Genotoxic

GENOTOXIC IMPURITIES
IN
PHARMACEUTICALS

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 The genetic change is referred to as a mutation and
the agent causing the change as a
mutagen. Genotoxicity is similar to mutagenicity.
 Genotoxicity is similar to mutagenicity except
that genotoxic effects are not necessarily always
associated with mutations.

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 Genotoxicity and
Genotoxic impurities

Any deleterious change in the genetic material(DNA)

regardless of the mechanism by which the change is
induced is called GENOTOXICITY.
Genotoxic impurities:
Chemical agents(Impurities) that damages the genetic
information within a cell causing mutations(changes in
DNA), which may lead to cancer.

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All that glitters is not…

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What is DNA?
DNA, or deoxyribonucleic acid, is the hereditary material
in humans and almost all other organisms. Nearly every
cell in a person’s body has the same DNA. Most DNA is
located in the cell nucleus (where it is called nuclear
DNA), but a small amount of DNA can also be found in
the mitochondria (where it is called mitochondrial
DNA or mtDNA). Mitochondria are structures within cells
that convert the energy from food into a form that cells
can use.
DNA contains the Nucleotides

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 DNA can be damaged due to these
impurities. Hence these impurities
should be controlled to an
acceptable level.
 Risk with this impurities :
 Cell damages
 Genetic disorder
 Leads to cancer

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 Damaged DNA :
The impurities reacts with DNA
elements, thereby damages the DNA

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 Dr. Paracelsus says
Father of modern Toxicology

“All substances are poisons, there is none which is not a

poison. The right dose differentiates from poison to
remedy”

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Assessment and control- How ?

Assessment and Control

of DNA Reactive (Mutagenic) Impurities
in Pharmaceuticals To Limit Potential
Carcinogenic Risk .
Some of the potential genotoxic impurities related
structures are published.

--ICH M7(R1)

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Potential Genotoxic Impurities based on structure.

These are
the Possible
Structures that
Can produce
Genotoxicity.

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 Few impurities comes under the
group Cohort of concern (COC)

 Some structural group of substances were identified

to be of high potent.
 This group of high potency mutagenic carcinogens,
referred to as the cohort of concern, comprises
aflatoxin-like-, N-nitroso-, and alkyl-azoxy compounds.

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Classification of Genotoxic impurities
Class Definition- Proposed action to control
Impurity
1 Known Mutagenic and Control at or below compound-specific
carcinogenic acceptable limit
Generate TD 50 Value to the specific substance

2 Known Mutagenic, Control at or below acceptable limits

Unknown carcinogens
3 Structural alerts- Not Control at or below acceptable limits
similar to API (appropriate TTC) or conduct bacterial
mutagenicity assay;
If non-mutagenic = Class 5
If mutagenic = Class 2
4 Structural alerts-similar
to API Not genotoxic

5 No Structural alerts Not genotoxic

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 Assessment and confirmation of an
impurity is a
Mutational or carcinogenic
 Computation assessment (Class 3,4,5)
 AMES Test (Class 3, 2)
 Vivo and vitro test(class-1)
 Long tem vivo test( Cohort of concern)

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 Limit of impurity
 Limit of impurity (ppm) : PDE or TTC or TD 50 (µg)
Max daily dose(g)

TTC : Threshold toxicological concern

(TTC=1.5µg for all the impurities with no sufficient data)

Note : Levels above 1.5µg shall be justified with clinical data or

toxicology data.
TTC concept is not applicable to Cohort of concern ( eg :Nitroso ) and
class-1 impurities

PDE: Permitted daily exposure is used for cohort of concern impurities.

TTC : This dose is applied to assess Class-2 and 3 impurities
TD 50 : This dose is applied to assess Class-1 impurities
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 TTC and TD50

 For class-2 and 3 impurities, TTC concept is applicable.

 Example to find the Limit :

If the dose is 10 grams, the limit = 1.5 µ/10 g =0.115ppm

 For class-1 impurities, TTC concept is not applicable, TD50

concept is to be followed.

 TD 50 is a dose of impurity given to group of animals(eg:rat or

mice), in which 50% of the tumor incidents observed.
 Its short term study( eg: 14 days)

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 Calculation of Ethylene oxide limit:
divide the value with safety factor

 TD50 values for ethylene oxide : 21.3 mg / kg

 Average human body weight : 50 kg
 Safety factor :50000
 Final TD 50 : (21.3 mg/ kg X 50 kg)
50000
= 21.3 µg
Further,
If the dose is 10 grams, the limit = 21.3µ/10 g =2.13ppm

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 Limits :

 Higher the maximum daily dose(MDD,

lesser will be the specification limits.
 As the drug dose goes higher into the body, the
impurity will also go higher into the body along with
the drug.

