Regulatory Toxicology and Pharmacology 114 (2020) 104666

Contents lists available at ScienceDirect

Regulatory Toxicology and Pharmacology

journal homepage: www.elsevier.com/locate/yrtph

Acute toxicity “six-pack” studies supporting approved new drug applications T

in the U.S., 2015–2018
Joseph Manuppello∗, Kristie Sullivan, Elizabeth Baker
Physicians Committee for Responsible Medicine, 5100 Wisconsin Ave., NW, Suite 400, Washington, DC, 20016-4131, USA

A R T I C LE I N FO A B S T R A C T

Keywords: The acute toxicity “six-pack” is a battery of animal tests used to evaluate acute systemic toxicity by three routes
Toxicity tests of exposure, skin and eye irritation/corrosion, and skin sensitization. A perception exists that these tests are not
Acute required for pharmaceuticals. For the four years from 2015 through 2018, we tallied the number of corre-
Animal testing alternatives sponding tests submitted by sponsors in approved, original new drug applications, as reported by the U.S. Food
Animal use alternatives
and Drug Administration (FDA) in publicly available reviews. In 125 reviews, we identified 228 single dose acute
United States Food and drug administration
Investigational new drug application
toxicity studies, 62 in vivo local tolerance studies, and 32 in vivo skin sensitization studies, as well as 37 in vitro or
Lethal dose 50 ex vivo local tolerance studies. A total of 4798 animals were used in these studies; however, FDA's reporting was
Local lymph node assay inconsistent, and we estimate the actual number of animals used to be 8998. For the evaluation of single dose
New approach methodology (NAM) acute toxicity, we accessed two guidance documents with conflicting recommendations regarding routes of
In silico administration and number of species to be used. For the evaluation of local tolerance and skin sensitization,
most studies examined were conducted by routes other than that intended for human administration. Non-
animal methods used to evaluate skin sensitization were not reported.

1. Introduction
Acute toxicity is the toxicity produced by a substance when it is
administered in one or more doses during a period not exceeding 24 h
(FDA CDER, 1996). Animals used in acute toxicity tests may suffer se-
vere pain and distress, as well as mortality, at the high dose levels
administered. A battery of animal tests that evaluates acute systemic
toxicity by the oral, topical, and inhalation routes of exposure, as well
as skin and eye irritation/corrosion, and skin sensitization, is commonly
referred to as the “six-pack”. Efforts to reduce the use of animals in
these tests have focused on their applications in the safety assessment of
agrochemicals and industrial chemicals. For the nonclinical assessment
of pharmaceuticals, a perception exists that these tests are generally not
required and only rarely conducted in specific cases (Strickland et al.,
2018; Prior et al., 2019). The U.S. Food and Drug Administration (FDA)
ceased to require the “classical” LD50 (median lethal dose) test more
than three decades ago, recognizing that the test, which used between
60 and 120 animals per test substance, was unnecessarily precise (FDA,
1988).
More recently, in its Predictive Toxicology Roadmap, FDA established
a comprehensive strategy to evaluate new methodologies and tech-
nologies for their potential both to expand predictive toxicology cap-
abilities and to reduce the use of animals in the assessment of regulated
products (FDA, 2017). Some FDA centers have also made efforts, either
internally or in collaboration with agency or industry partners, to de-
velop, validate, and implement such methodologies for specific end-
points. For example, in its guidance for industry and review staff,
Nonclinical Safety Evaluation of Reformulated Drug Products and Products
Intended for Administration by an Alternate Route, FDA's Center for Drug
Evaluation and Research (CDER) recommends that reformulated topical
drug products be evaluated for eye irritation/corrosion potential by
appropriate in vitro or ex vivo methods (FDA CDER, 2015). As discussed
below, such New Approach Methodologies (NAMs) are also available
for the evaluation of skin irritation/corrosion and skin sensitization. In

Abbreviations: BCOP, Bovine Corneal Opacity and Permeability; CBER, Center for Biologics Evaluation and Research; CDER, Center for Drug Evaluation and
Research; EPA, U.S. Environmental Protection Agency; FDA, U.S. Food and Drug Administration; GPMT, guinea pig maximization test; ICCVAM, Interagency
Coordinating Committee on the Validation of Alternative Methods; ICH, International Conference on Harmonisation; IDE, Investigational Device Exemptions; IND,
Investigational New Drug; LD50, median lethal dose; LLNA, Local Lymph Node Assay; LOEL, Lowest Observed Effect Level; MLD, Minimum Lethal Dose; MNL,
Maximum Nonlethal Dose; NAM, New Approach Methodology; NDA, New Drug Application; NOAEL, No Observed Adverse Effect Level; OECD, Organisation for
Economic Cooperation and Development
∗
Corresponding author.
