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10.5731/pdajpst.2014.00974Access the most recent version at doi: 221-23868, 2014 PDA J Pharm Sci and Tech
Sâmia Rocha de Oliveira Melo, Maurício Homem-de-Mello, Dâmaris Silveira, et al.
Toxicological Evaluation
Advice on Degradation Products in Pharmaceuticals: A
on May 27, 2014journal.pda.orgDownloaded from on May 27, 2014journal.pda.orgDownloaded from
REVIEW
Advice on Degradation Products in Pharmaceuticals: A
Toxicological Evaluation
SÂMIA ROCHA DE OLIVEIRA MELO, MAURI
´CIO HOMEM-DE-MELLO, DA
ˆMARIS SILVEIRA, and
LUIZ ALBERTO SIMEONI
Faculty of Health Sciences, University of Brasilia, Brası´lia, Brazil ©PDA, Inc. 2014
ABSTRACT Degradation products are unwanted chemicals that can develop during the manufacturing, transportation, and
storage of drug products and can affect the efficacy of pharmaceutical products. Moreover, even small amounts of
degradation products can affect pharmaceutical safety because of the potential to cause adverse effects in patients.
Consequently, it is crucial to focus on mechanistic understanding, formulation, storage conditions, and packaging to
prevent the formation of degradation products that can negatively affect the quality and safety of the drug product. In this
sense, databases and software that help predict the reactions involving the pharmaceutically active substance in the presence
of degradation conditions can be used to obtain information on major degradation routes and the main degradation products
formed during pharmaceutical product storage. In some cases, when the presence of a genotoxic degradation product is
verified, it is necessary to conduct more thorough assessments. It is important to consider the chemical structure to
distinguish between compounds with toxicologically alerting structures with associated toxic/genotoxic risks and com-
pounds without active structures that can be treated as ordinary impurities. Evaluating the levels of degradation products
based on a risk/benefit analysis is mandatory. Controlling critical variables during early development of drug products and
conducting a follow-up study of these impurities can prevent degradation impurities present at concentrations greater than
threshold values to ensure product quality. The definition of the impurity profile has become essential per various regulatory
requirements. Therefore, this review includes the international regulatory perspective on impurity documents and the
toxicological evaluation of degradation products. Additionally, some techniquesused in the investigation of degradation
products and stability-indicating assay methods are highlighted.
KEYWORDS: Degradation products, Toxicity, Regulatory guides and standards, Safety, Quality.
LAY ABSTRACT: Degradation products are impurities resulting from chemical changes that occur during drug
manufacturing. They can also form during storage and transportation in response to changes in light, temperature, pH,
and humidity, or due to inherent characteristics of the active pharmaceutical substance, such as their reaction with
excipients or on contact with the packaging. The presence of these chemicals can affect product safety and quality.
Therefore, it is necessary to know and follow the guidelines and standards regarding degradation products and
existing regulatory environments to assess the toxicity and risk related to their presence in pharmaceutical products.
Introduction
Degradation products are impurities that result from
chemical changes that occur during storage due to the
effects of light, temperature, pH, humidity, reaction
with excipients, or contact with primary packaging
(1). Often, the terms degradation products and impu-
rities are used as synonyms; however, there are some
conceptual differences. Impurities consist of any or-
ganic (raw materials, sub-products, intermediates,
degradation products, reagents, and catalysts) or inor-
ganic (reagents and catalysts, heavy metals or residues
of other metals, inorganic salts, and other materials
used in synthesis) source materials in the active phar-
maceutical substance or product. Degradation prod-
ucts are organic impurities resulting from the degra-
dation of the active pharmaceutical substance and/or
excipients (2).
*Corresponding Author: Saˆmia Rocha de Oliveira
Melo, MSc, SQN 205—Bloco C—Apto 206, 70843-
030 —Brası´lia—DF—Brasil, Tel.: ⫹55(61)34625442,
email: samiamelo@gmail.com
doi: 10.5731/pdajpst.2014.00974
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Most of the existing regulatory guidelines are appli-
cable to both impurities in pharmaceutical products
and active pharmaceutical substances in general. In
this work, some of these concepts will be extended to
organic impurities resulting from the degradation of
active substances present in the pharmaceutical prod-
uct; this will be referred to hereafter as degradation
products. The evaluation of degradation impurities for
the purposes of drug registration is consolidated in
international guidelines such as the International Con-
ference on Harmonization (ICH) (1), which has been
adopted by regulatory agencies in Europe, the United
States of America, and Japan.
Other guidelines include those determined by other
health authorities such as the Brazilian Health Surveil-
lance Agency (ANVISA) (3), the Canadian Health
Authority (Health Canada) (4), the Therapeutic Goods
Administration of the Australian Health Authority
(TGA) (1), and others. The regulatory concern is
based on the fact that the presence of these chemicals,
even in small amounts, can influence pharmaceutical
quality and safety (5); some of these substances may
have therapeutic or adverse activity at concentrations
even lower than those of the active substance in the
pharmaceutical product (6).
