Nitrosamine Impurities

Nitrosamines are included in the group of high potency mutagenic carcinogens. The main guideline dealing with assessment and control of DNA reactive (mutagenic) impurities in pharmaceuticals (ICH M7) identify any N-nitroso compound as part of the “Cohort of Concern (CoC)”, a group of highly reactive compounds known to be mutagenic and carcinogens.

The European Medicines Agency (EMA) and US-FDA guided the review process and started to collect the most common identified root sources: APIs, impurities, excipients, primary packaging. They could carry directly a nitrosamine but also their precursors (nitrosatables and nitrosating agent) interacting into the final drug product through the shelf life.

The investigation process prescribed to all Marketing Authorisation holders is a three-steps approach: Risk Assessment, Confirmatory testing and Remediation plan.

• During Step 1 the nitrosamines presence/formation risk is investigated for each product by gaining more knowledge of the manufacturing process through an initial risk assessment.
• During Step 2 nitrosamines presence/amount is confirmed with a sensitive and appropriately validated analytical method.
• In Step 3 the modifications identified as needed within the process are applied, to mitigate nitrosamines formation risk.

Each nitrosamine has different reactivity. Limits for Acceptable Intake are derived from scientific literature or derived by toxicologist: existing data and chemical structure of the molecule has huge impact on toxicity and thus on specification limits.

First discovery of N-Nitrosamines contamination in Pharmaceutical products went back to 2018, when the antihypertensive agent Valsartan was found to be contaminated by N-nitrosodimethylamine (NDMA).

Today, years after the first detection of nitrosamines, there are still challenges and discussions on the nitrosamine topic. A large proportion of marketed drugs contain secondary or tertiary amine as structural features that make them susceptible to nitrosation, resulting in thousands of different potential nitrosamine impurities. Concerns regarding potential drug shortage as a result of strict regulations should also requires a careful evaluation, together with a proper toxicological evaluation of the molecules.

The analysis of nitrosamines can be challenging. Ultra-low levels of these impurities must be quantified in diverse and complex matrices. The developed methods then need to be validated to conform to GMP requirements:

• We are equipped with all the required equipment to meet these limits, including, LC MSMS and GC MSMS systems.
• We have experience with nitrosamine testing, including validating methods for sartans and ranitidine, as well as screening testing methods.
• We can provide both toxicological and analytical support for risk evaluation.
• We are a network of BioPharma Product Testing laboratories with vast capacity at multiple sites globally performs this testing under GMP requirements.

Our laboratories specialise in method development and validation for highly sensitive and specific method to
assess carcinogenic or genotoxic impurities in drug products. We regularly face the challenges of low detection levels, difficult matrices and identification of unknown impurities during the pharmaceutical method development process.

In addition to experienced pharmaceutical impurity analysis, we can support MAH with toxicological risk
assessments. Our experienced toxicologists conduct risk assessments to address extractables & leachables,
elemental impurities (ICH Q3D).

Eurofins BioPharma Product Testing has already assisted many clients with meeting this timely new requirement with GMP-compliant services, including:

• Risk evaluation and expert support services (chemistry and toxicology)
• Acceptable intake and Limits calculation
• Validating methods for specific nitrosamine on drug substances and products, with LOD/ LOQ < Acceptable intakes
• Nitrates and nitrites testing on excipients
• Screening methods to support risk evaluation (on each matrix DS and/or DP)
• Confirmatory testing and release testing methods carried out using appropriately validated and sensitive methods.