NITROSAMINES RISKMITIGATION:

The critical role of excipients and supplier qualification
Alberto Berardi, Pauline H.M. Janssen, Bastiaan H.J. Dickhoff and Sander van Gessel
DFE Pharma, Goch, Germany

Introduction
The evaluation of nitrosamines in drug products has become a big challenge for many formulators in the pharmaceutical industry. In 2018, the European Medicines Agency (EMA)
reported the recall of several drugs containing Valsartan, because of contamination with anitrosamine impurity. Nitrosamines are potential genotoxic agents and some are classified
as probable or possible human carcinogens (Brambilla and Martelli, 2007). The maximum
acceptable intake limits for six species of nitrosamines commonly found in pharmaceuticalpreparations ranges between 26.5-96 ng/day (EMA, 2022a; US FDA, 2021).
Nitrosamine impurities typically originate either from the manufacture of drug substances or are formed in the formulation during shelf-life storage period. Nitrosamines that areformed during drug substance manufacture can typically be purged in subsequent steps, oradditional purification steps can be implemented. Nitrosamines that are formed in the final
drug product are however not possible to be purged. In case of vulnerable APIs, like secondary amines, nitrosamines can be formed by a reaction with a nitrosating agent, like nitrite
under appropriate conditions (for example elevated temperatures, acidic conditions, liquid
phase). Figure 1 describes the N-nitrosamine formation for secondary amines upon contact
with nitrite. Nitrite is the most important precursor of the ultimate nitrosating agents dinitrogen trioxide (N2O3) and nitrosyl ion (NO+). Nitrates are a secondary focus of attention,
given the possible conversion of nitrate to nitrite upon reduction (Boetzel et al., 2022; Lin,
1990; Nanda et al., 2021). Taking into consideration that nitrites are typically only present
in low concentration, the build-up of nitrosamines for sensitive APIs can be expected to be
proportional to the quantity of nitrite collectively present in the dosage form.

NO2- + H+

HNO2

NO2- + H+

HNO2

HNO2 + H+

H2NO2+

HNO2 + H+

NO++H2O

H2NO2 + NO2+

N2O3 + H2O

H2O H3O+(R)2NH + N2O3(R)2N - N=O + NO2

H2O H3O+(R)2NH + NO+(R)2N - N=O

Figure 1. Pathways of N-nitrosamine formation from secondary amines

1Market Authorization Holders (MAHs) are now requested by several regulatory authorities to reduce the presence of nitrosamine impurities in their drug products (EMA, 2022b,
2022a; US FDA, 2021). The FDA and the EMA both suggest a three-step mitigation strategy that both API and drug product manufacturers should follow. The first step includes theperformance of a risk assessment, the second is the confirmation of the identified risks bytesting, and the third consists of reporting changes implemented to prevent and reduce theformation of nitrosamine impurities in drug products.
Risk assessments by the drug product manufacturer should be designed to evaluate the potential sources of nitrosamine formation and contamination during manufacturing of drug
products. Excipients are considered as a potential risk factor during the drug product risk
assessment; the risk of the presence of nitrosamines is very low. However, many excipients contain traces of nitrites that can ultimately result in formation of nitrosamines under
specific conditions within the drug product. Excipient suppliers should provide information
about excipients to assist the MAH in their evaluation of the risk of the presence of nitrosamine impurities in the final drug product (IPEC Federation, 2022).
Multiple mitigation strategies are described in the guidance documents of the FDA and the
EMA, all aiming to reduce nitrosamines formation (EMA, 2022b; US FDA, 2021). Two key
strategies include formulation design and supplier qualification, although it is also encouraged to consider innovative mitigation strategies.

Formulation design
All formulation ingredients, including excipients can contain nitrosating precursor substances,like nitrites, which could contribute to the formation of nitrosamines in drug products. Reformulation towards different excipients with lower nitrite content could therefore be beneficial tosuppress the formation of nitrosamines. Another reformulation strategy could include the addition of antioxidants, such as ascorbic acid or alpha-tocopherol, to inhibit the reaction (Mergens,1982; Nanda et al., 2021; Ziebarth and Scheunig, 1976). Reformulation strategies with additionof inhibitors should however be considered with care, as inhibitors are typically highly reactive
species which could interact with the API or other chemical entities within the dosage form. It
is also thought that the rate of nitrosamine formation can be inhibited by increasing the pH of
the micro-environment, as the nitrosamine formation reaction is catalyzed by acidic conditions(Ziebarth and Scheunig, 1976). The incorporation of pH-modifiers, like sodium carbonate, could
therefore reduce the formation of nitrosamines as well. Also in this case, care should be takenthat pH-modifiers do not interfere with the stability of the API.

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Supplier qualification
Supplier qualification is a mitigation strategy that is also described in the FDA guidance andthat might have less impact on the registration dossier of the drug product. This is based on theobservation that nitrate and nitrite levels can significantly differ across excipient lots and suppliers (Boetzel et al., 2022). Many excipient suppliers provide nitrite information based upon risk
assessment only. Alternatively, when data is available, nitrite content might be measured withanalytical methods that are poorly sensitive (i.e. unable to detect low levels of nitrites), thus,
making differentiation between suppliers difficult.