This review describes the existing toxicological eval-
uation of degradation products in pharmaceuticals and
the relevant regulatory guidelines and standards.
International Regulatory Framework
In 1990, the ICH Q3A (R2) and Q3B (R2) guidelines
were published; they addressed the monitoring and
control of impurities in pharmaceutically active sub-
stances and products (7).
The ICH Q3B (R2) guideline directly addresses the
evaluation of degradation products in pharmaceuticals
but does not cover impurities due to the degradation of
excipients, biopharmaceuticals, animal products, fer-
mentation products, herbal products, peptides, radio-
pharmaceuticals, oligonucleotides, products used in
developing clinical trials, enantiomeric impurities,
polymorphic forms, and external contaminants (1, 8).
The guidelines do not apply to the assessment of
genotoxic or carcinogenic impurities (7). This guide-
line document suggests that the amount of degradation
products in pharmaceutical products be within certain
notification, identification, and qualification thresh-
olds (1, 8).
The ICH M7 draft guideline highlights the identification,
categorization, qualification, and control of mutagenic
impurities that are DNA-reactive, in addition to those
defined in the ICH Q3A (R2) and Q3B (R2) guidelines.
The ICH M7 draft guideline emphasizes the importance
of safety and quality risk management to establish levels
of mutagenic impurities that are expected to have negli-
gible carcinogenic risk (9).
According to the ICH, the reporting threshold is the
maximum permissible concentration of a specific
degradation product that does not need to be re-
ported; anything above this value should be re-
ported. The identification threshold is a limit above
which a degradation product should be chemically
identified. The qualification threshold is a limit
above which it is necessary to acquire and evaluate
data that establishes the biological safety of an
individual degradation product or a given degrada-
tion profile (1). Based on these assumptions, the
ICH Q3B (R2) guidelines specify numerical values
for the notification, identification, and qualification
thresholds shown in Table I.
TABLE I
Notification, Identification, and Qualification
Thresholds
Maximum
Daily
Dose
1
Threshold
1,2
Reporting ⱕ1 g 0.1%
⬎1 g 0.05%
Identification ⬍1 mg 1.0% or 5 g TDI, whichever
is lower
1–10 mg 0.5% or 20 g TDI,
whichever is lower
⬎10–2 g 0.2% or 2 mg TDI, whichever
is lower
⬎2 g 0.10%
Qualification ⬍10 mg 1.0% or 50 g TDI,
whichever is lower
10–100 mg 0.5% or 200 g TDI,
whichever is lower
⬎100 mg
to2g
0.2% or 3 mg TDI, whichever
is lower
⬎2 g 0.15%
Source: ICH, 2006 (1).
1
The amount of drug administered per day.
2
Thresholds for degradation products are expressed
either as a percentage of the drug or as total daily
intake (TDI) of the degradation product. Lower thresh-
olds may be appropriate if the degradation product is
unusually toxic.
3
Higher thresholds should be scientifically justified.
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A degradation product is considered to be qualified if
it meets one of the following conditions:
a) It is present in a marketed pharmaceutical product
(whether derived from the drug product itself or
not) in concentrations comparable to those that
have been evaluated in clinical studies that ensure
the safety of the pharmaceutical product, or
b) In situations where the amount is justified by the
scientific literature or when the level and proposed
acceptance criteria for impurities have been ade-
quately evaluated using in vitro comparative stud-
ies for genotoxicity (1, 10).
The ICHM7 draft guideline emphasizes that post-
approval alterations in marketed products that in-
clude changes in the drug substance chemistry, man-
ufacturing, and controls (including synthesis routes,
reagents, etc.) should be re-analyzed in terms of
degradation products. The objective is to determine
whether such modifications resulted in any new
mutagenic impurities or exceeded the highest accep-
tance criteria for existing mutagenic impurities. In
the same vein, post-approval submissions involving drug
products (changes in composition, dosage form, etc.)
should include the evaluation of the potential risk of any
new mutagenic degradants or higher values of compounds
that meet the acceptance criteria for previously identified
mutagenic degradants in comparison to the original product
(9).
Figure 1 shows a flow chart for making decisions
regarding a degradation product above the qualifica-
tion threshold. In addition to the ICH stability guide-
lines, which have been widely adopted by most regu-
latory agencies, other stability guidelines may also
contain information regarding degradation products
(Table II).
Degradation Products: Toxicity and Loss of Efficacy
From the time when the ICH guidelines were first
introduced, many changes have occurred, and the fo-
cus on the quality of pharmaceuticals is now on safety
considerations regarding the presence of impurities
and degradation products in finished pharmaceuticals
products. These guidelines are applied by several
countries for the evaluation of these drug products and
represent a major concern of regulatory agencies
worldwide (11).