E-mail address: jmanuppello@pcrm.org (J. Manuppello).

https://doi.org/10.1016/j.yrtph.2020.104666
Received 7 February 2020; Received in revised form 13 April 2020; Accepted 17 April 2020
Available online 23 April 2020
0273-2300/ © 2020 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/BY/4.0/).
J. Manuppello, et al.
addition, FDA participates in the Interagency Coordinating Committee
on the Validation of Alternative Methods (ICCVAM), which recently
published A Strategic Roadmap for Establishing New Approaches to Eval-
uate the Safety of Chemicals and Medical Products in the United States
(ICCVAM, 2018).
In order to measure any reduction in animal use, the number of
animals used must first be determined; however, this can be hindered
by the limited ability to quantify animals used for toxicity testing in the
U.S. (ICCVAM, 2018). For pharmaceuticals, FDA's reviews of approved
new drug applications (NDAs), which are publicly available, can pro-
vide insight into the types of studies that sponsors conduct and the
numbers and species of animals they use. For the four-year period from
2015 through 2018, we tallied the single dose acute (systemic) toxicity,
local tolerance (eye, skin, and vascular irritation/corrosion), and skin
sensitization studies that were submitted by sponsors in approved,
original NDAs, as reported by FDA in its reviews. For each study, we
also collected additional information, as described below, including the
number and species of animals used, if reported. We then compared our
results to the information needs and testing recommendations identi-
fied by FDA in its guidance to industry. Our results challenge the per-
ception that the acute toxicity “six pack” tests are only rarely conducted
for the nonclinical assessment of pharmaceuticals and identify needs for
clear guidance to industry and consistent reporting by FDA.
2. Materials and methods
On its Drugs@FDA: FDA-Approved Drugs web page (FDA, 2020a), we
searched FDA's reviews of approved, original NDAs by month from
January 2015 through December 2018. In the tables returned, for “Type
1" (new molecular entity) entries, we noted the approval date, drug
name, and company name in a Microsoft Excel workbook (Supple-
mentary File). We accessed each Type 1 entry and noted the route in-
tended for human administration. From the “Approval Date(s) and
History, Letters, Labels, Reviews” table, we accessed “Reviews”, and in
the drug approval package, we noted the availability of a Pharma-
cology/Toxicology Review either as a separate document or as a sum-
mary in a Multi-Discipline Review. We downloaded each of these re-
views and, for any review including scanned documents, recognized
text using Adobe Acrobat.
Most Pharmacology/Toxicology Reviews available as separate
documents included a “Studies Submitted” section with a “Studies Not
Reviewed” subsection; we noted whether studies that were submitted
but not reviewed were listed. If such studies were not listed, we copied
any explanatory text to a comment in the workbook (Supplementary
File, “List” sheet). Pharmacology/Toxicology Reviews summarized
within Multi-Discipline Reviews did not include a “Studies Submitted”
section, and studies not reviewed were not listed.
For each animal study listed, we noted the type of study (single dose
acute toxicity, local tolerance, or skin sensitization), whether the study
was not reviewed by FDA, the route of administration, and the species
and number of animals used, if reported.
In addition, for single dose acute toxicity studies, we noted whether
each study was described as “dose range finding,” “dose escalation,” or
similar and whether reporting included:
• no observed adverse effect level (NOAEL) or lowest observed effect
level (LOEL);
• minimum lethal dose (MLD) or maximum nonlethal dose (MNL);
• test substance-related lethality; and
• target organ(s) of toxicity.
For local tolerance studies, which include those for the evaluation of
skin and eye irritation/corrosion, we noted the type of irritation eval-
uated and, for in vitro or ex vivo studies, the method used. For skin
sensitization studies, we noted whether each study was a murine local
lymph node assay (LLNA) or a guinea pig maximization test (GPMT).
2
Regulatory Toxicology and Pharmacology 114 (2020) 104666
For each review in which multiple studies of the same type were
reported, we noted whether these studies differed in the test article,
species of animal, route of exposure, or experimental protocol used and
copied any relevant descriptive text to a comment in the workbook
(Supplementary File, "Multiples" sheet and individual review sheets).
