Risk Assessment Report on Nitrosamine Impurities in Dextrose Anhydrous

Dextrose anhydrous an active pharmaceutical ingredient manufactured by enzymatic hydrolysis of starch. Nitrosamines, recognized by IARC as probable human carcinogens (Group 2A), have prompted regulatory bodies to mandate risk evaluations for all human-use drugs.

Guidelines Used

EMA/189634/2019 & EMA/428592/2019

CHMP Opinion under Article 5(3) of Regulation (EC) No 726/2004

US FDA Guidance for Industry (2020)

Health Canada Notice (2021)

WHO/ICH guidance (Q3A, Q3B, Q3C, Q3D)

1. Introduction

This risk assessment evaluates the potential for N-nitrosamine impurities in dextrose anhydrous manufactured from edible corn starch via enzymatic hydrolysis. The purpose is to ensure that dextrose anhydrous is free of nitrosamine impurities or within acceptable thresholds per international regulatory standards.

The chemical substance, D-(+)-glucopyranose, is a simple monosaccharide with no intrinsic amine functionality, minimizing nitrosamine formation risks from the outset.

Nitrosamine impurities are a significant concern in pharmaceutical manufacturing due to their classification as probable human carcinogens. While Dextrose Anhydrous, a simple sugar used as an excipient, is not inherently prone to nitrosamine formation, contamination can occur through manufacturing processes or materials.

2. Objective and Scope

This assessment investigates:

• All materials, equipment, processing steps, and packaging involved.
• Potential sources and mechanisms of nitrosamine formation.
• Applicable control strategies and justifications in line with EMA, FDA, and WHO guidance.

3. Process Overview and Material Inputs

3.1 Flow Chart

3.2 Synthetic Route of Manufacturing Process

Glucose is produced via the enzymatic hydrolysis of starch. Dextrose anhydrous is produced through:

• Liquefaction of starch.
• Enzymatic saccharification using glucoamylase and α-amylase.
• Purification via membrane separation.
• Crystallization and drying to yield anhydrous glucose.

3.3 Starting materials

The Starting material used for the manufacturing process of Glucose is Starch granules. Following raw materials used in the manufacturing process:

• Starch granules
• Cold water
• Starch slurry
• α-Amylase
• Gelatinized Starch
• Purified water
• Liquefied starch
• Glucoamylase
• Pullulanase

4. Raw and Auxiliary Materials

All raw and auxiliary materials involved in the enzymatic production of dextrose anhydrous were reviewed to identify potential precursors for N-nitrosamine impurities, including the presence of amines, amides, or nitrosating agents (e.g., nitrites or nitrous acid). The table below outlines each input's function and its assessed nitrosamine risk profile.

Material	Function	Nitrosamine Risk Summary
Starch granules	Primary feedstock, carbohydrate source	Natural polysaccharide; no nitrogen functionality; no risk of nitrosamine formation.
Cold water	Suspension medium for starch	Water source tested for nitrite; compliant with GMP; no amine or nitrosating agents present.
Starch slurry	Intermediate (starch + water mixture)	No new chemicals added; same risk profile as individual components. No risk.
α-Amylase	Enzyme to hydrolyze starch to oligosaccharides	Protein-based; no secondary/tertiary amines in formulation. Sourced as food-grade; no nitrosating agents used.
Gelatinized starch	Intermediate (post-heating)	Result of physical transformation; no chemical modifications introducing risk; starch remains nitrogen-free.
Purified water	Processing aid and cleaning	Routinely tested for nitrites (per pharmacopeia); no contamination observed; meets conductivity and microbial limits.
Liquefied starch	Hydrolyzed starch solution	Product of α-amylase action; no nitrogen species introduced; no risk of nitrosamine generation.
Glucoamylase	Enzyme to convert oligosaccharides to glucose	Enzyme derived from GRAS microorganisms; no amine groups or nitrosating agents in formulation; validated source.
Pullulanase	Debranching enzyme to enhance hydrolysis	Enzyme typically derived from Bacillus or fungal species; protein-based, lacks nitrosamine precursors. Validated food-grade input.

