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Regulatory Highlights
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ABSTRACT: It is almost inevitable that any regulatory highlights review at present will be dominated by N-nitrosamines.
Therefore, “where are we?” and “what next?” are key questions that we explore within this review.
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s a reminder, it has now been nearly 3 years since the first
A recycled solvents, catalyst recycling, and poor cleaning
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reports of contamination of valsartan with an N-
nitrosamine, N-nitrosodimethylamine (NDMA).1−3 It quickly
became apparent that in relation to sartans there were two
separate root causes. The first was related to the inadvertent
creation of conditions whereby N-nitrosamines could be
generated with the process due to the presence of a secondary
amine impurity and a nitrosating agent within the same process
step. The presence of a nitrosating agent was the result of one
of a number of process changes made in relation to the
valsartan manufacturing process: sodium nitrite was introduced
in order to quench excess sodium azide. Sodium azide had
been introduced as a replacement for tributyltin azide in the
formation of the tetrazole ring (see Figure 1). Parallel to this,
Figure 1. Valsartan tetrazole synthesis.
the reaction solvent was also changed from xylene to N,N-
dimethylformamide (DMF). It was clearly not appreciated that
traces of the secondary amine dimethylamine (DMA), which
could be present in the DMF solvent either as an impurity or
as a degradant formed in situ during the reaction, could lead to
N-nitrosamine formation.
Unfortunately, and simultaneous to the above example,
other sartans and also non-sartans were found to contain N-
nitrosamines. In several examples, the presence of N-nitros-
amines could not be attributed to the processing chemistry
described but instead was related to poor manufacturing
practice that was linked to issues such as contaminated
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procedures.
As highlighted in previous reviews,4−7 this did not remain
isolated to issues relating to the chemistry and manufacturing
practice. N-Nitrosamines were found in other products,
including ranitidine, metformin, and several others. What is
interesting is that several additional root causes were seen:
1. Intermolecular reaction, in the case of ranitidine.8−11 To
investigate this, GlaxoSmithKline undertook a root-cause
analysis,12 which suggested that the presence of NDMA
results from slow degradation of the ranitidine molecule.
Analysis using suitably isotopically labeled ranitidine
hydrochloride confirmed the formation of NDMA solely
from an intermolecular reaction of ranitidine hydro-
chloride without the involvement of impurities. Factors
that influence the rate of degradation include heat,
humidity, and the crystal morphology of ranitidine
hydrochloride, with material exhibiting a columnar habit
showing a lower rate of degradation.
2. Formation in the drug product, typically resulting from
the reaction of a secondary amine active pharmaceutical
ingredient (API) with traces of nitrite found within
excipients.
3. Packaging-related risks due to the use of nitrocellulose-
based lidding foils.13
As a result of these, in September 2019 the European
Medicines Agency (EMA) triggered a review of all small-
molecule drug products,14 which was later followed by other
regulatory authorities, and the EMA further extended this
review to biologics in July 2020.15 The first phase of these
assessment processes ended in March 2021 for the majority of
authorities.
The aim of this review is not to revisit how we reached the
current position, as that is covered in previous reviews and
those of others,16 but instead to describe what happens next
and the challenges we still face.
Published: July 21, 2021
https://doi.org/10.1021/acs.oprd.1c00270
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Organic Process Research & Development
■
STEP 1 OUTCOMES
The aim of the step 1 EMA process was to identify risk/no risk
in the context of N-nitrosamines, but even this is challenging
because what constitutes “no risk” has not been defined. Is
there ever a situation that can be said to be “no risk”, especially
a hypothetical one? Indeed, the boundaries for this risk
assessment were never clear, e.g., should “no risk” be defined in
terms of inability to exceed a defined virtually safe dose (as is
the case in ICH M717) or no potential for formation
whatsoever, however fanciful the pathway?
The step 1 process was extremely complex and time-
consuming, hampered by challenges of obtaining the
potentially proprietary third-party information required to
complete the step 1 risk assessment, especially when
manufacturers believed their own risk assessment would
suffice. Levels of information sought ranged from processing
aspects for the registered route and the starting materials to the
quality of processing water and included Good Manufacturing
Practice (GMP) aspects of solvent recycling. Additional
challenges included the following:
1. The need to conduct extensive scientific investigations
to understand and quantify the risk associated with drug
product (DP) packaging.
2. Multiple reporting mechanisms, where each authority
required different information and formats.
The evidence shows that despite the above challenges,
substantive progress was made, and the vast majority of step 1
assessments were completed. So what was highlighted as a
potential risk? Anecdotal evidence has suggested that around
5−10% of all products identified a risk, and therefore, further
evaluation is required (i.e., progression to step 2). The above
numbers hide some of the realities of the situation. Given the
challenge of defining risk/no risk without a clear framework,
there is a considerable chance that companies will have
interpreted this differently, despite the efforts of EFPIA to
provide practical guidance.13
In addition, in cases where the license holder had insufficient
information, it is very likely that this led to testing, as many
companies will have taken a particularly conservative approach.