On FDA's Search for FDA Guidance Documents web page (FDA,
2020b), we filtered by product (Drugs) and topic (Pharm/Tox) and
downloaded all guidance documents returned. We also searched for and
downloaded any additional guidance documents listed in CDER's Re-
search List of Guidance Documents under the Pharmacology/Toxicology
heading (FDA, 2020c). We reviewed relevant general guidance docu-
ments in detail and searched all guidance documents for key terms
(single dose, acute, local tolerance, irritation, sensitization, and sensi-
tivity).
3. Results
For the four-year period from 2015 through 2018, we identified 125
FDA reviews of approved, original NDAs. Studies that were submitted
by sponsors, but not reviewed by FDA, were listed in 38 (30.4%) of
these reviews (Supplementary File, “List” sheet).
3.1. Single dose acute toxicity
In the 125 reviews examined, we identified 228 single dose acute
toxicity studies, 1.8 studies per review, on average. In 61 reviews, we
identified no such studies; in 15, we identified one study; and in 49, we
identified two through 12 studies. Twenty-two of these studies were
listed as submitted by sponsors but not reviewed by FDA
(Supplementary File, “List” sheet).
Oral administration was used in 112 (49.1%) of these studies, in-
travenous in 73 (32.0%), subcutaneous in 23 (10.1%), inhalation in
eight (3.5%), intraperitoneal in seven (3.0%), topical and intramuscular
in two each (0.9%), and intrathecal in one (0.4%). Rats were used in
112 studies (49.1%), mice in 56 (24.6%), dogs in 39 (17.1%), monkeys
in 19 (8.3%), and rabbits and cats in one each (0.4%). In addition, 163
studies (71.5%) were conducted by the route intended for human ad-
ministration, while 65 studies (28.5%) were conducted by other routes
(Supplementary File, “Summary” sheet).
Fig. 1 shows the number of single dose acute toxicity studies con-
ducted by species and route of administration as well as by routes in-
tended or unintended for human administration. Rats were used more
frequently than any other species in studies conducted by all routes,
except for intrathecal; monkeys were used in the only study conducted
by this route. Mice were used the next most frequently in studies by the
intravenous, subcutaneous, intraperitoneal and inhalation routes; mice
and dogs were used the same number of times in studies by the oral
route. Dogs were used the next most frequently in studies by the in-
travenous, subcutaneous, and inhalation routes. Monkeys were used the
next most frequently in studies by the oral and intravenous routes. In
addition, more studies were conducted by the route intended for human
administration in all species except rabbits; rabbits were used in only
one study, which was by the topical route.
For the evaluation of acute toxicity, we accessed two relevant
general guidance documents, Single Dose Acute Toxicity Testing for
Pharmaceuticals (FDA CDER, 1996) and M3(R2) Nonclinical Safety Stu-
dies for the Conduct of Human Clinical Trials and Marketing Authorization
for Pharmaceuticals (FDA CDER and FDA CBER, 2010). FDA CDER notes
that its 1996 guidance was originally published as part of a proposed
implementation document entitled U.S. FDA's Proposed Implementation
of International Conference on Harmonisation (ICH) Safety Working Group
Consensus Regarding New Drug Applications and that its approach is
considered to be in general agreement with the ICH position on acute
toxicity testing. FDA CDER and FDA CBER (Center for Biologics Eva-
luation and Research) note that the 2010 guidance is a revision of the
ICH M3 guidance originally published in 1997 and that the revisions
J. Manuppello, et al.
Fig. 1. The number of single dose toxicity studies reported by each route of administration and by routes intended or unintended for human administration, as well as
by species. “Other routes” combines studies conducted by topical, intramuscular, and intrathecal routes of administration.
further harmonize recommendations in a number of areas. As the re-
commendations cited below are identical in both the FDA (FDA CDER
and FDA CBER, 2010) and ICH (ICH, 2009) versions of this guidance,
we will refer to the latter for clarity.
In its 1996 guidance, FDA CDER states that the information ob-
tained from acute toxicity studies is useful in choosing doses for re-
peated dose studies and for providing preliminary identification of
target organs of toxicity. 34 single dose acute toxicity studies (14.9%)
were described as “dose range finding” or “dose escalation.” Target
organs of toxicity were identified in 8 studies (3.5%; Supplementary
File, “Summary” sheet). While FDA does not recommend specific test
guidelines for evaluating acute toxicity, it does recommend that the test
substance be administered to animals so as to identify doses causing no
adverse effects and doses causing major (life-threatening) toxicity.
