Regulatory Toxicology and Pharmacology 62 (2012) 449–455

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Regulatory Toxicology and Pharmacology

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Survey Report

In silico methods combined with expert knowledge rule out mutagenic potential
of pharmaceutical impurities: An industry survey
Krista L. Dobo a,⇑, Nigel Greene a, Charlotta Fred b, Susanne Glowienke c, James S. Harvey d,
Catrin Hasselgren e, Robert Jolly f, Michelle O. Kenyon a, Jennifer B. Munzner a, Wolfgang Muster g,
Robin Neft h, M. Vijayaraj Reddy i, Angela T. White d, Sandy Weiner j
a
Pfizer Global Research and Development, Eastern Point Road, Groton, CT 06340, USA
b
Safety Assessment, AstraZeneca R&D Södertälje, 151 85 Södertälje, Sweden
c
Novartis Pharma AG, Werk Klybeck, Klybeckstrasse 141, CH-4057 Basel, Switzerland
d
GlaxoSmithKline, Park Road, Ware, Herts SG12 0DP, UK
e
Global Safety Assessment, AstraZeneca R&D, S-431 83 Mölndal, Sweden
f
Eli Lilly and Company, Indianapolis, IN 46285, USA
g
F. Hoffmann-La Roche Ltd., Pharma Research and Early Development, CH-4070 Basel, Switzerland
h
Covance Laboratories Inc., 671 South Meridian Road, Greenfield, IN 46140, USA
i
Merck Research Laboratories, West Point, PA 19486, USA
j
Johnson & Johnson Drug Safety Sciences, Route 202 South, Raritan, NJ 08869, USA

a r t i c l e i n f o a b s t r a c t

Article history: With the increasing emphasis on identification and low level control of potentially genotoxic impurities
Received 12 January 2012 (GTIs), there has been increased use of structure-based assessments including application of computer-
Available online 31 January 2012 ized models. To date many publications have focused on the ability of computational models, either indi-
vidually or in combination, to accurately predict the mutagenic effects of a chemical in the Ames assay.
Keywords: Typically, these investigations take large numbers of compounds and use in silico tools to predict their
Genotoxic impurities activity with no human interpretation being made. However, this does not reflect how these assessments
In silico
are conducted in practice across the pharmaceutical industry. Current guidelines indicate that a struc-
(Q)SAR
Mutagenicity
tural assessment is sufficient to conclude that an impurity is non-mutagenic. To assess how confident
we can be in identifying non-mutagenic structures, eight companies were surveyed for their success rate.
The Negative Predictive Value (NPV) of the in silico approaches was 94%. When human interpretation of
in silico model predictions was conducted, the NPV increased substantially to 99%. The survey illustrates
the importance of expert interpretation of in silico predictions. The survey also suggests the use of multi-
ple computational models is not a significant factor in the success of these approaches with respect to
NPV.
Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction
In parallel with the development of the European Medicines
Evaluation Agency (EMEA) guidelines on the limits for genotoxic
impurities (GTIs) in pharmaceuticals (European Medicines Agency,
2006; United States Food and Drug Administration, 2008), strate-
gies for the assessment of GTIs have been developed within indus-
try to help identify, monitor and control these to within the
acceptable limits (Muller et al., 2006; Teasdale et al., 2011). Many
pharmaceutical companies have implemented systematic pro-
cesses for the identification of GTIs starting with the first clinical
supplies synthesized. Current practices for identification of GTIs
⇑ Corresponding author. Fax: +1 860 715 5878.
E-mail address: krista.l.dobo@pfizer.com (K.L. Dobo).
often rely on the use of in silico methods to predict the mutagenic
potential of novel chemicals based on their chemical structure
(Dobo et al., 2006).