The toxicological activity of degradation products can
be observed in many pharmaceutically active sub-
stances and products and, in some cases, can compro-
mise therapeutic action. Some examples of degrada-
tion situations that can affect the safety of drug
products are listed in Table III.
Failures of products to comply with stringent limits on
degradation products result in recalls of large quanti-
ties of pharmaceutical products from the market. Some
examples of recall notifications from September 2011
to March 2012 issued by the Food and Drug Admin-
istration (FDA) are listed in Singh et al. (11). To solve
problems related to degradation products, the industry
today is forced to invest in tools that can predict the
early formation of degradation products (11). Some of
these tools also allow the evaluation of the toxicity of
some structures.
Forced Degradation Studies and Stability-Indicating
Assay Methods
A tool commonly used by pharmaceutical companies
to predict the formation of degradation products is
forced degradation or stress testing. These tests pro-
vide information on the major degradation routes and
the main degradation products formed during storage
of pharmaceutical products. Stress tests usually in-
clude subjecting pharmaceutical products to thermo-
lytic, hydrolytic, oxidative and photolytic conditions,
and pH changes (12–15).
Some databases may be useful in defining the degra-
dation routes of the active pharmaceutical being in-
vestigated. An example is the Drug Degradation Da-
tabase (16) created by Pfizer, Eli Lilly, and Amgen,
which contains over 300 active pharmaceutical sub-
stances with registered degradation routes. Stress tests
are important for defining and validating analytical
methodologies as well for determining stability-indi-
cating assay methods that are able to measure the
amount of the active pharmaceutical substance in the
presence of other contaminants such as degradation
impurities (17, 18).
In a study by Garg et al. (19), a forced degradation of
the active pharmaceutical ingredient (API) fentanyl,
an opioid analgesic, was performed using light, acid,
base, heat, and oxidation. Under acidic conditions,
fentanyl degraded to N-phenyl-1-(2-phenylethyl)-
piperidin-4-amine (PPA), a potentially genotoxic
compound due to the aniline moiety in the structure.
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In this evaluation, a stability-indicating high-perfor-
mance liquid chromatography (HPLC) method eval-
uating fentanyl and its related compounds was val-
idated and demonstrated to be specific, precise,
linear, accurate, sensitive, robust, and suitable for
the intended use (19). Figure 2 depicts the path to
define a stability-indicating methodology.
Many forced degradation studies use mass balance to
assess the extent of degradation. This practice may
only be acceptable in early or clinical development
and not at the registration phase. In mass balance
studies, the amount of active pharmaceutical substance
is compared to that of the degradation products formed
(20). Due to the lack of structural information regard-
ing the degradation products and their unavailability
in the early stages of new drug development, degra-
dation is usually quantified using the normalization
percent area (area %). In this technique, it is normal to
use an identical response factor (area relative to
Figure 1
Decision tree for pharmaceutical degradation products. Figure adapted from ICH (1).
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amount) for the parent drug and the degradation prod-
uct (1, 21). However, it is not always possible to
obtain a suitable mass balance; in some cases, the
degradation product is volatile and is lost before the
analysis is completed. In other cases, there are differ-
ences in the molecular weight and the response de-
pending on the detector used, among other limiting
factors (21).
Investigation of Degradation Impurities: Technical
Orthogonal Assessments
Pharmaceutical products need to be quantitatively and
qualitatively assessed for impurities. These products
may be evaluated using HPLC coupled with diode
array detection (HPLC-DAD), liquid chromatogra-
phy–mass spectrometry (LC-MS), or liquid chroma-
tography coupled with nuclear magnetic resonance
(LC-NMR), among other techniques. In many cases, it
is necessary to use orthogonal analytical methods to be
confident that impurity detection and identification are
not confused by false positives caused by interference.
For example, in the case of pramlintide, a 37-amino
acid peptide used to treat people with type 1 and
insulin-using type 2 diabetes, the complexity of quan-
tifying low levels of structurally related impurities and
degradation products requires the application of highly
selective HPLC techniques and orthogonal separation
modes (22).
In the case of hydrazine, the principal hydrolytic deg-
radation product of the anti-tuberculosis drug isonia-
zid and a known degradant of hydralazine, a drug for
treating hypertension, developing and validating
methods for accurate determination at the parts-per-
million level in the presence of API or drug product
formulation excipients could be challenging due its
polarity and low molecular weight combined with its
susceptibility to oxidation.A wide variety of tech-
niques can be employed in this assessment (23, 24).
