting et al (2022), J. Med. Chem., 65, 15584-15607
 ICH M7 Workflow

Class Brief Definition Proposed action for control

1 Known mutagenic carcinogen Control to compound-specific limit

2 Known mutagen, carcinogenic potential not Control to acceptable limits (i.e. appropriate
determined Threshold of Toxicological Concern (TTC))
3 Alerting structure, unrelated to structure of Control to appropriate TTC
the drug substance, no mutagenicity data
-OR-

conduct Ames test: If negative, treat as class

5; if positive, treat as class 2
4 Alerting structure, but the alert matches Treat as non-mutagenic impurity
that for the drug substance or related
compounds and these have been tested
and shown to be non-mutagenic
5 No structural alerts, or alerting structure Treat as non-mutagenic impurity
with sufficient negative mutagenicity or
carcinogenicity data

Figure from: Ponting et al, “Use of Lhasa Limited products for the in silico prediction of drug toxicity”, ch. 17 of In Silico Methods for Predicting Drug Toxicity, ed. Benfenati (2nd Edition), Springer
 Expert Systems – e.g. Derek Nexus
• Activate Derek alerts 007 (mutagenicity) and 070 (carcinogenicity)
• All N-nitroso except aromatic N-nitroso (other alert) and known negatives
• Toxicophore highlighted
• Alert description
• Examples
• References

Statistical Systems – e.g. Sarah Nexus

• Sarah has positive hypotheses for most nitrosamines
• Model based on public data
• Training set available for inspection
• Can augment with private data
• Those similar enough to known negatives report negative

Software versions: Nexus 2.5.2, Derek Nexus 6.2.1 with Knowledge version 2022.2, Sarah Nexus 3.2.1 with model version 2022.2
 Comparison of classification methods
• Two methods have recently been published:
• Structural features1
• Set of elements which can occur in a nitrosamine
• Each compound can contain many features
• Developed from small-molecule carcinogenicity data
• Structural classes2
• Set of orthogonal classes
• Each compound should fit into exactly one category
• Developed to cover specific chemical space fully
• Multiple methods of setting an AI from these exist
• Lowest reliable TD50 in class/with feature concerned2
• 5th percentile of all compounds with feature3
• Select structurally closest analogue within class1

1: Cross and Ponting (2021), Comput. Toxicol., 20, 100186; Thomas et al (2022), Chem. Res. Toxicol., 35, 1997-2013;
2: Dobo et al (2022), Chem. Res. Toxicol., 35, 475-489
3: Thomas et al (2021), Regul. Toxicol. Pharmacol., 121, 104875
 Feature-based predictions
• The features described can have statistically-significant effects
• When treated independently via a Bayesian model
• Can use features to assign potency categories
• And thence propose limits

Thomas et al. (2022), Chem. Res. Toxicol., 35, 1997-2013; Ponting et al (2022), J. Med. Chem., 65, 15584-15607
 ICH M7 Workflow

Class Brief Definition Proposed action for control

1 Known mutagenic carcinogen Control to compound-specific limit

2 Known mutagen, carcinogenic potential not Control to acceptable limits (i.e. appropriate
determined Threshold of Toxicological Concern (TTC))
3 Alerting structure, unrelated to structure of Control to appropriate TTC
the drug substance, no mutagenicity data
-OR-

conduct Ames test: If negative, treat as class

5; if positive, treat as class 2
4 Alerting structure, but the alert matches Treat as non-mutagenic impurity
that for the drug substance or related
compounds and these have been tested
and shown to be non-mutagenic
5 No structural alerts, or alerting structure Treat as non-mutagenic impurity
with sufficient negative mutagenicity or
carcinogenicity data

Ponting et al (2022), “Use of Lhasa Limited products for the in silico prediction of drug toxicity”, ch. 17 of In Silico Methods for Predicting Drug Toxicity, ed. Benfenati (2nd Edition), Springer,
What about Expert Review?
• Required for in silico predictions under ICH M7 & is essential for each
impurity that is processed
• Used to ensure predictions are relevant & accurate
• Used to conclude assessment of activity based on predictions

• Often straightforward, but some situations are harder to resolve

• How do I conclude if Derek and Sarah disagree?
• How do I find relevant information from the software to support my conclusion?
• How do I document this in a concise way for a regulator?

• Often completed with recycled arguments for common prediction scenarios

• How can I make expert review consistent and efficient to save time?
Nexus 2.3 – Expert Review workflow

Following an ICH M7 prediction, the results from Derek & Sarah are
evaluated & arguments relevant to those predictions are presented to
the user, guiding the expert review process.