 Therefore, higher the MDD, lesser the control limits

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 Assessment- Mutational

 A computational toxicology assessment should be

performed using Quantitative Structure-Activity
Relationship ((Q)SAR) methodologies
 To confirm the mutation of an impurity evident from
computational, bacterial mutagenicity assay shall be
done(Microbial test).

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 Confirm with two methods.

 Two (Q)SAR prediction methodologies that

complement each other should be applied. One
methodology should be expert rule-based.
 The second methodology should be statistical-based.

(Q) SAR :
Quantitative Structure-
Activity Relationship
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Nitrosoamine-ToxTree Software
assessment

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 Evaluation order:
From class 5 to class1

 If no structural alert – No concern- class 5

 If structural alert is given, but similar to API- No concern.
 Cant control to 1.5µg, conduct AMES test(Mutagenicity
confirmation test).
 If ames test is negative- No concern –class 5
 If ames test shows positive- Mutagenicity is confirmed.
class 2 –control to 1.5
 If can not be controlled- studies shall be done to set limit –
Compound specific limit. conduct rodent study and
determine TD 50 values.
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 TTC for Less than life time usage
drugs
TTC for multiple impurities in a drug

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 Control strategy-A

 Keep a control test in API

And skip test is recommended (when few batches
shows the results below 30% of the specification limit.

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 Control strategy-B

 Keep a control test in Raw material or intermediate or

in-process
Set the specification higher than the API limit and show
the control/purge of the impurity.

In this case avoid the control test in API, by showing

absence study.

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 Control strategy-C

 Avoid control test in API or RM/INT

Understand process parameters and impact on residual
impurity levels (including fate and purge knowledge)
with sufficient confidence that the level of the impurity
in the drug substance will be below the acceptable limit
.

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Potential impurities-But no evidence
on its presence

 Avoid control test in API or RM/INT

Understand process parameters and impact on residual
impurity levels (including fate and purge knowledge)
with sufficient confidence that the level of the impurity
in the drug substance will be below the acceptable limit
.
Absence study on 3 to 6 batches should show below
30% of the specification

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 Analytical challenges
 Concentration <0.1 ppm detection is not possible using
regular HPLC, GC methods.

 LC-MS or GC-MS methods are highly sensitive and both LC-

MS and GC-MS methods are stability indicative. These are
mass specific.
In MS detectors, Q3(Triple quadrapole) are highly sensitive. i.e
LC-MS/MS , GC-MS/MS

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Carbozole with 0.825 ppm by LC-MS

Even very Low level detection is possible by LC-MS

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Nitrosoamines Risk identification
points

 Nitrosoamines are poentical mutagenic and carcinogenic.

 Formation : nitrous acid ot its salts reacts with secondary
amines under acidic condtions leads to formation of
nitrosoamine impurities.
 The limits should be <0.03 ppm , effective from Feb-2021.
 Mean while little higher limits are acceptable. But threaten
to be under FDA inspection and warning letter scope.

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Nitrosoamines Risk identification
points

 Presence of reagents with nitrous compounds.

 Presence of secondary amines.
 Recovery solvents from other sites are used.
 Contaminated nitroso impurities.

 Risk assessment is mandatory to all products for its

absence or presence and report should be shared to
authorities by 26th march 2020
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Steps for characterization and risk
minimization.

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Why nitrosoamines should be <0.03
ppm, Even though it has higher
limits.

 These impurities are under cohort of concern class, as

per ICH M7, The kind of impurities should be
controlled to less than the original limit.

 This type of limits are called “As low as reasonably

practicable (ALARP)”.

 This is not applicable to class-1 to class-5 impurities.

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 Can TTC be MORE than 1.5µg

 TTC values for genotoxic impurities above 1.5 µg /day

will be treated on a case-by-case basis. For short-
duration treatments, the acceptability of higher levels
will be acceptable. (slide 28 of the ppt)

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More than one impurity-different
structures

 When more than one genotoxic impurity is present in

the drug substance, the TTC value of 1.5 µg/day can be
applied to each individual impurity only if the
impurities are structurally unrelated.

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 Not required in specification

 If a potential genotoxic impurity is just a theoretical

impurity i.e. based on theoretical considerations but
not found in practice as demonstrated by studies
during development of the manufacture, the impurity
does not need to be included in the drug substance
specification.

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