Ultimately, does this lack of knowledge lead us toward
continued testing, where actually a bit more knowledge would
have provided a “no identified risk” conclusion from step 1?
A further concern is that for older generic products, one
potential scenario is that one company may have identified a
potential risk whereas another may have identified no risk for
the same API/DP. How will this be addressed? Will authorities
simply revert to the lowest-risk scenario and request test data
in all cases? Reports of regulatory questions of this nature have
already been shared.
■
STEP 2 CHALLENGES
Analysis. The apparent fact that 10% of all products require
further testing presents in itself a huge challenge. The analysis
of N-nitrosamines at typically parts per billion levels is at the
cutting edge of technology and practical expertise. Does the
capacity in terms of both equipment and expertise exist to do
this? We already know of challenges of contamination, false
positives,7 and sensitivity faced in such analysis. These are
further compounded by other issues such as analyte stability
and complex sample matrices. All of these are exacerbated by
the extremely conservative limits applied, a point that is further
examined below.
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The magnitude of the task was further increased by the
regulatory requirement to examine 10% of all annual drug
product batches and to show that the N-nitrosamine levels are
<10% of the acceptable intake.15
ICH M7. Most, if not all, of the guidelines relating to N-
nitrosamines make reference to ICH M7 and the need to
follow the principles of this guideline. The problem is that the
N-nitrosamine guidelines themselves reference additional
considerations. Specific examples include the following:
1. The ambiguous challenge to the use of ICH M7 control
options for N-nitrosamine impurities, in particular
control option 4.
2. The use of Haber’s law and the principles of adjusted
less-than-lifetime (LTL) limits.
3. How to calculate an actual limit? The agencies (as
further illustrated below) have issued their own limits,
but are these aligned/justified with the principles of ICH
M7?
4. Perhaps the most concerning of all, the use of an Ames
test to confirm the mutagenic potential of an N-
nitrosamine.
Many of these challenges are clearly safety-related, but they
impact directly on the quality, specifically the analysis of
products during step 2. They drive analytical testing limits that,
particularly for high-dose products, are close to unachievable,
especially when there is a need to show that the levels are
below 10% of the acceptable limit to avoid longer-term testing
as stipulated in the EMA Q&A process.15
■ SAFETY CHALLENGES
There are numerous safety challenges associated with nitros-
amines, and a proportion of these are further discussed below.
Arguably the biggest single issue is the uncertainty around
the acceptability of the Ames test to mitigate the risk
associated with N-nitrosamines. At a recent U.S. Food and
Drug Administration (FDA) webinar,18 this was called into
sharp focus. The clear indication was that the bacterial reverse
mutation assay (Ames test) was considered by some at least to
be inadmissible and that for N-nitrosamines it may provide
insufficient proof of absence of concern. However, much of
this appeared to be based on previous experiences with poorly
designed Ames tests conducted on N-nitrosamines dating back
more than two decades. While there was some agreement that
a “well-engineered” Ames test would be appropriate to
understand potential mutagenicity, no clear position was
reached. What are the specific concerns with use of the
Ames test to understand potential mutagenicity of N-
nitrosamines? Comments have been made about various
factors of the Ames test that impact its performance regarding
N-nitrosamines. These include the following:
• The use of DMSO as the solvent, which can suppress
P450 enzymes.
• The use of preincubation.
• The use of rat S9 (the indication being that this should
be substituted in preference for hamster S9).
While there have been historical issues with the use of the
Ames test, there is a lack of clear evidence that the Ames test
would miss N-nitrosamines as a potent cohort of concern. To
reinforce this, Thresher et al.19 showed the correlation between
mutagenicity and carcinogenicity to be equivalent to if not
better than that seen in any other class of mutagen.
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Also of concern is the suggestion that the Ames test can
simply be substituted for the alternative transgenic study. This
clearly contradicts the testing paradigms defined in the
international guidelines ICH S2 (R1) (Genotoxicity Testing
and Data Interpretation for Pharmaceuticals Intended for
Human Use)20 and ICH M7 (Assessment and Control of
DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to
Limit Potential Carcinogenic Risk).17
Other areas of concern involve limits. The EMA guideline15
contains the limits shown in Table 1. However, it is not clear
Table 1. Limits Presented in the EMA Guideline15
N-nitrosamine (CAS number) limit (ng/day)a
NDMA (62-75-9) 96.0b
NDEA (55-18-5) 26.5b
EIPNA (16339-04-1) 26.5c
DIPNA (601-77-4) 26.5c
NMBA (61445-55-4) 96.0c
MeNP (16339-07-4) 26.5c
NDBA (924-16-3) 26.5c
NMPA (614-00-6) 34.3b
a
These limits are applicable only if a finished product (FP) contains a
single N-nitrosamine. The conversion to a specification limit in parts
per million for a particular medicinal product is calculated by dividing
the limit in this table (in ng/day) by the maximum daily dose (in mg)
of a given product as reflected in the Summary of Product
Characteristics (SmPC). bLimit calculated on the basis of the
harmonic-mean TD50 derived from the Carcinogenic Potency
Database (CPDB). cLimit derived using the structure−activity
relationship (SAR)/read-across approach.
how these limits were derived and whether they are correct. As
an example, for N-nitrosomethylphenylamine (NMPA)
(Figure 2), the EMA reports the limit as 34.3 ng/day, and
the FDA requests a limit of 26.5 ng/day, whereas the Lhasa
database21 recommends a higher limit of 106 ng/day. Which of
these is the “correct” value? ICH M7 needed an addendum
Figure 2. Discrepancies in potential limits for NMPA.
