Introduction
Genotoxic impurities (GTIs) are chemical substances that can damage DNA, potentially causing mutations and cancer. Even at very low levels, GTIs pose a safety risk, so they are strictly controlled in pharmaceuticals.
Unlike normal impurities (which are usually controlled as a percentage of the drug substance, e.g., 0.10% or 0.15%), GTIs are controlled in ppm (µg/g) or µg/day exposure, because even trace levels of these impurities can be harmful.
This article explains:
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How to decide whether an impurity is genotoxic.
The TTC principle used for setting limits.
3. A simple formula to calculate genotoxic impurity limits with examples.
Step 1: How to Determine Whether an Impurity is Genotoxic
Before setting a limit, we must identify whether the impurity has DNA-reactive potential or not . The common approach is:
1. Structural Alert Screening:
Check the impurity’s chemical structure for functional groups known to be genotoxic (e.g., nitrosamines, alkyl halides, epoxides, aziridines, hydrazines).
Use in-silico tools like QSAR (Quantitative Structure–Activity Relationship) to predict genotoxicity.
2. In-Vitro Testing
If QSAR gives a structural alert → perform a bacterial reverse mutation test (Ames test).
Positive Ames test = impurity considered genotoxic → must apply TTC principle.
3. If No Structural Alert
If no alert in QSAR and negative Ames test → impurity is non-genotoxic → then it can be controlled like ordinary impurities (per ICH Q3A/Q3B).
Summary Rule:
Alert/Positive result → Genotoxic impurity (control at ppm).
No alert/Negative → Non-genotoxic impurity (control as normal).
Regulatory classification:
EMA guideline “Guideline on the Limits of Genotoxic Impurities” classifies impurities into categori tv aa,es:
Class 1: Known potent carcinogens (e.g., nitrosamines).
Class 2: Probable genotoxic carcinogens.
Class 3: Alerting structure, unknown carcinogenicity.
Class 4/5: No concern.
👉 If classified as genotoxic (Class 1–3) → apply TTC/AI limit.
Step 2: TTC Principle (Threshold of Toxicological Concern)
Default limit for GTIs: 1.5 µg/day exposure
This corresponds to a lifetime cancer risk of 1person in 100,000.
If carcinogenic potency data exists (like nitrosamines), a compound-specific AI (Acceptable Intake) may be lower than 1.5 µg/day.
Step 3: Simple Formula for Genotoxic Impurity Limit
To set genotoxic impurity limit like other impurities limit you need to know the maximum daily dose of the drug, If you know the maximum daily dose in mg convert it the gram
If the maximum daily dose (MDD) of the drug is expressed in grams, dived the TTC limit in (µg/day) with the MDD in gram.
And the formula becomes very simple as below:
GTIs Limit (ppm)
=TTC (µg/day)/Daily Dose (g/day)
Step 4: Examples
Example 1: Olaparib (Maximum daily dose is 600 mg/day = 0.6 g/day)
Limit=1.5/0.6=2.5 ppm
Example 2: Paracetamol (4000 mg/day = 4 g/day)
Limit=1.5/4=0.375 ppm
Observation:
Higher daily dose → Stricter (lower) ppm limit.
Lower daily dose → Relaxed (higher) ppm limit.
By this way we can set the limit for genotoxic impurities but for Nitrosamine approach is little bit different as these impurities need more stringent control.
Special Case – Nitrosamines
Nitrosamines are extremely potent as they are known carcinogens, so they do not use the TTC of 1.5 µg/day. Instead, they have individual Average daily intake (AI) values:
To set limit for these impurities First convert Their AI value from ng/day to µg/day by just dividing ng/day value with 1000, like for NDMA AI value is 96 ng/day. After dividing with 1000 it will become 0.096 µg/day.
NDMA = 96 ng/day
NDEA = 26.5 ng/day
DIPNA = 26.5 ng/day, etc.
Example1: NDMA in a 1 g/day drug
Limit=0.096/1=0.096ppm
(much stricter than 1.5 µg/day TTC).
Example 2: NDMA in a 0.5 g/day Drug (500 mg/day)
Limit=0.096/0.5=0.192ppm
Example 3: NDEA in a 2 g/day Drug
Limit=0.0265/2=0.013
Normally, genotoxic impurity limits are based on lifetime exposure (around 70 years).
But if the drug is only taken for a shorter duration (e.g., 24 months for oncology therapy), regulators allow higher daily exposure because the total cumulative risk is lower.
Step 1: The principle
Default TTC (lifetime use) = 1.5 µg/day (≈ 70 years).
For shorter duration use, TTC is adjusted upward using scaling factors.
EMA/ICH guidance gives the following approximate acceptable intakes (AI):
Treatment Duration AI (µg/day)
Lifetime (≥10 years). 1.5 µg/day
1–10 years 10 µg/day
1–12 months 60 µg/day
≤1 month 120 µg/day
Step 2: For 24 months (2 years) exposure
This falls in the 1–10 years category.
👉 Acceptable Intake (AI) = 10 µg/day instead of 1.5 µg/day.
Step 3: Formula for ppm limit
Limit (ppm)=AI (µg/day)/Daily Dose (g/day)
Step 4: Example Calculations:
Example 1: Drug dose = 1 g/day, exposure = 24 months
Limit=10/1=10ppm
Example 2: Drug dose = 0.5 g/day, exposure = 24 months
Limit=10/0.5=20ppm
Conclusion
Step 1: Determine genotoxicity (QSAR + Ames test).
Step 2: If genotoxic, apply TTC (1.5 µg/day) unless a compound-specific AI is available.
Step 3: Use the simplified formula:
Limit (ppm)=
TTC or AI (µg/day)/Daily Dose (g/day)
Step 4: For nitrosamines, use their very low AI values instead of TTC.
This approach ensures both patient safety and regulatory compliance when setting genotoxic impurity limits.