Benchmarking excipient types and suppliers on nitrites levels
Whether MAHs opt for the strategy of reformulation with different excipients or supplier qualification, the scientific literature can serve as a valuable tool to appreciate the extent of excipient-to-excipient, supplier-to-supplier and batch-to-batch variability in nitrite levels. In an early
investigation, Wu et al. screened the levels of nitrites and nitrates of various fillers, superdisintegrants, binders, lubricants and glidants (Wu et al., 2011). Results showed that these impurities areomnipresent in excipients, although the levels seemed highly dependent on the both the excipient type and the supplier. It was suggested that this is due to minor manufacturing differences,as processing water, acid titration steps, bleaching and oxidation of air during drying, which could
all contribute to the presence of these impurities in the excipients. More recently, Lhasa Limited
has built a “Nitrites in Excipients” database and some of the results are shared in the article ofBoetzel et al. The main findings were that 1) both average and variance in nitrites levels are highly
different among excipients; and 2) with the same excipient type, different suppliers show majordifferences in values (Boetzel et al., 2022).
Excipients for which reformulation (i.e. changing to an alternative excipient) or supplier qualification (i.e. changing to an alternative supplier) is more important are those that are present inlarge percentage in the formulation, as is the case of fillers. The contribution of impurities in these
excipients is relatively higher to that of low-dosed ingredients such as disintegrants, glidants
and lubricants. However, even for the low-dosed excipients, selection shall also be considered insome specific cases. For example, the superdisintegrant crospovidone showed a mean level ofnitrites which was 9 to 12-fold higher than that of the fillers microcrystalline cellulose and lactose.
Therefore, the overall contribution of nitrites coming from crospovidone shall be considered high,despite its relatively low concentration in the formulation. Replacement of crospovidone withcroscarmellose sodium (15-fold lower mean value of nitrite) could be a viable option for formulations containing sensitive APIs (Boetzel et al., 2022).
Within the same excipient type, different suppliers provide excipients with distinctively different
levels of impurities. Table 1 shows the median nitrite levels of eight suppliers of MCC, as reported
by Boetzel et al. It should be noted that the number of samples tested is different for each supplier. For more details readers can refer to the original article (Boetzel et al., 2022). The mediannitrite level of different suppliers can vary substantially, up to a factor of more than 16.

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Table 1. Nitrite levels (median) in MCC from different vendors (Boetzel et al., 2022). ppm = µg/g

Supplier
Nitrite

#1

#2

#3

#4

#5

#6

#7

#8

1 ppm

0.1 ppm

1 ppm

0.2 ppm

1 ppm

1.2 ppm

0.2 ppm

1.6 ppm

Nitrites and nitrates in DFE Pharma excipients
DFE Pharma is one supplier of excipients that from the early beginning has tried to understandthe issue of nitrosamines formation. The basis of their expertise relates to the knowledge gainedby the mother company FrieslandCampina. This dairy company already had experience in nitrosating agents, because of the strict regulations regarding contaminants in (infant) food (European Union, 2006). The filled IPEC Europe questionnaire was the first step of DFE Pharma to
support MAHs with their nitrosamine risk assessment. This questionnaire includes a matrix toconsider the structure and the origin of the excipient as a first indication of risk. Factors considered were the presence of nitrosating agents in the production process, the type of water that isused, the use of recycled/recovered solvents, the presence of amines, amides, primary amines orammonium salts and the multipurpose use of the equipment. It was concluded that the probability that nitrosamines and nitrosating agents are present in their excipients is very low. Analytical
data confirmed that nitrosamine, nitrate and nitrite content were below the detection limits.
Given that the initial method of quantification was not sensitive enough to detect the low levelsof nitrites and nitrates present in the excipients, a more sensitive method was identified (ISO14673-2: 2004 (E): segmented flow analysis). This method is validated for milk and milk products
and not specifically for excipients. Table 2 shows the content of nitrites and nitrates in the excipients of DFE Pharma.
Table 2. Nitrites and nitrate levels in excipients from DFE Pharma as measured by the ISO-14673-2: 2004 (E):segmented flow analysis. Limit of detection (LOD) and limit of quantification (LOQ) are indicated.
SSG = sodium starch glycolate and CCS = croscarmellose sodium

Product class

Brand name

Nitrate(LOD=0.1ppm;
LOQ=0.3ppm)

MCC

Pharmacel®

0.8 ppm<0.1 ppm

Milled and sieved
lactosePharmatose®

0.7 ppm<0.1 ppm

Lactochem

1.4 ppm<0.1 ppm

Direct compression
lactoseSuperTab

0.7 ppm<0.1 ppm

SuperTab® NZ

7.7 ppm

0.7 ppm

Inhalation lactose

Respitose

0.7 ppm<0.1 ppm

Primojel® (SSG)

2.0 ppm<0.1 ppm

Primellose (CCS)