NOAELs or LOELs were reported for 38 single dose acute toxicity stu-
dies (16.7%), while MLDs or MNLs were reported for 54 studies (23.7%;
Supplementary File, “Summary” sheet). In its 2009 guidance, ICH states
that lethality should not be an intended endpoint in studies assessing
acute toxicity (ICH, 2009). Substance-related lethality was reported in
63 studies (27.6%; Supplementary File, “Summary” sheet).
In its 1996 guidance, FDA CDER recommends that acute toxicity
studies in animals ordinarily be conducted using two routes of drug
administration, the route intended for human administration and in-
travenous, if feasible, as well as in at least two mammalian species,
including a nonrodent species, when reasonable (FDA CDER, 1996).
However, in its 2009 guidance, ICH recommends that acute toxicity
information be obtained from appropriately conducted dose-escalation
studies or short-duration dose-ranging studies, and that when this in-
formation is available from any study, separate single-dose studies are
not recommended. In addition, ICH notes that while historically, acute
toxicity information has been obtained from studies in two mammalian
species, using both the clinical and a parenteral route of administration,
it now recommends that studies providing this information be limited
to the general toxicity test species and to the route intended for human
administration only (ICH, 2009).
Of the 64 reviews in which single dose acute toxicity studies were
reported, 56 (87.5%) included studies conducted by the route intended
for human administration,1 and 27 (42.2%) included studies by a dif-
ferent route. The intended route was the only route reported to have
been used in 37 (57.8%) of these reviews. Of the 47 reviews in which
3
Regulatory Toxicology and Pharmacology 114 (2020) 104666
studies conducted by the intended route were reported, and this route
was not intravenous, additional studies by the intravenous route were
reported in 14 reviews (29.8%). Overall, studies conducted by only one
route were reported in 45 reviews (70.3%), while studies conducted by
more than one route were reported in 19 reviews (29.7%). In addition,
studies conducted in only one species were reported in 15 reviews
(23.4%), while studies conducted in more than one species were re-
ported in 49 reviews (76.6%; Supplementary File, “Summary” sheet).
With regard to animal use, in its 1996 guidance, FDA CDER notes
that studies should be designed so that the maximum amount of in-
formation is obtained from the smallest number of animals (FDA CDER,
1996). In the 141 studies for which animal numbers were reported,
4279 animals were used, an average of 30 animals per study across
species (Supplementary File, “Summary” sheet). Table 1 shows the total
and average (per study) number of animals used, by species, as well as
the estimated total number of animals used when these average num-
bers are applied to studies for which the number of animals used was
not reported. Extrapolation raises the estimated total to 7034 animals.
3.2. Local tolerance
In the 125 reviews examined, we identified 62 local tolerance stu-
dies, 0.5 studies per review, on average. In 87 reviews, we identified no
such studies; in 23, we identified one study; and in 15, we identified
two through five studies. 12 of these studies were listed as submitted by
sponsors but not reviewed by FDA (Supplementary File, “Summary”
sheet).
Eye irritation was evaluated in nine of these studies (14.5%), skin
irritation in 18 (29.0%), and vascular irritation in 35 (56.5%). In ad-
dition, 21 studies (33.9%) were conducted by the route intended for
1
Of the remaining eight reviews in which toxicity was reported to be eval-
uated only by a route other than that intended for human administration, the
intended route was ophthalmic in three, and toxicity was evaluated by the
intravenous route in each. In another three of these reviews, the intended route
was oral, and toxicity was evaluated by the intravenous route in two and by the
topical route in one. In one of these reviews, the intended route was vaginal,
and toxicity was evaluated by the subcutaneous route. In the last of these re-
views, the intended route was topical, and toxicity was evaluated by the in-
travenous route.
J. Manuppello, et al. Regulatory Toxicology and Pharmacology 114 (2020) 104666

Table 1 epithelium model EpiOcular (OECD, 2019a) in one study, and with
Reported animal use in single dose acute toxicity studies. human corneal epithelial cells in one study. Skin irritation was eval-
Studies Animal Animal Studies Not Animal Nos. uated with reconstructed human epidermis models in seven studies
Reporting Nos. Total, Nos. Per Reporting Total, with EpiDerm™ and six with EpiSkin™ (OECD, 2014). Mucous mem-
Animal Nos. Reported Study, Animal Nos. Extrapolated brane irritation was evaluated in one hen's egg chorioallantoic mem-
Average brane test (Supplementary File, “Summary” sheet).