Another example is clopidogrel hydrogen sulfate, an
oral antiplatelet agent used to inhibit blood clots that
develops a methyl-ester salt structure with various
acidic counter ions such as HCl or besylates. These
acids have the potential to react with the methyl ester
either directly in an anhydrous environment or with
residual methanol generated by hydrolysis when ex-
posed to moisture. The resulting reaction products are
methyl chlorideor methyl besylate, respectively,
which are structures with genotoxicity concerns. The
presence of genotoxic degradants at low levels is
typically undetectable when conventional analytical
tools for degradation studies are used. It is therefore
Table II
Regulatory Authorities and Economic Blocs That Include the Subject “Degradation Products” in Their
Guidelines
Entity Guidelines Reference
WHO Stability testing of active pharmaceutical ingredients and finished
pharmaceutical products
(61)
ASEAN
b
ASEAN guidelines on stability studies of drug products (62)
GCC
c
The GCC Guidelines for Stability Testing of Drug Substances and
Pharmaceutical Products
(63)
MCC
d
Stability (64)
FDA
e
TGA
f
Health Canada
EMA
g
Q3B (R2). Impurities in New Drug Products (1)
Q3A (R2). Impurities in New Drug Substances (65)
Q1A (R2). Stability Testing of New Drug Substances and Products (66)
Q1B. Photostability Testing of New Drug Substances and Products (67)
Q1C. Stability Testing for New Dosage Forms (68)
Q1E. Evaluation of Stability Data (69)
ANVISA
h
Resolution RE-01, July 29, 2005. Guideline on Stability Studies. (70)
a
WHO, World Health Organization;
b
ASEAN, Association of South East Asian Nations;
c
GCC, Cooperation Council
for The Arab States of the Gulf;
d
MCC, Medicine Control Council;
e
FDA, Food and Drug Administration;
f
TGA,
Therapeutic Goods Administration;
g
EMA, European Medicines Agency;
h
ANVISA, Brazilian Health Surveillance
Agency.
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TABLE III
Degradation Situations That Can Affect the Safety of Drug Products
Product Degradation Effect Reference
Tetracycline Epimerization under acidic conditions forms
anhydrotetracycline and 4-epitetracycline
Reversible Fanconi syndrome (71, 72)
Meropenem
(solution)
Alkaline conditions and thermal degradation may
yield 4-methyl-3- (1H-pyrrol-3-ylsulfanyl)-5H-
pyrrole-2-carboxylic acid and other degradants
High concentrations of degraded samples
demonstrate in vitro cytotoxic activity in
mononuclear cells after 48h
(73)
Chloramphenicol Photo-oxidation Inhibition of antibiotic activity (74)
Isoniazid Hydrolytic degradation at high temperatures forms
hydrazine
Genotoxicity and carcinogenicity in animal
models
(75, 32)
Ibuprofen Oxidation and photodegradation can result in the
generation of 1-(4-isobutylphenyl)-1-ethanol
Cytotoxicity to fibroblasts at concentrations
above 1mM
(76, 77)
Ascorbic acid Dehydration and hydrolysis under anaerobic
conditions may yield furfural
Hepatotoxity in rats (78)
Enalapril maleate
(tablets)
High temperature and humidity can form enalaprilat
and a diketopiperazine derivative
Change in the dissolution profile, impairment of
drug bioavailability and decrease in the
effectiveness
(79)
Pethidine Hydrolytic degradation of an ester group can generate
N-methyl-4-phenyl-1,2,3,6 tetrahydropyridine
(MPTP)
Severe and irreversible symptoms of
Parkinson’s disease
(80)
Lidocaine Hydrolytic degradation generates 2.6-xylidine Anoxia and damage to the hematopoietic system (81–83)
Gabapentin Intramolecular cyclization can form the -lactam
3,3-pentamethylene-4-butyrolactam(2-
azaspiro[4,5]decan-3-one)
Induction of seizures in animal models (84)
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prudent to consider more selective, sensitive detectors,
such as mass spectrometry (MS), and conduct targeted
analysis if the risk is high based on systematic risk
assessment. Otherwise, risks from genotoxic degra-
dants could remain undetected due to analytical limi-
tations (23).
The concept of orthogonality covers a multidimen-
sional separation system. Two analytical methods can
be considered orthogonal when the separation mech-
anisms are independent such that the selectivity ob-
tained using the first method is not correlated with that
obtained using the second method (25).
Two strategies were reported by Argentine et al.
(25) for the development of orthogonal analytical
methods to fully characterize pharmaceutically ac-
tive substances. The first strategy involves the use
of reversed-phase liquid chromatography (RP-LC),
which maximizes selectivity by evaluating chro-
matographic supports while using various organic
solvents and mobile phase components that cover a
wide pH range. The second strategy involves the use
of different separation mechanisms, such as capil-
lary electrophoresis (CE), supercritical fluid chroma-
tography (SFC), thin layer chromatography (TLC), gas
chromatography (GC), normal phase chromatography
(NP-HPLC) and hydrophilic interaction liquid chroma-
tography (HILIC). A three-dimensional impurity pro-
file can be visualized if orthogonal detection principles
are utilized (25). Reversed-phase chromatography
(RP-LC) and a UV detector can be utilized in associ-
ation with CE coupled to MS to obtain as much
information as possible. This technique can be ex-
tended to other detection options such as photodiode
array detection (PAD) and evaporative light scattering
(ELS) (25).