The user may add their own custom arguments, for example if they
have proprietary knowledge that is relevant to the review.
 Integrating Derek & Sarah

When an ICH M7 prediction is run, specific information relating to Derek & Sarah is highlighted in the Sarah prediction results:
• Do the Sarah training examples activate Derek mutagenicity in vitro alerts?
• Do the Sarah hypotheses relate to any activated Derek mutagenicity in vitro alerts?
• Have the Sarah training examples which are non-mutagenic been tested in the most appropriate strains?
Nexus 2.4 – Cohort of Concern flags

New arguments alert the user to compounds which are expected to

belong to a Cohort of Concern. These indicate to the user that they
are required to undertake a compound-specific risk assessment for the
compound to comply with ICH M7 and regulatory requirements.
Selection of an argument shows that this has been addressed
specifically by the user.
Nexus 2.5 - refining the Cohort of Concern
• N-N=O substructure does not always imply potency
• α-Hydrogen is required for diazonium mechanism and thus high potency
• If the amine nitrogen is aromatic, disruption of aromatic system is unlikely
• Can still be mutagenic (but not CoC) via alternative mechanism
• Nitrosated primary amines considered likely to be unstable
• Hetero-substituted amines (e.g. nitrosated hydroxylamines) weak
• Now implemented into Nexus 2.5
Aromatic
Different mechanism

No α-hydrogen No α-hydrogen
Very weak carcinogen Aromatic
Different mechanism? No α-hydrogen
Nitrosohydroxylamine
No α-hydrogen Primary amine Weak carcinogen
Carcinogenicity negative Unstable Different mechanism?
Publications so far
Acknowledgements
• Lhasa Limited
• Rachael Tennant, Rob Thomas, Susanne Stalford, Rob Foster, Andrew Thresher, Grace
Kocks, Anne-Laure Werner, Fernanda Waechter, Carolina Martins-Avila, Ash Ali, Mike
Burns, Ro Lopez-Rodriguez, Anax Oliveira, Rich Williams
• Key Collaborators
• Kevin Cross, Melisa Masuda-Herrera, Alejandra Trejo-Martin, Joel Bercu, Krista Dobo,
Amit Kalgutkar, Joerg Schlingemann, Andy Teasdale, Graham Smith, Ant Lynch, Jim
Harvey, Andreas Czich, Raphael Nudelman, Naiffer Romero
• And many, many more!

Lhasa Limited +44(0)113 394 6020

Granary Wharf House, 2 Canal Wharf [email protected]
Leeds, LS11 5PS www.lhasalimited.org
Registered Charity (290866)

Company Registration Number 01765239

 FDA Overview
Control of Nitrosamine Impurities
in Human Drugs

Andre Raw, PhD

Associate Director for Science and Communication
Office Of Lifecycle Drug Products
Office of Pharmaceutical Quality

1
Pharmaceutical Quality
A quality product of any kind consistently
meets the expectations of the user.

www.fda.gov

2
Pharmaceutical Quality
A quality product of any kind consistently
meets the expectations of the user.

Drugs are no different.

www.fda.gov

3
Patients expect safe and effective
medicine with every dose they take.

www.fda.gov

4
 Pharmaceutical quality is
assuring every dose is safe and
effective, free of contamination
and defects.

www.fda.gov

5
 It is what gives patients confidence
in their next dose of medicine.

www.fda.gov

6
The U.S. Food and Drug Administration is alerting health care professionals and patients of a voluntary recall of
several drug products containing the active ingredient valsartan, used to treat high blood pressure and heart
failure. This recall is due to an impurity, N-nitrosodimethylamine (NDMA), which was found in the recalled
products. However, not all products containing valsartan are being recalled. NDMA is classified as a probable
human carcinogen (a substance that could cause cancer) based on results from laboratory tests. The presence of
NDMA was unexpected and is thought to be related to changes in the way the active substance was manufactured.

7
 Over the past several years, industry and regulators have
learned a lot about what factors lead to the risk of nitrosamine
impurities in pharmaceuticals

8
 What are Nitrosamines?
• What are Nitrosamines?
H
R1 R1
HNO2 -HX
N H R1 N N O X N N O
N O
R2 R2
R2
Secondary, tertiary,
or quaternary amines

• Nitrosamines are
 Probable or possible human carcinogens
 Potent genotoxic agents
 “Cohort of concern” compounds in the ICH M7(R1)
ICH M7 (R1) Guidance: Assessment and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals To
Limit Potential Carcinogenic Risk (March 2018)
www.fda.gov https://www.fda.gov/RegulatoryInformation/Guidances/default.htm. 9
 Cohort of Concern with
Stringent Intake Limits
• Acceptable Intake Limits (AI)
Table 1. AI Limits for Nitrosamines in Drug Products