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table to resolve this,17 and it is likely that the same process will
be needed for N-nitrosamines.
Perhaps a greater challenge is the EMA limit of 18 ng/day
prescribed in the absence of any specific carcinogenicity data
for a novel N-nitrosamine. It is difficult to see the logic of this
value on the basis of the available carcinogenicity data,
especially in the context of larger “API-like” N-nitrosamines
that may potentially be formed during the formulation process,
especially where the drug itself is a secondary amine. While a
definitive correlation among molecular weight and steric,
electronic, and other factors is yet to be established or
confirmed, there is clear evidence to show that potency is
related to these factors. Indeed, the limit of 18 ng/day for a
novel N-nitrosamine may not be supported by the evidence,
and its continued application runs the real risk of impacting the
availability of several well-established products. It is therefore
critical that an additional weight-of-evidence rationale can be
developed in order to establish an acceptable daily intake for
novel N-nitrosamines that takes into account the above factors
and derives a more accurate safety control figure.
Another area of significant concern relates to the use of
Haber’s law and its application to N-nitrosamines. The most
recent release of the Q&A document15 (June 29, 2021, EMA/
409815/2020 Rev.4) states that adjustments based on duration
cannot be made for N-nitrosamines. This is believed due to
concerns that higher short-term doses may overload repair
mechanisms. One may take the view that such a precautionary
approach was expedient, but is it required from a safety
perspective? On the basis of a comprehensive study of NDEA,
Bercu et al.22 showed that data correlating to exposure
duration (as a percentage of lifespan) and cancer incidence in
rodent bioassays indicate that the LTL acceptable intake (AI)
as derived using the ICH M7 framework17 would not exceed a
negligible additional risk of cancer. Therefore, controlling N-
nitrosamines to an LTL AI based on the ICH M7 framework is
demonstrated to be protective for potential carcinogenic risk to
patients over the typical exposure durations of clinical trials
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Organic Process Research & Development
and many prescribed medicines. However, the question is
whether this new evidence will change current regulatory
policy.
■
WHAT/WHERE NEXT?
The next step for those products defined as “at risk” and thus
part of step 2 is to determine the extent of the risk. Will it be
possible to take science into account in conducting step 2? For
example, if the risk is related to the synthesis, can testing be
focused solely on the API? If the risk is related to drug product
stability, can this be factored into the testing program or will it
simply have to remain focused on 10% of drug product batches
on an annual basis?
Allied to this, of course, is the question of the limits as
described above. Who decides these limits? For a novel N-
nitrosamine, this would surely need to be the maximum
allowable amount (MAA). However, what if a limit is proposed
that authorities do not support? Would this invalidate the step
2 testing results? Industry will likely analyze to the most
conservative levels provided by regulators, but what is the
approach if the analytical capability is not available to achieve
this, particularly for higher-dose compounds? Will there be an
insistence to analytically confirm absence at a higher level for a
potential impurity that was only ever a “hypothetical risk”?
Arguably, though, the biggest problem from a practical
perspective is the scale of the challenge. Time will tell whether
this is practically feasible.
Understanding of the science will be key. Significant
progress has already been made, as highlighted in the paper
by Bercu et al.22 and other papers (e.g., the risk associated with
nitrite in water published by Ashworth et al.23). We are
confident that industry will look to replace assumptions with
facts and work together to share experiences. To do so is
critical. The validity of the Ames test for confirming
mutagenicity (or not) of an N-nitrosamine needs resolution,
as it brings significant and sometimes enormous complications
to the management of this issue. It is to be hoped that with
increased scientific understanding of both safety and quality
factors, current regulatory guidance can be aligned though the
appropriate regulatory consultative dialogue. What happens
next in relation to ICH M7? It is to be hoped that ICH M7
guidance will ultimately be further updated to capture effective
and appropriate control of N-nitrosamines, which after all are
potentially mutagenic and thus very much in scope of the
guidance and its principles.
Whatever the future holds, it is clear that this issue is far
from resolved and that bumps in the road are inevitable. Let us
all hope that some context remains in the balance between risk
and critical medicines and their supply.
Andrew Teasdale
Michael W. Urquhart orcid.org/0000-0002-8626-0123
■
AUTHOR INFORMATION
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.oprd.1c00270
■
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