Control of Genotoxic Impurities
In some cases, when the presence of a genotoxic
degradation product is verified, it is necessary to con-
duct more thorough assessments. To ensure greater
control of genotoxic impurities, the European Medi-
cines Agency (EMA) published the 2006 “Guideline
on the limits of genotoxic impurities”, which advised
that any genotoxic impurities should be identified us-
ing both existing genotoxicity data and “structural
alerts” (based on the chemical constituents of pharma-
ceutical drug substances known to be associated with
particular types of toxic effects, e.g., mutagenicity)
(7). This guideline was made available online at the
start of 2007 and applies to new active substances and
applications. It is also useful to evaluate variations of
existing active substances when the assessment of the
synthesis route, process control, and impurity profile
does not ensure the prevention of the introduction of
new or higher levels of genotoxic impurities compared
Figure 2
Flow chart for defining stability indicating assays. Source: Bakshi and Singh (85).
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with products containing the same active substances
that are currently authorized in the Europe (26).
Genotoxic impurities are chemicals capable of causing
changes in gene expression and direct or indirect dam-
age to DNA or chromosomes (27, 28). To establish
acceptable levels of exposure to genotoxic and/or car-
cinogenic compounds, the EMA guidelines consider
the following categories of genotoxic impurities.
Genotoxic Compounds with Sufficient Evidence for a
Threshold-Related Mechanism
Compounds in this class have an established level of
exposure that can be genotoxic, and the same proce-
dure defined for class 2 residual solvents described in
the ICH Q3C(R5) guideline, which establishes permit-
ted daily exposure (PDE) limits, is applicable (29). If
the assessed values are above the threshold, it is
necessary to reduce the compound in the pharmaceu-
tical product to safe levels below the PDE.
Genotoxic Compounds without Sufficient Evidence for
a Threshold-Related Mechanism
This category consists of compounds for which there
are clearly defined genotoxic mechanisms, requiring
pharmaceutical and toxicological evaluations to estab-
lish safe exposure levels. In general, pharmaceutical
product evaluation should be guided by a policy of
reducing substances to a level “as low as reasonably
practicable” (ALARP) when it is not possible to avoid
them. It is impossible to define a safe exposure level
for genotoxic carcinogens without a threshold, and due
to the difficulty of completely eliminating genotoxic
impurities, it is common to define an acceptable risk
level, that is, an estimate of daily exposure below
which there is negligible risk to human health (30).
Thus, the EMA and ICH M7 draft guideline adopts the
concept of a “threshold of toxicological concern tox-
icity” (TTC) for genotoxic impurities (9, 30, 31).
Figure 3 illustrates the decision tree used to evaluate
genotoxic impurities.
An example of a compound in this category is hydra-
zine, which has been shown to cause gene mutations
and chromosome aberrations and to induce cancer in
some animal studies but is lacking any convincing
evidence for carcinogenicity in humans. Hydrazine
has been classified as a probable human carcinogen
and has to be controlled with a staged TTC system
(32).
The TTC can be defined as the dose (g/day) of any
unstudied chemical for which the probability of cancer
incidence is insignificant. The TTC was initially set by
the FDA at an estimated value of 0.5 ppb (equivalent
to 1.5 g/patient/day or 0.025 g/kg/day) (33). This
value corresponds to a 10
–5
lifetime probability of
developing cancer for pharmaceuticals that show some
benefit with intentional exposure (28) and corresponds
toa10
–6
risk in situations in which there is no
theoretical benefit involved (1, 9). In line with the
guidelines proposed, the Pharmaceutical Research and
Manufacturing Association (PhRMA) has proposed a
risk level of 10
–6
for clinical trial therapies lasting
fewer than 12 months that have no pharmacological
benefit for volunteers. For studies lasting longer than
12 months, a risk of 10
–5
has been defined because
these studies usually have a drug benefit for volunteers
(28). TTC values greater than 1.5 g/person/day may
be acceptable in situations such as short-term exposure
to treatments when life expectancy is less than 5 years
or where the impurity is a known substance for which
much greater human exposure occurs from other
sources, for example, food (1).
The TTC concept is applicable to impurities that have
negligible risk of carcinogenic or toxic effects; it is not
applied to compounds presenting risks of developing
cancer even at doses below the TTC (30, 33–35).
However, exceeding the TTC is not necessarily asso-
ciated with an increase in cancer risk (9).