Over the last two decades there have been numerous publica-
tions comparing and contrasting the different commercial systems
commonly used within the pharmaceutical industry (Greene,
2002; Hansen et al., 2009; Hillebrecht et al., 2011; Naven et al.,
2010; Snyder, 2009; Snyder et al., 2004; White et al., 2003). These
reviews assessed a large set of molecules that have been previously
tested in the Ames assay and compared the predictions of the in
silico system to the results of the biological assay. The relative sen-
sitivity and specificity of each system was then reported and used
for comparison purposes. In general, the commercial systems per-
formed adequately when identifying Ames positive compounds in
sets of chemicals from the public domain but often did not perform

0273-2300/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.yrtph.2012.01.007
450 K.L. Dobo et al. / Regulatory Toxicology and Pharmacology 62 (2012) 449–455
as well for proprietary active compound sets from pharmaceutical
companies. This discrepancy arises from the fact that pharmaceu-
tical compounds tend to consist of complex molecular structures
which generally do not contain reactive functional groups such
as alkylating agents, aromatic nitro groups and epoxides. This is
not the case for public domain data sets. In contrast to the active
drug substance, starting materials and intermediates are smaller
and often reactive molecules that are more likely to have common
features with mutagens in public databases. As such, in silico sys-
tems should have greater success at predicting the mutagenic po-
tential of pharmaceutical impurities.
To increase the sensitivity of computational models for pharma-
ceutical compounds, i.e. their ability to correctly identify Ames po-
sitive compounds, some authors have combined the results from
more than one model, developing a consensus result (Hillebrecht
et al., 2011; Matthews et al., 2008; Pearl et al., 2001; White
et al., 2003). These consensus approaches have shown greater sen-
sitivity but have the disadvantage that certain compounds fall out-
side the applicability domain (i.e. are not well represented by the
training set) of at least one of the models used. In such cases, the
model would not be useful alone or in combination and a consen-
sus result would be misleading. More importantly, these studies
did not take into account any human interpretation of each of
the predictions made and as a result do not accurately reflect the
process that has been adopted by most pharmaceutical companies.
In April 2011, the Drug Information Association (http://
www.diahome.org) organized a conference held in Washington,
DC which focused on the use of in silico tools for the identification
of GTIs. A specific topic of interest was how likely it is that struc-
tural assessments will result in a false negative prediction, i.e. an
Ames positive compound is mistakenly predicted non-mutagenic.
Given that in silico analyses have been used for several years to
identify potential GTIs, a cross-industry survey of practical experi-
ence was conducted. The survey was intended to evaluate the rate
of success in predicting a negative result in the Ames assay. That is,
where an in silico assessment of a starting material, synthetic inter-
mediate or impurity predicted no potential for mutagenicity and
the material was later tested in the Ames assay, how frequently
was there agreement between the negative prediction and the
assay outcome. Ames testing is often done on process intermediates
Table 1
Classification scheme for impurities in drug products.
Class 1 Known to be both mutagenic and carcinogenic
Class 2 Known to be mutagenic but unknown carcinogenic
potential
Class 3 Structurally alerting compound, unrelated to the
active pharmaceutical ingredient (API) and of
unknown mutagenic potential
Class 4 Structurally alerting compound related to the API
Class 5 No structural alerts or sufficient evidence for
absence of mutagenicity
Table 2
Common computational systems and publicly available databases used to classify compounds by survey participants.
Name Abbreviation
Derek for Windows DfW
MCASE MC4PC MCASE
TOXNET TOXNET
Vitic Vitic
SciFinder SciFinder
NTP NTP
ExPub ExPub
for reasons other than control of impurities, such as providing data
for occupational health exposure controls or shipping regulations.
Predictions were made prior to the generation of the Ames data
and therefore was not used to ‘‘train’’ the in silico systems.
Eight companies participated in the survey. Each survey partic-
ipant provided a step by step description of the process used for
evaluation of structures of starting material intermediates and
impurities, as well as summary data comparing concordance of
negative predictions vs. the Ames result. This manuscript describes
the methods used by each of the companies surveyed and how suc-
cessful each company has been with correctly predicting a negative
result in the Ames assay. For the current survey, no attempt was
made to evaluate the overall sensitivity and accuracy of the struc-
ture assessment approaches, since the question most critical to
protecting patients relates to correct negative predictions. Com-
pounds considered to be potentially mutagenic based on the struc-
tural assessment will either be controlled within the final product
and/or tested in the Ames assay.
2. Methods
In general, most organizations conduct structural assessments
of all starting materials, intermediates and identified impurities
for mutagenic potential early in the development of a drug candi-
date (prior to the first in human clinical investigation). As changes
in synthetic process are made new starting materials, intermedi-
ates and impurities are also evaluated using in silico methods.