0.6 ppm<0.1 ppm

Solani Amylum

0.5 ppm<0.1 ppm

Prejel

0.6 ppm<0.1 ppm

BioHale sucrose

0.4 ppm<0.1 ppm

BioHale trehalose

0.4 ppm<0.1 ppm

Superdisintegrants
Starches

®

®

®

®

®

Stabilizers®

Nitrite(LOD= 0.03ppm;
LOQ= 0.1ppm)

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Values of nitrites are typically lower than the LOQ of 0.1 ppm. The only limitation of this approach isthe fact that most products had levels below the LOQ of the detection method. Therefore, the value of <0.1 ppm nitrites indicated for most products is an overestimated worst-case scenario and it isunknown how low are the exact values. Precise data will be collected once a more sensitive methodof quantification will be available for testing. Despite the different quantification methods, a comparison of the obtained data with those reported in the publication of Boetzel et al. can be attempted.
It appears that for MCC, lactose and superdisintegrants, DFE Pharma belongs to the suppliers withthe lowest levels of nitrites. Only the direct compression lactose produced in Kapuni, New Zealand,has values slightly higher than 0.54 ppm, which is reported as mean value for lactose across different
suppliers (Boetzel et al., 2022).

Future outlook
The regulatory health authorities and the pharmaceutical industry together are taking important
steps to reduce the risk of nitrosamine exposure to patients. Excipient suppliers have a crucial rolein the pharmaceutical supply chain and have the responsibility to control the content of nitrosating
agents in their products. Ideally, excipient suppliers should be able to supply high quality and facilitate
safe use of their excipients by maintaining low nitrite levels, combined with an implemented control
strategy to ensure these low levels are obtained consistently. A current challenge to implement thisapproach, however, is the capability of analytical methods to detect low levels of nitrites (Wang et al.,2017). The availability of analytical methods with quantification levels down to part-per-billion (ppb)levels would be desirable. For high-risk formulations, improved analytical methods could lead to nitrite
specification limits agreed between excipients suppliers and drug product manufacturers. The reasonable expectation is that to have excipients low on nitrites and nitrates. Claims of nitrites and nitrates
free excipients are however unrealistic because minimal traces in excipients are inherently present in
the raw materials used to manufacture the excipients and cannot thus be removed. Altogether, theproposed steps can support end product manufacturers to reduce nitrosamine impurities in their drug
products – allowing us to move jointly towards a healthier world.

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References
Boetzel, R., Schlingemann, J., Hickert, S., Korn, C., Kocks, G., Luck, B., Blom, G., Harrison, M., François, M.,
Allain, L., 2022. A Nitrite Excipient Database: A useful Tool to Support N-Nitrosamine Risk Assessments
for Drug Products. J. Pharm. Sci.
Brambilla, G., Martelli, A., 2007. Genotoxic and carcinogenic risk to humans of drug–nitrite interaction
products. Mutat. Res. Mutat. Res. 635, 17–52.
EMA, 2022a. Questions and answers for marketing authorisation holders/applicants on the CHMP Opinion for the Article 5(3) of Regulation (EC) No 726/2004 referral on nitrosamine impurities in human medicinal products. EMA/409815/2020 Rev.11.
EMA, 2022b. Nitrosamine impurities.
European Union, 2006. Setting maximum levels for certain contaminants in foodstuffs.
IPEC Federation, 2022. The Role of Excipients in Determining N-Nitrosamine Risks for Drug Products.
Lin, J.-K., 1990. Nitrosamines as potential environmental carcinogens in man. Clin. Biochem. 23, 67–71.
Mergens, W.J., 1982. Efficacy of vitamin E to prevent nitrosamine formation. Ann. N. Y. Acad. Sci. 393,
61–69.
Nanda, K.K., Tignor, S., Clancy, J., Marota, M.J., Allain, L.R., D’Addio, S.M., 2021. Inhibition of N-nitrosamine
formation in drug products: a model study. J. Pharm. Sci. 110, 3773–3775.
Poulsen, K.P., Rubin, J., 1985. Drying of foods with flue gas from natural gas risk/benefit, in: Drying’85.
Springer, pp. 308–311.
Sen, N.P., 2018. Formation and occurrence of nitrosamines in food. Diet, Nutr. Cancer A Crit. Eval. 135–160.
US FDA, 2021. Control of Nitrosamine Impurities in Human Drugs: Guidance for Industry. Cent. Drug Eval.
Res. Silver Spring, MD, USA.
Wang, Q.-H., Yu, L.-J., Liu, Y., Lin, L., Lu, R., Zhu, J., He, L., Lu, Z.-L., 2017. Methods for the detection anddetermination of nitrite and nitrate: A review. Talanta 165, 709–720.
Wu, Y., Levons, J., Narang, A.S., Raghavan, K., Rao, V.M., 2011. Reactive impurities in excipients: profiling,
identification and mitigation of drug–excipient incompatibility. Aaps Pharmscitech 12, 1248–1263.
Ziebarth, D., Scheunig, G., 1976. Effects of some inhibitors on the nitrosation of drugs in human gastricjuice. IARC Sci. Publ. 279–290.

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To learn more about how DFE Pharma can help you
to mitigate nitrosamines related risk, please contact
Alberto Berardi: alberto.berardi@dfepharma.com.

dfepharma.com

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