Nitrosamine AI Limit (ng/day)1,2

NDMA 96
NDEA 26.5
NMBA 96
NMPA 26.5
NIPEA 26.5
NDIPA 26.5

1 The AI limit is a daily exposure to a compound that approximates a 1:100,000 cancer risk after 70 years of
exposure.
2 The conversion of the AI limit into ppm varies by product and is calculated based on a drug’s maximum daily

dose (MDD) as reflected in the drug label (ppm = AI (ng)/MDD (mg)). 10

 Root Causes of Nitrosamine Impurities in APIs
and Drug Products
 Properties of the  Use of recovered or
starting materials, Process Supply recycled materials or
intermediates or Related Chain other intermediates
drug substance contaminated with
 Specific process nitrosamines
conditions Nitrosamines  Cross-contamination
in the Drug in multi-purpose
 Impurities in or Substance
reactions with and/or Drug facilities
raw materials Product

 Stability of drug substance or drug product

11

Stability  Excipient compatibility

- 11 -
Potential Nitrosamine Impurities Generated
During the Synthesis of Drug Substances
H
N N
N N Br - O
O N

DMF TEA DIPEA TBAB NMP

R1 R2
N N
+ Na
O O
H

O OH

N N N N N
N N
O N N N
O O O N N
O O
NDMA NDEA NEIPA NDIPA
NMBA NDBA 12
 Nitrosamine Drug Substance Related Impurities (NDSRIs)
From Synthesis of Drug Substances

R1 R2 R5 R6
Nitrite Source R3 R4 N
N N
H H
H
Starting Material/ Active Ingredient
Intermediate
+
+

R3 R4 R5 R6
N Carry-over in synthesis N

N N
O O

Undesired Impurity NDSRI

13
 Synthesis of Drug Substances

Lessons Learned: Hidden sources of precursors

• Substantial quantity of sodium nitrite in sodium azide.

• Contaminating amines in bases/catalysts.

• Degradation of amide solvents that generate secondary amines.

• Amine contaminants present in starting materials or intermediates.

• Secondary and tertiary amine functional groups on intermediates and API molecules.

14
 Root Causes of Nitrosamine Impurities in APIs
and Drug Products
 Properties of the  Use of recovered or
starting materials, Process Supply recycled materials or
intermediates or Related Chain other intermediates
drug substance contaminated with
 Specific process nitrosamines
conditions Nitrosamines  Cross-contamination
 Impurities in or in the Drug in multi-purpose
reactions with raw Substance
and/or Drug facilities
materials Product

 Stability of drug substance or drug product

15

Stability  Excipient compatibility

- 15 -
Lessons Learned: Solvents

• Use solvents of appropriate grade.

– Exercise due diligence when choosing vendors
– Is vendor recycling solvents?
– How are tankers cleaned?

• Process understanding should extend to recovered solvents.

• Analytics: Attention to “new unknown” peaks

16
 Root Causes of Nitrosamine Impurities in APIs
and Drug Products
 Properties of the  Use of recovered or
starting materials, Process Supply recycled materials or
intermediates or Related Chain other intermediates
drug substance contaminated with
 Specific process nitrosamines
conditions Nitrosamines  Cross-contamination
 Impurities in or in the Drug
Substance in multi-purpose
reactions with raw and/or Drug facilities
materials Product

17
 Stability of drug substance/drug product
Stability  Excipient compatibility
- 17 -
 Stability Failure Modes
Evaluate Inherent Propensity of the
Active Ingredient to Generate Nitrosamines
O
O NH N
+ N
- N S N
O N O
H

Ranitidine NDMA

FDA Requests Removal of All Ranitidine Product (Zantac) from the Market
https://www.fda.gov/news-events/press-announcements/fda-requests-removal-all-ranitidine-
products-zantac-market
18
 NDSRIs Formed in Drug Product
During Manufacturing and/or Shelf-Life

O
H N
Residual Nitrites in Drug
Product Formulation
N N
R1 R2 R1 R2

NDSRI
Active Ingredient Derivative of Active Ingredient Structure

Processing Steps to purge NDSRIs is not possible for those generated in drug products

19
 Excipients/Water: Common Source of Nitrite

Possible Nitrite Source: Processing water, processing steps requiring acid

titration, bleaching, and oxidation of air as excipient is being heated in a drying process

www.fda.gov
Wu, et al. AAPS PharmSciTech, 2011, 12(4), 1248-1263 20
 Risk Assessment Should Consider this Failure
Mode that Leads to NDSRIs in Drug Products
From FDA Nitrosamine Guidance

Nitrites are common nitrosating impurities that have been reported

in many excipients at ppm levels. Nitrite impurities are found in a
range of commonly used excipients, which may lead to nitrosamine
impurities forming in drug products during the drug product
manufacturing process and shelf-life storage period.