Principles for the classification of GTIs first published by Muller
et al. (2006) have been widely adopted across the pharmaceutical
industry (Table 1). Most companies in this survey typically used
one or more in silico system to identify structural features associ-
ated with mutagenicity and carcinogenicity. Structurally alerting
features of the starting materials and intermediates were then
compared to those of the API at many companies. Where starting
materials or intermediates contained alerting features that were
present in the API in the same chemical context (i.e. similar steric
and electronic environments) then these compounds were consid-
ered to be qualified as Class 4. That is, if the respective API was
negative in an Ames mutagenicity assay, then the compound in
question was considered non-mutagenic.
Further evaluation of the compounds was, in most cases, con-
ducted using structure-searchable databases. These databases were
used to identify the compound in question, any structurally similar
compounds with the same alerting features, and respective litera-
ture references regarding genotoxic and/or carcinogenic precedent.
In addition, a proprietary structure-searchable database was some-
times used to identify compounds with similar alerting features and
any corresponding genotoxicity data. Finally, in some instances, the
drug safety scientist consulted with a medicinal chemist to better
understand how various substitutions would affect the reactivity
of an alerting sub-structure (e.g. electron withdrawing or donating
Supplier, URL and selected references
Lhasa Ltd., http://www.lhasalimited.org/ (Greene, 2002;
Greene et al., 1999;Ridings et al., 1996; Sanderson and Earnshaw, 1991)
Multicase Inc., http://www.multicase.com/
(Matthews and Contrera, 1998; Rosenkranz and Klopman, 1990;
Rosenkranz and Klopman, 1993; Saiakhov and Klopman, 2010)
http://toxnet.nlm.nih.gov/
Lhasa Ltd., http://www.lhasalimited.org/
Chemical Abstracts Service, http://www.cas.org/
http://ntp-server.niehs.nih.gov
http://www.expub.com
 K.L. Dobo et al. / Regulatory Toxicology and Pharmacology 62 (2012) 449–455
effects). Computational systems and databases commonly used by
companies that participated in this survey are listed in Table 2.
In some organizations, although not all, molecules that have a
structural alert or concern were submitted to an Ames test in order
to verify or reject the structure-based prediction. In other compa-
nies the decision to test structurally alerting impurities and degra-
dants was taken on a case by case basis and was limited to those
structures which were actually observed in final API or from degra-
dation studies rather than all those that were simply predicted
using in silico tools.
Later in development, when a project reaches milestones such
as of proof of concept, many starting materials and intermediates
are tested in the Ames assay to help support the setting of occupa-
tional exposure limits in the workplace. These practices make it is
possible to evaluate the predicted outcome of the Ames test which
was made previously based on structural analysis, with the actual
outcome of the Ames assay.
While most companies have similar strategies for reviewing
impurities, that include expert judgment as well as in silico tools,
the actual methods and approaches for structure-based assess-
ments vary from one organization to the next. To exemplify these
differences, the methods employed by organizations that partici-
pated in the survey are presented in Appendix A.
3. Results and discussion
The primary objective of this investigation was unique in that it
was a survey of practical experience in the use of structure-based
methods to evaluate GTIs. There was no attempt to compare and
contrast the performance of various commercial or proprietary sys-
tems. Rather, the survey was intended to understand the basic
methodology being used across pharmaceutical companies to con-
duct a structure-based assessment and the rate of success in pre-
dicting a negative result in the Ames assay. That is, for those
cases where a structure-based assessment of a starting material,
synthetic intermediate or impurity resulted in the conclusion of
no potential for mutagenicity and the material was tested at some
point later in time in the Ames assay, how frequently was there
Table 3
Summary of process used by each company to complete a structure-based assessment.
a
DEREK computational system with commercially available rules as well as rules that have been developed and implemented by the respective company.
b
MCASE computational system with commercially available rules as well as rules that have been developed and implemented by the respective company.
c
(Q)SAR (Quatitative) Structure Activity Relationship.
d
The structure of a starting material, intermediate or impurity that has not been tested in the Ames assay is compared to the structure of the API which has been tested in the
Ames assay for structural similarity.
451
agreement between the prediction and the assay outcome. When
conducting structure based assessments of impurities, confidence
in negative predictions is of greatest concern for assuring human
safety, as a false negative prediction could result in human expo-
sure several orders of magnitude greater than acceptable exposure
limits for mutagenic impurities. In contrast, false positive predic-
tions are of low concern, as this would result in low level impurity
exposures to non-mutagenic impurities.