Classification of Impurities According to PhRMA
PhRMA envisions classifying genotoxic pharmaceuti-
cal impurities into five specific categories defined in a
document that establishes procedures for testing, qual-
ifying, and reporting toxicological risks (31). Figure 4
shows the five classes of impurities classified by
PhRMA.
Class 1 compounds contain impurities that have high
risks of genotoxicity. It is appropriate to eliminate
these compounds or ensure their presence at levels
below the safety threshold (e.g., TTC) (7). Class 2
compounds contain genotoxic impurities but have un-
known carcinogenicity. Class 3 compounds contain a
chemical alert group unrelated to the parent com-
pound. Compounds in classes 2 and 3 must be at or
below acceptable limits (generic or adjusted TTC) (9).
Exceptions occur in cases where there is evidence for
a threshold-related mechanism of genotoxicity, in
which case the limit set by the PDE is used (28).
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Compounds included in Classes 4 (with the same alert
structures as the parent drug substance) and 5, in
which the active pharmaceutical substance is not
genotoxic, are treated as non-mutagenic impurities
based on ICH Q3A (R2) and Q3B (R2) guidelines (9).
Thus, the treatment of impurities involves the identi-
fication and classification of structural alert groups for
the pharmaceutical active substance and its impurities;
these are then grouped into one of the five classes
defined above, and an appropriate threshold is set for
each case (28).
In a summary, the alerting genotoxic drug degradant
structure can come from a parent drug that already con-
tains a genotoxic alert or from a parent drug with no
alerting structures. Some examples follow. Oxybupro-
caine has an alert for aromatic amines that form degra-
dants via hydrolysis that have the same alert structure as
Figure 3
Flow chart defining genotoxic-indicating assays. Source: CHMP (30).
1
Impurities with a structural relationship to high potency carcinogens are excluded from the TTC approach.
2
If carcinogenicity data are available: Does intake exceed the calculated 10
–5
cancer lifetime risk?
3
Case-by-case assessments should include the duration of treatment, indication, patient population, etc.
*Abbreviations: NOEL/UF, No Observed Effect Level/Uncertainty Factor; PDE, Permitted Daily Exposure;
TTC, Threshold of Toxicological Concern.
** Use alternatives (different synthetic routes, formulations, starting materials, etc.).
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the parent. This acid degradant can be qualified by stan-
dardized mutagenicity data obtained from the parent
molecule. Acetaminophen contains a structural alert for
N-acylatedaminoaryls, which form p-aminophenol, a
compound that has a structural alert for an aromatic
amine, which is different from the alert in parent struc-
ture. Propofol does not have any alert structures but
degrades via oxidation into a dimeric degradation prod-
uct with alerts for mutagenicity (36).
The existence of structural alert groups in an impurity
alone is insufficient to trigger follow-up measures,
unless it is a structure in the cohort of concern (a
group of highly potent mutagenic carcinogens) or in
the case of a new relevant impurity for which hazard
data were generated after the overall control strategy
and specifications for market authorization were es-
tablished (9).
Databases and Software Used To Predict the
Formation of Degradation Compounds and Alerting
Toxic/Genotoxic Structures
Software that helps predict the reactions that occur
with the pharmaceutically active substance in the pres-
ence of certain reagents, starting materials, and deg-
radation conditions can be used to obtain information
regarding degradation products. Zeneth (a degradation
Figure 4
Classification of impurities by PhRMA. Source: Muller et al. (28), Dolan et al. (33), Dobo et al. (40, 86).
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expert system) by Lhasa is a program that provides
detailed descriptions of degradation pathways with
supporting literature references (37).
In addition to programs that predict degradation reac-
tions, programs are also used to evaluate alert struc-
tures for genotoxicity (Figure 5). Most pharmaceutical
companies use these programs in combination with
other suitable tests for predicting compound toxicity/
genotoxicity (38). Assessments that use software pro-
grams to evaluate the toxic effects of compounds
before synthesis or testing are referred to as in silico
assessments.Software programs that use this principle
are available as described by Muster et al. (39).
DEREK (Deductive Estimation of Risk from Existing
Knowledge), MCASE (Multiple Computer-Automated
Structure Evaluation), and TOPKAT (TOxicity Pre-
diction by Komputer-Assisted Technology) are among
the most frequently used.
DEREK recognizes the alert structures for genotoxic-
ity in the tested agent using a bacterial mutagenicity
assay, in vitro cytogenetic testing, and bibliographic
references. MCASE is an organizational model based
on data obtained from bacterial mutagenicity studies
conducted on a series of compounds. TOPKAT is a
program that uses data obtained from bacterial muta-
genicity tests to evaluate similarities between the
tested molecule and other molecules that exist in its
database, excluding those that do not have significant
evidence for mutagenicity (39). These programs are
often used in combination to increase specificity while
evaluating genotoxicity. For instance, the CASETOX
(Computer Automated Structure Evaluation for Toxi-
cology) programs, DEREK and TOPKAT, have spec-
ificities of 78% to 82%, respectively, when used alone,
and 92% to 100% when used in combination (38). In
Dobo et al. (40), eight companies were surveyed for
their success rate in identifying non-mutagenic struc-
tures. The negative predictive value (NPV) of the in
silico approaches was 94%. When human interpreta-
tion of in silico model predictions was conducted, the
NPV substantially increased to 99%. This survey also
suggests that the use of multiple computational models
Figure 5
Chemical structures that have functional alert groups for genotoxicity. Figure adapted from Muller et al. (28).