Mice 36 1348 37 20 2097
Rats 65 2418 37 47 4166 3.3. Skin sensitization
Dogs 24 335 14 15 544
Monkeys 14 136 10 5 185 In the 125 reviews examined, we identified 32 skin sensitization
Rabbits 1 10 10 0 10
studies, 0.3 studies per review, on average. In 101 reviews, we identi-
Cats 1 32 32 0 32
fied no such studies; in 18, we identified one study; and in six, we
identified two through four. Eight of these studies were listed as sub-
human administration, while 41 studies (66.1%) were conducted by mitted by sponsors but not reviewed by FDA. Skin sensitization was
other routes (Supplementary File, “Summary” sheet).2 evaluated in 28 murine LLNAs, and four GPMTs (Supplementary File,
Fig. 2 shows the number of eye, skin, and vascular irritation studies “Summary” sheet).
by species as well as by routes intended or unintended for human ad- We found no general guidance for the evaluation of skin sensitiza-
ministration. Rabbits were used more frequently than any other species tion; however, in Immunotoxicology Evaluation of Investigational New
for all study types. In all of the local tolerance studies in which species Drugs, FDA CDER recommends that when a drug is intended for topical
other than rabbits were used, vascular irritation was evaluated. In ad- administration, its sensitizing potential be evaluated using an appro-
dition, more studies were conducted by routes other than that intended priate assay (FDA CDER, 2002). Only two of the 24 reviews (8.3%) in
for human administration in rabbits and rats; in all other species, stu- which the results of skin sensitization studies were reported are for
dies were conducted by the intended route only. drugs intended for topical administration (Supplementary File, “Sum-
For the evaluation of local tolerance, we accessed one relevant mary” sheet). FDA CDER identifies the most common methods for
general guidance document, M3(R2) Nonclinical Safety Studies for the evaluating the skin sensitizing potential of drugs to be the Buehler assay
Conduct of Human Clinical Trials and Marketing Authorization for and the GPMT, both of which it considers to be reliable and to have a
Pharmaceuticals, the same as that cited above for single dose acute high correlation with known human skin sensitizers. We found no re-
toxicity (FDA CDER and FDA CBER, 2010; ICH, 2009). ICH notes that it ports of Buehler assays in the reviews examined. FDA CDER also notes
is preferable to evaluate local tolerance by the route intended for that results obtained with the murine LLNA correlate well with tradi-
human administration as part of general toxicity studies, and stand- tional guinea pig tests, the results are quantitative rather than sub-
alone studies are generally not recommended (ICH, 2009). All of the jective, and CDER accepts this method for evaluating the skin sensi-
local tolerance studies identified were stand-alone studies.3 In addition, tizing potential of drugs intended for topical use.
ICH recommends that, to support limited human administration by In the six skin sensitization studies for which animal numbers were
nontherapeutic routes (for example, a single intravenous dose to assist reported, 215 animals were used, an average of 36 animals per study.
in the determination of absolute bioavailability of an oral drug), a local Extrapolation to studies for which the number of animals used was not
tolerance study in a single species is considered appropriate (ICH, reported raises the estimated total to 1147 animals (Supplementary
2009). File, “Summary” sheet).
Of the 38 reviews in which local tolerance studies were reported, 13
(34.2%) included studies in which irritation was evaluated by the route 4. Discussion
intended for human administration, and 29 (76.3%) included studies by
a different route. The intended route was the only route reported to We identified gaps in the information reported by FDA in many of
have been used in nine of these reviews (23.6%). Overall, studies its reviews of approved NDAs which likely resulted in underestimates of
conducted by only one route were reported in 30 reviews (78.9%), the number of single dose acute toxicity, local tolerance, and sensiti-
while studies conducted by more than one route were reported in eight zation studies submitted by sponsors as well as in the number of ani-
reviews (21.1%). In addition, studies conducted in only one species of mals used in these studies. FDA listed studies that were submitted but
animal were reported in 34 (89.5%) reviews, while studies conducted in not reviewed in only 30% of its reviews; if such studies were not listed,
more than one species were reported in four reviews (10.5%; we copied any explanatory text to a comment in the Microsoft Excel
Supplementary File, “Summary” sheet). workbook (Supplementary File, “List” sheet). In many cases, this text is
In the 22 local tolerance studies for which animal numbers were simply “Studies Not Reviewed: None” or similar. While it is possible
reported, 304 animals were used, an average of 14 animals per study. that in some cases, this indicates that all submitted studies were re-
Extrapolation to studies for which the number of animals used was not viewed, there are reasons to doubt this interpretation. Instead, it may
reported raises the estimated total to 817 animals (Supplementary File, indicate only that all studies judged to be “pivotal” were reviewed.