3.1. Comparison of methodology
The in silico methods routinely used by the eight companies
surveyed are summarized in Table 3. There are two key observa-
tions from this survey:
First, all eight companies surveyed initiated their in silico assess-
ment by processing structures through a (Q)SAR system or systems
(vendor and/or in-house) to identify sub-structures that may confer
mutagenic potential. It is evident that the number and combination
of (Q)SAR systems utilized across the eight companies were not
identical however, all eight companies surveyed used DfW. Despite
the differences, the underlying principles and practices used to con-
duct an in silico assessment were highly similar.
Second, seven of the eight companies further evaluated all
structures that did not contain any in silico alerts (as defined by
output of (Q)SAR tools). This included taking into consideration
their own working knowledge of mutagenic structure activity rela-
tionships of specific structural classes, structural similarity of an
impurity to the respective API, database searches (public and/or
proprietary), and in some cases consultation with medicinal chem-
ists to better understand the impact of various substitutions on
chemical reactivity. All of this information was taken into consid-
eration before arriving at a conclusion that a structure was not
mutagenic. Therefore, when it comes to practical application of
(Q)SAR tools for the identification of potentially mutagenic impu-
rities, the majority of companies surveyed relied on an expert for
the final interpretation, which may or may not agree with the out-
put of the (Q)SAR tools.
452 K.L. Dobo et al. / Regulatory Toxicology and Pharmacology 62 (2012) 449–455

Table 4a
Individual company concordance. Negative predictions based on (Q)SAR tool output alone vs. Ames assay result.

Data assessment Company 1 Company 2 Company 3 Company 4 Company 5 Company 6 Company 7 Company 8
a
Ames Neg/Predicted Neg 29/30 60/66 91/94 180/196 55/60 47/50 NA 69/70
NPV (%)b 97 91 97 92 92 94 NA 99
a
Number of compounds that produced a negative result in the Ames test/number of compounds that were predicted negative.
b
Negative Predictive Value.

Table 4b
Individual company concordance. Negative predictions based on (Q)SAR tool output plus expert evaluation vs. Ames assay result.

Data Assessment Company 1 Company 2 Company 3 Company 4 Company 5 Company 6 Company 7 Company 8
Ames Neg/Predicted Nega 29/29 60/62 NA 180/180 55/57 47/48 32/32 NA
NPV (%)b 100 97 NA 100 96 98 100 NA
a
Number of compounds that produced a negative result in the Ames test/number of compounds that were predicted negative.
b
Negative Predictive Value.

Table 5
(Q)SAR vs. Q(SAR) plus expert evaluation. Summary of concordance.

Structural assessment Predicted Ames result Number of compoundsa Number of Ames positiveb (%) Number of Ames negativec (%)
(Q)SAR Negative 566 35 (6) 531 (94)
(Q)SAR + expert evaluation Negative 408 5 (1) 403 (99)
a
Total number of compounds predicted negative.
b
The number of compounds predicted negative that produced a positive response in the Ames assay.
c
The number of compounds predicted negative that produced a negative response in the Ames assay.

3.2. Concordance assessment
The concordance between negative predictions and the outcome
of the Ames assay, that is, the Negative Predictive Value (NPV) was
very high when each company’s data were considered individually.
For each company the NPV was evaluated using two approaches. In
the first analysis, the prediction of mutagenicity based solely on the
output of QSAR or SAR tools was compared to the outcome of the
Ames assay (Table 4a). Seven of eight companies provided data
for this comparison. Company number seven did not routinely cap-
ture mutagenicity predictions based solely on (Q)SAR and therefore
data were not provided. The number of compounds evaluated ran-
ged from 30 to 196 per individual company and concordance was
very similar across all companies, ranging from 91% to 99%. There-
fore, despite the differences in the number and type of (Q)SAR tools
that were used in practice the ability to correctly predict a negative
result in the Ames assay was very high (based solely on the output
of the computational system prediction).
In the second analysis, the prediction of mutagenicity based on
(Q)SAR output together with additional information gathered by
the assessor was compared to the outcome of the Ames assay (Ta-
ble 4b). Six of eight companies provided data for this comparison.