A: Alkyl, Aryl or H
X: F, Cl, Br, I
EWG: Electron-Withdrawing Group (CN, CⴝO, ester, etc.)
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is not a significant factor in the success of these
approaches with respect to NPV (40).
Other promising computational methods can be used
in the toxicological assessment of genotoxic impuri-
ties. In this context, the application of promising com-
putational methods, for example, QSARs (Quantita-
tive Structure–Activity Relationships) and SARs
(Structure–Activity Relationships) is needed, espe-
cially when very limited information on impurities is
available (41). QSARs can identify structural alerts for
known and expected impurities present at levels below
qualified thresholds. It provides the information nec-
essary to establish the practical use of a new in silico
toxicology model to predict the Salmonella t. muta-
genicity (Ames assay outcome) of drug impurities and
other chemicals with high sensitivity (81%) and high
negative predictivity (81%) based on external valida-
tion with 2368 compounds foreign to the model and
with known mutagenicity (42). An important aspect of
SARs as predictive toxicity tools is that they are
derived directly from mechanistic knowledge. Mech-
anistic knowledge provides a basis for interaction and
dialogue between model developers, toxicologists, and
regulators, and it permits the integration of the QSARs
results into a wider regulatory framework in which
different types of evidence and data concur or com-
plement each other and are used as a basis for making
decisions and taking action (43).
The ICH M7 guideline describes the use of two
QSARs prediction methodologies that complement
each other; one should be expert rule-based and the
second should be statistically based. The absence of
structural alerts from two complementary QSARs
methodologies is sufficient to classify the impurity as
no concern (9).
The genotoxic and carcinogenic properties of various
substances can also be found in databases such as
TOXNET (TOXicological data NETwork), NIOSH
(the National Institute for Occupational Safety &
Health), GESTIS (information system on hazardous
substances of the German), Discovery Gate (SYMX),
and Pharmapendium (Elsevier) (44–48).
Toxicological Assays Used To Qualify Impurities
In the potential presence of chemical structures con-
taining alerts for genotoxicity, risk should be evalu-
ated by conducting toxicological tests. Currently, it is
not possible to register a new medication without
having information regarding its mutagenicity (49).
Most existing guidelines on the subject adopted by
regulatory agencies in Brazil (50), Japan, the United
States and the European community support the ne-
cessity of a battery of tests for genotoxicity, which
increases the sensitivity and broadens the spectrum of
genetic events detected during the toxicity evaluation
of a particular chemical (49). A standard battery of
tests includes bacterial mutagenicity tests (51, 52), in
vitro chromosomal damage with mammalian cells or
in vitro mouse lymphoma thymidine kinase (TK) as-
says (53, 54), and in vivo tests for chromosomal
damage using rodent hematopoietic cells (55). In vitro
assays play an important role in genotoxic assessment
because of their high sensitivity and rapid toxicity
evaluation; in vivo tests are used to measure the ef-
fects of exposure route, treatment duration, metabo-
lism, and the organs affected (49).
Negative test results from this battery are generally
sufficient to ensure the absence of genotoxic activity.
If a compound demonstrates positive results for any of
these tests, depending on its therapeutic use, it is
necessary to perform more than one test to determine
its toxicity profile (56). In cases where a mutagenic
compound is a non-carcinogen in a rodent bioassay,
there would be no predicted increase in cancer risk (9).
Some of these tests were demonstrated in Vijayan et
al. (57) to provide information about the genotoxic
activity of the main degradation product of piperacil-
lin, piperacillin impurity-A. This degradation product
appears during manufacturing and storage processes
of the antibiotic penicillin, and its level failed to pass
the validation criteria of the computer-assisted toxicity
prediction carried out by TOPKAT software. A bac-
terial mutagenicity test and an in vitro chromosomal
aberration study were performed to establish the safety
profile and qualification. The results of these studies
indicated that piperacillin impurity-A is non-muta-
genic in the Ames test and non-clastogenic in the
chromosomal aberration study (57).
Because a single test is not capable of detecting all
relevant genotoxic substances, some situations require
changes in the set of genotoxicity tests used. This is
especially critical for compounds that have one or
more of the following characteristics: high toxicity,
poor or no absorption, structural alerts but with neg-
ative results in the standard battery of genotoxic tests,
showing evidence of tumor response or possessing
structurally unique chemical classes (49).