All materials listed above are of natural origin or food/pharmaceutical grade, and no amines, nitrosating agents, or residual nitrites are present. The enzymatic and aqueous nature of the process precludes the formation of nitrosamines. Moreover, no secondary processing conditions (e.g., extreme pH, use of nitrite salts, high temperatures under acidic conditions) are conducive to nitrosamine formation.

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5. Evaluation of Nitrosamine Formation Pathways

5.1 Nitrosating Agents

Nitrites/nitrous acid are not used at any point in the process. Purified water, raw materials, and reagents are verified to be nitrite-free through validated testing.

5.2 Amine Sources

No primary, secondary, tertiary amines, quaternary ammonium salts, or amides are used or generated. Glucoamylase and α-amylase are protein-based enzymes, but their structure does not lead to nitrosamine precursors under processing conditions.

5.3 Reaction Conditions

The entire production process is aqueous-based, conducted at mild pH and temperature. No acidic nitrosation conditions or oxidizing environments are present.

6. Recycled Material & Mother Liquor

• No recovery or reprocessing of degraded batches
• Mother liquor is retained only as API solution and not recycled for future batches
• No carry-over risk of nitrosamine precursors

7. Equipment and Water System

7.1 Manufacturing Equipment

• Made of 316L stainless steel
• Filter materials: Polypropylene → no nitrosamine leachates
• Passivation: Avoids nitric acid

7.2 Purified Water

• Controlled via passivation, pasteurization and validation per USP.
• Nitrite monitoring: Performed routinely; no detectable levels
• Conductivity: < 1.3 μS/cm, compliant with pharmacopeial specs

8. Packaging Evaluation

The packaging system for dextrose anhydrous has been critically assessed to evaluate the potential for introduction, formation, or migration of N-nitrosamine impurities during packaging, storage, or distribution.

Dextrose anhydrous is packed using the following materials and procedure:

• The product is filled into clean, double-lined polyethylene bags, which serve as the primary contact packaging.
• Prior to sealing, the bags are flushed with nitrogen to displace atmospheric oxygen, minimizing oxidative degradation or formation of reactive nitrogen species.
• The polyethylene liners are then tightly sealed and placed into 25 kg high-density polyethylene (HDPE) drums.
• These HDPE drums are equipped with multiple security and tamper-evident mechanisms, including:

1. Stainless steel (SS) tamperproof seal
2. Tech lock anti-theft device
3. Drum locking seal

• Each drum is clearly labeled on both the body and lid for traceability and regulatory compliance.

8.1 Nitrosamine Risk Assessment of Packaging Materials: 

Component	Material	Assessment Summary
Inner liners	Polyethylene (PE)	Chemically inert; no amines, nitrosating agents, or nitrosamine degradation potential identified.
Outer drum	HDPE	Does not contain nitrocellulose, amine-based additives, or reactive nitrogen components. Stable under storage conditions.
Seals & Locks	Stainless steel & polymeric materials	Mechanically functional components with no direct product contact; no risk of nitrosamine migration.
Nitrogen flushing	Inert gas (N₂)	Reduces oxygen and moisture; prevents oxidative reactions. Promotes chemical stability; does not contribute to nitrosation.

Accordingly, the risk of N-nitrosamine contamination from packaging materials is negligible and fully controlled.

9. Residual Solvents and Impurities

Independent analysis confirms:

• No organic impurities, residual solvents, or degradation products are structurally related to nitrosamines.
• Risk assessment compliant with ICH Q3A/B/C for all residuals and impurities.

10. Control Measures and Testing

• Nitrosamine-specific testing is not required based on this risk assessment. However, if required in future, analytical capability (e.g., LC-MS/MS) can be implemented.
• Change control measures are in place for any process or material modifications, triggering reassessment.
• All suppliers of raw materials are qualified and subject to periodic audits for nitrosamine control.

11. Conclusion

Based on a thorough evaluation of materials, equipment, process chemistry, utilities, and packaging, the manufacturing of dextrose anhydrous poses no credible risk of nitrosamine contamination.

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