Company 3 did not routinely rely on expert evaluation for interpre-
tation of (Q)SAR output. Although company 8 routinely uses expert
evaluation for interpretation of (Q)SAR output, when data were
collected for the purposes of the survey, only compounds that were
predicted negative based solely on (Q)SAR output could be readily
identified. The number of compounds evaluated ranged from 29 to
180 per individual company and NPV was even higher, ranging
from 96% to 100%. In all cases, when an expert evaluated the
(Q)SAR output and put this into context with other information,
the NPV increased. As a result, this further minimized the occur-
rence of cases where a compound was predicted negative and
was then found to be mutagenic in the Ames assay. The data in Ta-
ble 4b illustrate that the number of predicted negative compounds
was reduced after taking into account expert evaluation based on
knowledge and the literature.
When possible survey participants provided some detail of the
issues identified during the expert evaluation, that were not
flagged by the (Q)SAR system(s). Three companies indicated that
boronic acids accounted for one or more compounds that were
identified as potentially mutagenic via the expert evaluation.
Two companies indicated that certain types of aromatic amines
were flagged as potentially mutagenic. No other specific classes
of compounds were noted.
After considering each company’s data individually, the data
from all eight companies were pooled together to evaluate overall
concordance based on a large number of molecules (Table 5). The
correlation between negative predictions and the outcome of the
Ames assay was very high when all the data were considered col-
lectively. From a total of 566 compounds that were predicted neg-
ative based solely on (Q)SAR, 531 (94%) produced negative results
in the Ames assay. Given the fact that many compounds associated
with pharmaceutical synthesis are chemically reactive by design,
the incidence of Ames positive compounds in a data set comprised
of such chemicals should be higher than that for APIs, which for
marketed pharmaceuticals has been estimated to be around 7%
(Snyder, 2009). Based on a previously published retrospective anal-
ysis (Dobo et al., 2006), the incidence of Ames positive compounds
in any given set of intermediates and impurities could be as high as
20–25%. However, not all structural alerting compounds are tested
in the Ames assay as they may not be present above the allowable
TTC or their levels are easily controlled during synthesis of the API.
Therefore, the exact ratio of Ames positives to negatives is un-
known. Nonetheless, expert review of (Q)SAR output increased
overall NPV. Of the 408 compounds that were predicted negative
based on (Q)SAR and an expert review, 403 (99%) showed no evi-
dence of mutagenicity in the Ames assay.
Of the eight companies surveyed three companies used one in
silico tool to support predictions and five companies used two or
more. The NPVs for companies using one in silico tool (companies
2, 3 and 7; Table 3), ranged from 91% to 97% (Table 4a). In compar-
ison, the NPVs for companies using two or more tools (companies
1, 4, 5, 6, and 8; Table 3), the NPVs ranged from 92% to 99%
 K.L. Dobo et al. / Regulatory Toxicology and Pharmacology 62 (2012) 449–455
(Table 4a). Thus, there is no apparent relationship between the
number of in silico systems used and degree of concordance
achieved between negative predictions and Ames assay outcome.
4. Conclusions
It is clear from the survey that the processes companies employ
for GTI evaluation are highly similar. Prediction processes probably
over predict the potential for mutagenicity (higher false positives)
in order to support more accurate prediction of no potential for
mutagenicity (low false negatives). This survey of practical experi-
ence indicates that in silico analysis alone predicts a negative out-
come correctly 94% of the time. There is no apparent relationship
between the number or combination of in silico tools used and con-
cordance with a negative outcome in the Ames. These results also
demonstrate that an analysis that relies on the use of an in silico
system in conjunction with expert evaluation results in even fewer
false negative conclusions. Taken together, results from the survey
argue that current processes for evaluation of GTIs are more than
adequate in preventing false negatives and protecting patients.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Appendix A
A.1. Company 1 methods
The structure-based assessment was initiated by submitting the
structures of starting materials, intermediates and the respective
API to DfW for identification of structural features alerting for
mutagenicity. The compounds were also analyzed using MCASE
using the AZ2 module for Ames mutagenicity and the internal
‘‘Genetox Warning System’’ (GWS) (Glowienke and Hasselgren,
2010). Both of these tools provide a QSAR prediction of the activity
in addition to structural alerts.