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Discussion
The presence of even small amounts of degradation
products can affect pharmaceutical safety because of
their potential to cause adverse effects in patients. For
this reason, their assessment is necessary to determine
shelf life and for medicine registration by national
health authorities.
The United States Pharmacopoeia (USP) recently in-
cluded the concept of degradation products under the
topic “Impurities in Drugs and Pharmaceutical Forms”
in USP 33–National Formulary (NF) 28, thus demon-
strating the evolution of regulatory issues and con-
cerns regarding this issue (58). Much of the existing
regulatory material regarding degradation is contained
in international guidelines such as those of the ICH,
FDA, and EMA.
When the concentration of a degradation product is
above the qualification threshold but qualifying data
are lacking, it is necessary to conduct toxicological
studies that take the maximum dose ingested, target
population, and route and duration of drug adminis-
tration into consideration (1). Degradation product
evaluations should consider the chemical structure to
distinguish between compounds with toxicological
alert structures with associated toxic/genotoxic risks
and compounds without alert structures that can be
treated as ordinary impurities. Existing computer pro-
grams that perform predictive assessments based on
chemical structure and compound activity are very
useful in determining toxicological responses (38, 39).
Pharmaceutical industry research and development
(R&D) has its own characteristics and requires signif-
icant investment. There is consensus that R&D con-
sumes resources and is considered highly complex. In
developing countries, R&D is hampered by a number
of factors, such as the lack of supportive policies,
incentives, infrastructure, and qualified staff. More-
over, the formation of specialized human resources in
key areas of pharmaceutical production is often not a
priority. All these factors coupled with negligible in-
teraction between pharmaceutical companies and uni-
versities/research centers negatively affects degrada-
tion research in developing countries (59). Research
that focuses on the assessment of degradation products
is normally initiated during drug development and
informs the choice of excipients, active ingredients,
packaging material, and appropriate stability-indicat-
ing assay methods.
Once it has been determined that degradation products
exist at concentrations above the limits set by the ICH
or belong to a genotoxic category, the pharmaceutical
development team should assess whether it is worth-
while to proceed with the proposed formulation. In
most cases, the assessment of degradation products
during development includes forced degradation stud-
ies, which allow the definition of factors, such as the
selectivity of the analytical method used. It is not
always possible to find reference standards for degra-
dation products that need to be identified, quantified,
and qualified. Often, it is necessary to characterize and
synthesize the products to conduct the studies. Time
and cost are limiting factors in this process.
When degradation products requiring qualification are
present, it may be simpler and cheaper to reduce
impurities to concentrations below the qualification
threshold (6). In this way, applying the concept of
quality by design is very desirable because it allows
one to reduce to desired levels or eliminate the impu-
rities that arise by controlling the critical parameters.
Quality by design refers to the use of a systems ap-
proach to pharmaceutical development, that is, design-
ing and developing formulations to ensure pre-defined
parameters of quality. It includes the definition of a
quality profile, the identification of critical quality
attributes, parameters, and sources of variability to
obtain consistent quality over time and thus allow
the understanding and control of variables in phar-
maceutical formulation (60). This concept can be
applied to the evaluation of degradation impurities,
including those that are genotoxic, using an inte-
grated control strategy. The strategy focuses on
mechanistic understanding, formulation, storage
conditions, and packaging.
If, under the proposed packaging and storage condi-
tions, it is anticipated that the degradation product will
be formed at levels approaching the acceptable limit,
the formation of degradation products must be con-
trolled (9). Controlling critical variables and conduct-
ing a follow-up study of these impurities can exclude
degradation impurities that are present at concentra-
tions greater than threshold values, thus ensuring
product quality. This approach can be applied during
early development stages and involves appropriate
identification methods (45). If it is anticipated that
formulation development and packaging design op-
tions are unable to bring mutagenic degradant levels to
less than the acceptable limits or to a level as low as
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reasonably practicable, the highest limit can be justi-
fied based on a risk/benefit analysis (9).
Investing in research is still the easiest and cheapest
alternative for the pharmaceutical industry to ensure
the development of robust formulations. When geno-
toxic degradation compounds are discovered after
manufacturing, the drug design process may have been
wasted, and the discovery may result in product recall.
Such outcomes are detrimental for both the manufac-
turing industry and the exposed patients.
Conclusion
In the current regulatory environment, there is in-
creasing overlap of drug registration rules among
countries. Existing drug products are frequently si-
multaneously registered and marketed in more than
one country, and regulatory agencies have sought to
harmonize regulations that ensure pharmaceutical
quality and safety. Stringent regulatory standards
and an industry-wide commitment to the issue of
degradation are essential to ensure pharmaceutical
safety, efficacy, and quality.
Declaration of Interest Statement
The authors report no conflict of interest.
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