“Summary” sheet). For example, among the explanations for why studies were not re-
Local tolerance was evaluated by in vitro or ex vivo methods in 23 viewed, we find:
studies for eye irritation, 13 studies for skin irritation, and one study for
mucous membrane irritation. Eye irritation was evaluated with the • “Studies considered irrelevant for the nonclinical safety assessment
bovine corneal opacity and permeability (BCOP) test method (OECD, were not reviewed; "
2017) in 21 studies, with the reconstructed human cornea-like • “Studies Briefly/Not Reviewed: Selected ADME studies; studies by
IV or IP route of administration; several non-pivotal studies; "
• “Preliminary dose-range finding studies and non-pivotal studies for
2 deciding nonclinical safety … were not reviewed in detail; "
• “Dose range finding studies and non-pivotal studies were not re-
If the route intended for human administration was intravenous, sub-
cutaneous, intramuscular, or intraperitoneal, any vascular irritation study was
considered to be by the intended route. viewed; "
3
We did not note when local tolerance was evaluated as part of general • “Dose-ranging and non-GLP studies were examined, but not for-
toxicity studies, because this would have required examining all general toxi- mally reviewed; "
city studies reviewed, which is outside the scope of this article. • “Dose range-finding studies have not been reviewed."
4
J. Manuppello, et al.
Fig. 2. The number of local tolerance studies by type of irritation evaluated and by routes intended or unintended for human administration, as well as by species.
Fig. 3. Pharmacology/Toxicology Reviews summarized as a section within Multi-Discipline Reviews, or available as a separate document, in each month examined
beginning December 2016.
In addition, among the explanations for why reviews in which
submitted but not reviewed studies were listed, we find:
• “The following studies were not reviewed as they are non-pivotal,
less relevant or redundant at the current stage; "
• “The studies listed in the following table … were not reviewed, as
they were not pivotal to the nonclinical assessment …; "
• “The studies listed below were … not reviewed, as they were not
pivotal to the nonclinical assessment.…" (Supplementary File, “List”
sheet).
From these explanations, it seems clear that dose range finding
studies, in particular, were frequently submitted by sponsors but not
reviewed by FDA. These studies are relevant to this analysis, because
FDA CDER includes choosing doses for repeated dose studies among the
useful information that can be obtained from acute toxicity studies, and
ICH recommends that acute toxicity information be obtained from dose-
escalation studies or dose-ranging studies.
Studies that were listed as submitted by sponsors but not reviewed
by FDA included 22 single dose studies, 12 local tolerance studies, and
eight sensitization studies. If similar numbers of studies were submitted
5
Regulatory Toxicology and Pharmacology 114 (2020) 104666
but not reviewed in those reviews in which such studies were not listed,
we estimate the number of submitted studies missed in this analysis to
be 50 single dose studies, 27 local tolerance studies, and 18 skin sen-
sitization studies.
A total of 4798 rats, mice, dogs, monkeys, rabbits, pigs, guinea pigs,
minipigs, and cats were reported to have been used in the studies ex-
amined. However, the number of animals used was reported in only
62% of single dose acute toxicity studies, 35% of local tolerance studies,
and 19% of skin sensitization studies. If, on average, the number of
animals used for each study type was similar in studies for which the
number of animals used was not reported, we estimate an additional
2755 animals were used for single dose studies, 513 animals for local
tolerance studies, and 932 animals for skin sensitization studies, raising
the estimated total number of animals used in the reviews examined to
8998.
In order to measure any reduction in the number of animals used in
the testing of new drugs, the number of animals used in that testing
must be accurately determined. Unfortunately, FDA's reviews of ap-
proved NDAs became less helpful in this regard during the four years
examined. As noted above in Section 2.0, Pharmacology/Toxicology
Reviews summarized as a section within Multi-Discipline Reviews did
J. Manuppello, et al.
not include a “Studies Submitted” section, and studies not reviewed
were not listed in these reviews. The first Multi-Discipline Review we
identified was for an NDA approved in December 2016, and the
availability of this type of review, rather than a Pharmacology/Tox-
icology Review as a separate document, became more frequent over
time (Fig. 3).
Finally, only reviews of approved NDAs are publicly available.
While for the time period examined, we found 125 such reviews for new
molecular entities, according to FDA-TRACK, FDA's agency-wide per-
formance management system, FDA received 1970 original investiga-
tional new drug (IND) applications during the same time period, nearly
16 times the number of available NDA reviews (FDA, 2020d). There-
fore, it seems certain that the single dose acute toxicity, local tolerance,
and skin sensitization studies examined represent only a small part of
all such studies conducted to support new drugs during this time period
and that the number of animals used in the studies examined represents
a correspondingly small part of the total number of animals used. Based
on this analysis, the total number of animals used in studies to support
new drugs during this time period can be roughly estimated to be
140,000.