Positive predictions or a structural alert leads to further inves-
tigation or qualification of the impurity in a standard 5-strain
Ames test by testing of neat material. Structurally alerting features
of the starting materials and intermediates identified using any of
the tools were compared to that of the API. Starting materials or
intermediates that had alerting features that were present in the
API in the same context (i.e. similar steric and electronic context)
were considered to be non-unique and qualified via testing of the
API in standard genotoxicity tests (Class 4). That is, if the respective
API was negative in an Ames mutagenicity assay, then the com-
pound in question would be considered non-mutagenic.
Compounds predicted to be inactive by MCASE and the GWS
QSAR models and had no structural alerts from any system were
further evaluated by read-across. This was conducted by examin-
ing the output from the GWS which automatically searches for
experimentally tested structural near neighbors of the query com-
pound(s). The GWS includes data reported in the CCRIS database,
the NTP database, the internal database and Ames data included
in the MCASE software. Any structurally similar compounds were
evaluated in terms of genotoxic and/or carcinogenic precedent
and their chemical/structural relevance to the query compound.
Negative predictions were sometimes questioned if the training
set of the models lack similar structures and such compounds were
usually tested experimentally. Based on all available evidence for
mutagenic and carcinogenic potential collected, each starting
material, intermediate and impurity was classified according to
the definitions published in Muller et al. (2006).
For this survey of practical experience, all starting materials,
intermediates and impurities that were predicted to be non-muta-
453
genic (Class 5) and that were subsequently tested in the Ames as-
say were included. This set represents a subset of all compounds
that are negatively predicted by all models but it is a suitable set
for this analysis as it represents those predictions that were con-
sidered least reliable.
A.2. Company 2 methods
The structure-based assessment presented here was initiated
by submitting the structures of synthetic intermediates and the
respective API to DfW v12 for identification of structural features
alerting for mutagenicity. Additionally each synthetic intermediate
was assessed according to both the definitions published in Muller
et al. (2006) and through existing knowledge by comparison with
structures in an internal proprietary structure-searchable data-
base. If each of these assessments failed to highlight a structural
concern then the synthetic intermediate was considered to be
non-SAR alerting.
Where a CAS number was available further evaluation was con-
ducted using the structure-searchable databases, TOXNET and Vitic
as well as NTP, Japan Existing Chemical Data Base (http://dra4.-
nihs.go.jp/mhlw_data/jsp/SearchPageENG.jsp), ExPub, BG Chimie
(http://www.bgchemie.de) and Scopus (http://www.scopus.com/
home.url). The databases were used to identify literature regarding
genotoxicity and/or carcinogenicity data for each synthetic inter-
mediate or any structurally similar compounds with the same
alerting features.
Synthetic impurities with alerting features that were present in
the API in the same context (i.e. similar steric and electronic con-
text) were considered to be non-unique and qualified via testing
of the API in standard genotoxicity tests (Class 4). That is, if the
respective API was negative in an Ames mutagenicity assay, then
the compound in question would be considered non-mutagenic.
In some instances a consultation with a Computational Chemist
was performed to confirm the reactivity of an alerting sub-struc-
ture was similar between parent and synthetic impurity.
For this survey, synthetic intermediates from a subset of 29 post
proof of concept drug candidates were identified. All synthetic
intermediates that were predicted to be non-mutagenic and where
Ames data was available were included in the survey, that is, those
compounds which were non-SAR alerting (Class 5) or showed
structural similarity to the parent (Class 4). ICH Q3C solvents were
excluded from the analysis.
A.3. Company 3 methods
Structural assessment of all starting materials, intermediates,
and identified impurities for mutagenic potential were initiated
early in the development of a drug candidate (prior to the first in
human clinical investigation). As changes in synthetic process were
made new starting materials, intermediates and impurities were
also evaluated using in silico methods. Later in development, when
a project reached the milestone of proof of concept, many starting
materials and intermediates were tested in the Ames assay to help
support the setting of occupational exposure limits in the work-
place. These practices made it possible to evaluate the predicted
outcome of the Ames test which was made years previously based
on structural analysis, with the actual outcome of the Ames assay.
The structure-based assessment was initiated by submitting the
structures of starting materials, intermediates and the respective
API to DfW for identification of structural features alerting for
mutagenicity. For this survey of practical experience, all starting
materials, intermediates and impurities that were predicted to be
non-mutagenic and subsequently tested in the Ames assay were
included.
