This document discusses genotoxic impurities and their analysis. It defines different types of DNA damage including single-base alterations, two-base alterations, chain breaks, and cross-linkages. It also discusses types of genetic damage such as mutagenic, senescence, apoptosis, and clastogenic effects. The document reviews ICH guidelines on impurities and how subsequent guidelines addressed genotoxic impurities. It outlines the process for assessing impurities based on ICH M7, including classifying impurities, identifying alerting structures, and using structure-activity relationships or the Ames test. Detection techniques for impurities including ICP-MS and ICP-AES are also summarized.

1. Genotoxic Impurities
and Analysis
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Prepared By :
Mahendra G S
M-Pharm,Pharmaceutical
Chemistry
JSSCP, MYSURU

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5. 1. Single-base alteration
 Depurination
 Deamination of cytosine to uracil
 Deamination of adenine to hypoxanthine
 Alkylation of base
 Insertion or deletion of nucleotide
 Base-analog incorporation
2. Two-base alteration
– UV light–induced thymine-thymine (pyrimidine) dimer
3. Chain breaks
a. Ionizing radiation
b. Radioactive disintegration of backbone element
c. Oxidative free radical formation
4. Cross-linkage
a. Between bases in same or opposite strands
b. Between DNA and protein molecules (eg, histones)
Types of DNA damage
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6. Single-base alteration
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7. Two-base alteration
Pyrimidine Dimers
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8. Keto – Enol Tautomerization
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9. • Point mutations: The replacement of one base
pair by another results in point mutation. They
are of two sub-types.
– Transitions: In this case, a purine (or a pyrimidine) is
replaced by another.
– Transversions: These are characterized by
replacement of a purine by a pyrimidine or vice versa.
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10. Types of DNA damage
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12. Types of Genetic Damage
• Mutagenic (not all DNA damage results in mutation)
– Causing change in the genetic information
• Senescence –irreversible dormancy
• Apoptosis –programmed cell death
• unregulated cell division
• Clastogenic
– Causing change in the chromosome
• Structural (breaks)
• Numerical (aneuploidy) –changes in the number in a cell
• Carcinogenic
– Causing or facilitating propagation of cancer
– Sufficient mutagenic and/or clastogenic activity =
carcinogenicity
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14. ICH Q3 Impurities
• Q3A Drug Substances– 1995 (step 4), R1 (2002),
R2 (2006)
• Q3B Drug Products– 1996 (step 4), R1 (2003), R2
(2006)
• Q3C Residual Solvents– 1997, R1-5 (2002, 2005,
2009, 2011)
• Most ICH guidelines on impurities in drug
substances and drug products are >15 years old
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15. Filling the Gaps
• ICH Q3D Elemental Impurities – Step 4 (Dec. 2014) - USP ,
• Establishment of Allowable Concentration of Genotoxic
Impurities in Drug Substance and Product 2005, PhRMA
Position Paper
• EMA, Guideline on the limit of Genotoxic Impurities 2006
• FDA Draft: Genotoxic and Carcinogenic Impurities in Drug
Substance and Products: Recommended Approaches and
Acceptable Limits -2008 (allined with EMA)
• ICH M7 – Genotoxic Impurities – Step 4 (June 2014) - changes
from EMA and FDA guidance
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16. ICH M7 – Genotoxic Impurities
• Filling the ICH Q3 A/B gap for “impurities that are
expected to be unusually potent, producing toxic
or pharmacological effects at a level not more
than (≤) the identification threshold.”
– Identification of "unusually potent" impurities not
described and
– No threshold of concern given
– EMA guideline and FDA draft guidance (earlier to ICH
M7 guidelines followed for GTIs)
– Threshold of Toxicological Concern (TTC), 1.5 µg/day
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17. Assessing Impurities – ICH M7
• All impurities (actual and potential), where
the structures are known, should be evaluated
for mutagenic potential.
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18. Classifying impurities – PhRMA paper
recommendation, adopted in M7
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19. Alerting Structures – examples
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20. Assessing Impurities – ICH M7
• Is an impurity potentially genotoxic?
• Search databases and literature for
carcinogenicity and bacterial mutagenicity data in
order to classify impurity as Class 1, 2, or 5
• When data are not available
• Use Structure-Activity Relationships (SAR) that
focus on bacterial mutagenicity predictions. This
could lead to a classification into Class 3, 4, or 5.
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23. AMES TEST
• The Ames test is a biological assay to assess
the mutagenic potential of chemical
compounds.
• Negative result classifies compound as normal
ICH impurity and overrides a positive in silico
prediction for genotoxicity
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24. Procedure for AMES TEST
The mutagenicity of a substance is proportional to the number of
colonies observed.
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Inductively coupled plasma mass spectrometry (ICP-MS) –detection capability is at
concentrations as low as one part in 1015

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Inductively coupled plasma atomic emission spectroscopy (ICP-
AES), also referred to as inductively coupled plasma optical
emission spectrometry (ICP-OES), is an analytical technique used
for the detection of trace metals.

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