These observations suggest a need for FDA to adopt a standard
format for its reviews of approved NDAs that includes a list of all studies
submitted as well as the species and number of animals used in those
studies, preferably both for studies that were and were not reviewed.
Ideally, such minimal animal use information would be made public not
only for reviews of approved NDAs but also for reviews of INDs.
We found little association between the recommendations made by
FDA CDER in its guidance to industry and the information reported in
reviewed studies. In its 1996 guidance, FDA CDER states that the in-
formation obtained from acute toxicity studies is useful in choosing
doses for repeated dose studies and in providing preliminary identifi-
cation of target organs of toxicity. Further, FDA CDER recommends that
the test substance be administered to animals so as to identify doses
causing no adverse effects and doses causing major (life-threatening)
toxicity. However, this information is reported for relatively few of the
studies examined. Only 15% of these studies were described as “dose
range finding” or “dose escalation” (as noted above, it seems certain
that such studies were underreported); target organs of toxicity were
identified in 4%; NOAELs or LOELs were reported in 17%; and MLDs or
MNLs were reported in 24%. This raises the question of why the ma-
jority of these studies were conducted, if not to provide the information
that FDA CDER identifies as useful. Indeed, the more recent ICH gui-
dance states that when acute toxicity information is available from any
study, separate single-dose studies are not recommended. This guidance
also states that lethality should not be an intended endpoint in studies
evaluating acute toxicity; however, test substance-related lethality was
reported in 28% of the studies examined (Supplementary File,
“Summary” sheet). These observations are consistent with those of
Robinson et al. (2008) who found that acute toxicity data was generally
not used to terminate drugs from development, to support dose selec-
tion for repeat dose studies in animals, or to set doses for the first
clinical trials. These authors concluded that acute toxicity studies are
not needed prior to first clinical trials in humans (Robinson et al.,
2008), and ICH cites this article in its 2009 guidance. In addition,
Chapman et al. (2010) found that acute toxicity studies conducted later
in drug development are not used for managing overdose of pharma-
ceuticals and are of little value in treating human poisoning from
chemicals.
Oral and intravenous routes of administration were used more fre-
quently than other routes in the single dose acute studies examined,
accounting for 49% and 32% of studies, respectively. In its 1996 gui-
dance, FDA CDER recommends that acute toxicity studies in animals
ordinarily be conducted using two routes of administration, the route
intended for human administration and intravenous, if feasible, while
ICH recommends that such studies be limited to the intended route
only. Of the reviews in which such studies were reported, 30% included
6
Regulatory Toxicology and Pharmacology 114 (2020) 104666
studies by more than one route. In addition, of the reviews for which
the intended route was not intravenous, 30% included studies by the
intravenous route in addition to the intended route. FDA also re-
commends that acute toxicity studies be conducted in at least two
mammalian species. Of the reviews in which such studies were re-
ported, 77% included studies in more than one species. These ob-
servations suggest that many sponsors are following recommendations
made by FDA CDER in its 1996 guidance rather than those made by ICH
in its 2009 guidance.
FDA CDER also notes that studies should be designed so that the
maximum amount of information is obtained from the smallest number
of animals. FDA cites its LD50 test policy, in which it states that the
“classical” LD50 test requires from 60 to more than 120 animals per test
substance to attain a statistically precise median number (FDA, 1988).
FDA recognized that this test was unnecessarily precise and ceased to
require it. We calculate the average number of both mice and rats used
in those studies for which animal numbers were reported to be 37.
While this is lower than that required by the classical LD50 test, as
described by FDA, it is still a relatively large number compared to that
used to meet other regulatory requirements. For example, in its Health
Effects Test Guidelines; OPPTS 870.1100; Acute Oral Toxicity, the U.S.
Environmental Protection Agency (EPA) recommends use of the Up-
and-Down Procedure for the evaluation of acute systemic toxicity, with
which for most applications, testing will be completed with only four
animals after the initial reversal in animal outcome, or between six and
fifteen animals in total (EPA, 2002).4
We must emphasize that the two general guidance documents ac-
cessible for evaluating single dose acute toxicity include conflicting
recommendations, and many sponsors appear to be following re-
commendations made in the older guidance. Clearly, the availability of
two conflicting guidance documents can only increase sponsors' un-
certainty over FDA's expectations. We recommend that FDA CDER re-
vise its 1996 guidance to be consistent with the more recent ICH gui-
dance, in particular with regard to reducing the overall number of
stand-alone studies by encouraging the use of acute toxicity information
obtained from other studies, using no more than one route of admin-
istration and one species of animal if stand-alone studies are conducted,
and discouraging the inclusion of lethality as an intended endpoint.