454 K.L. Dobo et al. / Regulatory Toxicology and Pharmacology 62 (2012) 449–455
A.4. Company 4 methods
Company 4 had used MC and expert opinion for the interme-
diates submitted for this survey but recently switched to DfW
and compounds were retested using that program. Highly concor-
dant results were obtained with MC when compared to DfW and
when expert opinion was included the results for all intermedi-
ates were equivalent. As this was a survey of current practice,
the DfW results were used for this analysis. A structure-based
assessment was initiated by submitting the structures of starting
materials and intermediates of the respective API to DfW for
identification of structural features alerting for genotoxicity and
mutagenicity. The penultimate compounds were tested in Ames
regardless of the in silico prediction. The remaining starting
materials and intermediates were further evaluated using the
structure-searchable external databases such as TOXNET, ExPub,
VITIC and SciFinder. The databases were used to identify the com-
pound in question, any structurally similar compounds with the
same alerting features, respective literature references and any
corresponding genotoxicity data. Internal proprietary databases
were also searched to ascertain whether genotoxicity data already
exist for the compounds being evaluated or close congeners. In
addition, intermediates and reagents that were negative in DfW
were further evaluated using internal proprietary SAR tools and
the drug safety scientist consulted with a medicinal chemist to
better understand how various substitutions would affect the
reactivity of an alerting sub-structure (i.e. electron withdrawing
or donating effects). Based on these data, each starting material
and intermediate is predicted to be genotoxic or non-genotoxic
and a recommendation is made whether to test in the Ames as-
say. Appropriate limits for the compounds in final API are then
provided to process chemists.
For those cases where a structure-based assessment of a start-
ing material, synthetic intermediate or impurity resulted in the
conclusion of no potential for mutagenicity and the material was
tested in the Ames assay, this survey assessed how frequently
there was agreement between the negative prediction and the as-
say outcome.
A.5. Company 5 methods
All process intermediates, reagents, and identified impurities
were structurally assessed for their potential mutagenicity using
the in silico tools, DEREK (Lhasa Ltd., Leeds, UK) and MCASE (Mul-
tiCASE Inc., Cleveland, OH). The assessment was performed on all
synthetic routes used for the preparation of API (Active Pharma-
ceutical Ingredient) for clinical use. Those non-alerting in DEREK
and MCASE were further evaluated for the presence of literature
based alerts (Ashby and Tennant, 1991) and ensured that these
were adequately covered in the in silico tools. If the same alerting
sub-structure was also present in the API in a similar chemical con-
text and the API was negative in the Ames assay, then the alerting
structure was considered qualified and treated as an ordinary
impurity.
A.6. Company 6 methods
The structure-based assessment was initiated by submitting
the structures of starting materials, intermediates and the
respective API to DfW, MCASE and an internal SAR system for
the identification of structural features alerting for Salmonella
mutagenicity. Structurally alerting features of the starting mate-
rials and intermediates identified were compared to that of the
API. Starting materials or intermediates that had alerting fea-
tures that were present in the API in a similar structural context
(i.e. similar chemical environment of the alerting moiety) were
considered to be non-unique and qualified via testing of the
API in standard genotoxicity tests (Class 4). Thus, if the respec-
tive API was negative in an Ames mutagenicity assay, then the
compound in question was considered non-mutagenic. Usually
no additional structure-based evaluation was conducted for
these compounds.
For alerting and non-alerting starting materials and intermedi-
ates, further evaluations were conducted (if applicable, e.g. as indi-
cated by the presence of a CAS number) using mainly the
structure-searchable databases, TOXNET and Vitic but also, if con-
sidered informative, others such SciFinder or by general Google
searches. These databases were used to identify the compound in
question, in some cases to identify structurally similar compounds
with the same alerting fragments, and respective literature refer-
ences regarding genotoxic and/or carcinogenic precedent. In addi-
tion, an internal proprietary structure-searchable database was
also used to identify compounds or structural analogues already
tested internally in an Ames test. In some instances, the drug safety
scientist consulted with a medicinal chemist to better understand
how various substitutions would affect the reactivity of an alerting
sub-structure (e.g. electron withdrawing or donating effects).
Based on all the available evidence for mutagenic and carcinogenic
potential collected, each starting material, intermediate and impu-
rity was classified according to the definitions published by Muller
et al. (2006).