21
 If Risk for Creation of NDSRIs in Drug Product

Considerations for Risk Mitigation based upon Control/Design

(Not All-Inclusive List)
 Control of Formulation Inputs

● Work with your excipient supplier to control residual nitrites

From FDA Nitrosamine Guidance: The supplier qualification program should take into
account that nitrite impurities vary across excipient lots and may vary by supplier.
Drug product manufacturers should also be aware that nitrite and nitrosamine
impurities may be present in potable water.

● During Development: Selection of formulation excipients less likely to contain nitrites.

23
 Formulation Design (Additive Inhibitors)
Environmental N-Nitroso Compounds Analysis and Formation
IARC Scientific Publication No. 14 (1976), Ziebarth, D. and Scheunig, G. pages 279-290
O
NH 0.4 µmol Sodium Nitrite
N
HN N
25 mL Gastric Juice
NH
Piperazine 60 min at 37 C

24
 Formulation Design Mitigation
Inhibition of N-Nitrosamine Formation in Drug Products: A Model Study
Nanda et al. Journal of Pharmaceutical Sciences (August 2021)
NH Manufacture Tablets (100 mg with 10% 4-PPHCl )
HCl Common Excipients (known to contain nitrite)
Spike with Anti-Oxidant Inhibitors (0.1% wt, 1 wt%)

Stress at 50 C/75% RH for 1 month

4-phenylpiperidine hydrochloride (4-PPHCl)

Inhibitor Level Growth on nitrosamine (ppb) Inhibition Efficiency (%)

No inhibitor 345 N/A

Ascorbic Acid 0.57 µmole (0.1 wt%) 283 17.9

5.7 µmole (1.0 wt%) -72 120.9

Sodium Ascorbate 0.57 µmole 344 0.3

5.7 µmole 30 91.3

Ferulic Acid 0.57 µmole 137 60.3

Caffeic Acid 0.57 µmole 129 62.6

5.7 µmole -72 120.9

α- Tocopherol 0.57 µmole 148 57.1

5.7 µmole 64 81.5 25

 Formulation Design Mitigation (Addition of Anti-Oxidant)
NDMA Formation in Experimental Batches of Metformin Film Coated Tablets .

Effect of Anti-Oxidants
50 C, 75% RH, 25 day

Without BHT BHA Propyl

Gallate

N-Nitrosodimethylamine formation in metformin hydrochloride sustained-release tablets: Effects of metformin

and hypromellose used in drug product formulation
Hao et, al Journal of Pharmaceutical and Biomedical Analysis, 222 (2023) 26
 Formulation Design (Impact of pH)
Environmental N-Nitroso Compounds Analysis and Formation
IARC Scientific Publication No. 14 (1976), Ziebarth, D. and Scheunig, G., pages 279-290
O
NH 0.4 µmol Sodium Nitrite
N
HN N
25 mL Gastric Juice
NH
Piperazine 60 min at 37 C

27
 Formulation Design Mitigation
NDMA Formation in Experimental Batches of Metformin Film Coated Tablets .

NDMA NDMA
Inital T =0 60 oC/75% RH, 7 days
Control < LOQ 31 ppb

H2O2 (400 ppm) < LOQ 33 ppb

0.5% Na2CO3 + H2O2 (400 ppm) < LOQ < LOQ

H2O2 (400 ppm) + dimethylamine HCl (500 ppm) < LOQ 43 ppb
0.5% Na2CO3 + H2O2 (400 ppm) + dimethylamine HCl (500 ppm) < LOQ < LOQ

“pH modification of the tablets by the addition of Na2CO3 was proven to be effective in terms of removing
the DMA precursor from the tablets and stopping N-nitrosation completely, no matter the pathway”

Inhibition of N-Nitrosamine Formation in Drug Products: A Model Study

Jires et. al. Journal of Pharmaceutical and Biomedical Analysis, 218 (2022) 28
 FDA Communication Nov. 18, 2021
Discusses these Possible Mitigation Strategies for NDSRIs

29
 Acknowledgements

• Colleagues from OPQ (7 sub-offices)

• Colleagues from OND, OGD, ORA
• OPQ Nitrosamine Workgroup
• CDER Task Force Workgroup

30
Thank You!
 Empowering a healthy tomorrow

Don’t ignore the small peaks.

Tackle impurities NOW.