For this survey all starting materials, intermediates and impuri-
ties that were predicted to be non-mutagenic and subsequently
tested in the Ames assay were included.
A.7. Company 7 methods
The structure-based assessment was initiated by submitting
the structures of starting materials, intermediates and the
respective API to DfW for identification of structural features
alerting for mutagenicity and carcinogenicity. Structural alerting
features of the starting materials and intermediates identified
using DfW were compared to that of the API. Starting materials
or intermediates that had alerting features that were present in
the API in the same context (i.e. similar steric and electronic
context) were considered to be non-unique and qualified via
testing of the API in standard genotoxicity tests (Class 4). That
is, if the respective API was negative in an Ames mutagenicity
assay, then the compound in question was considered non-
mutagenic. No additional structure-based evaluation was con-
ducted for these compounds.
For the remainder of starting materials and intermediates, fur-
ther evaluation was conducted using the structure-searchable
databases, TOXNET, VITIC and SciFinder. The databases were used
to identify the compound in question, any structurally similar
compounds with the same alerting features that were identified
using DfW, and respective literature references regarding geno-
toxic and/or carcinogenic precedent. In addition, an internal pro-
prietary structure-searchable database was also used to identify
compounds with similar alerting features and any corresponding
genotoxicity data. In some instances, the drug safety scientist
consulted with a medicinal chemist to better understand how
various substitutions would affect the reactivity of an alerting
sub-structure (e.g. electron withdrawing or donating effects).
Based on all available evidence for mutagenic and carcinogenic
potential collected, each starting material, intermediate and
impurity was classified according to the definitions published in
Muller et al. (2006). For this survey of practical experience, all
starting materials, intermediates and impurities that were pre-
dicted to be non-mutagenic and subsequently tested in the Ames
assay were included.
 K.L. Dobo et al. / Regulatory Toxicology and Pharmacology 62 (2012) 449–455
A.8. Company 8 methods
The genotoxic impurities to be monitored were selected after
careful review of the chemical process. All chemical entities that
may be present in the chemical process were tabulated: (a) raw
materials, intermediates and reagents, generally depicted in the
synthesis scheme, (b) impurities detected in the drug substance
(identified impurities) and (c) so-called readily predictable by-
products, potentially arising from side reactions. Reasons to ex-
clude structures from further evaluation were chemical purging.
The level of detail increased with the stage of the development.
Knowledge established on mutagenic/carcinogenic properties
or, if such knowledge was not available, the in silico assessment
for alerting sub-structures was used to differentiate the structures
into five classes of decreasing toxicological concern. All chemical
entities listed were evaluated in two independent in silico systems,
DfW and MCASE, and assigned to one of the five classes defined in
the publication of Muller et al. (2006). Assignment to Class 3 trig-
gered genotoxicity (Ames) testing, the results of which directed the
compound either to Class 5 or 2.
The DfW rulebase applied contains all rules with the superend-
points mutagenicity and genotoxicity that were commercially
available as well as ones that have been developed internally (pro-
prietary). Structural alerts in DfW were considered relevant if the
uncertainty term was ‘plausible’, ‘probable’ or ‘certain’. The MCASE
analysis was conducted by using the commercial A2H module for
Ames test results. A chemical structure was regarded as ‘alerting’
(Class 3) if either DfW or MCASE gave a positive prediction. In
the case of intermediates which were predicted positive, but con-
tained the same structural alert(s) as the respective API, structures
were compared and classification in some cases was changed from
3 to 4.
In addition, all structures were further evaluated by searching
for genotoxicity and carcinogenicity data for the specific chemical
or structurally similar compounds in a number of structure search-
able databases (e.g. TOXNET, Vitic, SciFinder, in-house database).
By applying in-house rules based on mutagenicity testing within
the company-specific chemical space and carefully reviewing the
existing knowledge of close structural analogues, the sensitivity
of the pure in silico prediction was significantly improved.
All available information was summarized in an ‘in silico assess-
ment sheet’ and carefully reviewed by an expert panel consisting of
all involved parties (in silico toxicology, genotoxicology, chemistry,
process chemistry, analytics) deciding on qualification, specifica-
tion and necessary action items.
After each synthesis/process changed with an impact on poten-
tial genotoxic impurities (e.g. new intermediate or critical reagent),
or if additional impurities were identified in newly synthesized
batches, the process described was repeated.
455
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