Alternatively, FDA CDER could simply retire its 1996 guidance in favor
of the more recent ICH guidance.
FDA could further reduce the number of animals used for the eva-
luation of acute toxicity by recommending the use of NAMs to meet its
regulatory needs. For example, Prieto et al. (2013) found that the in
vitro cytotoxicity test based on neutral red uptake by BALB/c 3T3 cells
has high sensitivity, although the model does not perform well with
chemicals needing biotransformation or acting via specific mechanisms
including neurotoxicity or cardiotoxicity. Other organ-specific models
may be used to refine this approach, including those based on HPCT-
1E3 hepatocytoma cells (Kneuer et al., 2007), human BJ fibroblasts
(Mannerström et al., 2017), and a variety of neuronal cells (Forsby
et al., 2009). Organ-specific toxicity is better investigated with human-
relevant in vitro or in silico models or a combination of both; cardio-
toxicity is an area that has seen particularly fruitful work (e.g., Sirenko
et al., 2017; Passini et al., 2017; Kurokawa et al., 2018). In addition,
first-in-human microdosing studies can be used to determine kinetic
properties (Burt et al., 2018), and microphysiological systems show
great promise in determining both toxicity and kinetics (e.g., Jang
et al., 2019). Finally, ICCVAM recently organized a global collaboration
4
The test consists of a single ordered dose progression in which animals are
dosed, one at a time, at a minimum of 48-h intervals. The first animal receives a
dose a step below the level of the best estimate of the median lethal dose. If the
animal survives, the dose for the next animal is increased by 3.2 times the
original dose; if it dies, the dose for the next animal is decreased by a similar
dose progression.
J. Manuppello, et al.
to build predictive in silico models for acute oral systemic toxicity based
on a large dataset of rodent studies and targeted towards regulatory
needs identified across federal agencies. When the models were com-
bined, consensus predictions demonstrated excellent performance when
compared to the animal data (Kleinstreuer et al., 2018a). The Colla-
borative Acute Toxicity Modeling Suite is available both as standalone
software and through EPA's CompTox Dashboard (EPA, 2020).
The only relevant general guidance accessible for the evaluation of
local tolerance was M3(R2) Nonclinical Safety Studies for the Conduct of
Human Clinical Trials and Marketing Authorization for Pharmaceuticals
(ICH, 2009). ICH notes that it is preferable to evaluate local tolerance
by the route intended for human administration as part of general
toxicity studies, and stand-alone studies are generally not re-
commended. We found that the intended route was the only route used
in just 24% of reviews in which local tolerance studies were reported.
While we did not note when local tolerance was evaluated as part of
general toxicity studies, we identified a total of 62 stand-alone local
tolerance studies, raising concern that sponsors frequently overlook this
recommendation. ICH also recommends that, to support limited human
administration by nontherapeutic routes, a local tolerance study in a
single species is considered appropriate. While it is possible that this
exception accounts for the 41 studies conducted by routes other than
those intended for human administration, it seems unlikely that this is
the case. In addition, more than one species was reported to be used for
the evaluation of local tolerance in four reviews (Supplementary File,
“Summary” sheet).
In Nonclinical Safety Evaluation of Reformulated Drug Products and
Products Intended for Administration by an Alternate Route, FDA CDER
recommends that for reformulated topical drug products that contain
substances that have not been evaluated for eye irritation/corrosion
potential, that potential should be evaluated by appropriate in vitro or
ex vivo methods (FDA CDER, 2015). Specifically, the in vivo rabbit
ocular irritation test method is no longer recommended. While this
recommendation is limited to a specific class of drug products, we
found that the BCOP test, an ex vivo method, was used more than twice
as frequently as the rabbit test in the reviews examined. In addition,
almost as many skin irritation tests used reconstructed human epi-
dermis as used rabbits (Fig. 4).
FDA addresses the evaluation of skin sensitization only in its gui-
dance, Immunotoxicology Evaluation of Investigational New Drugs, in
which it recommends that when a drug is intended for topical admin-
istration, its sensitizing potential be evaluated using an appropriate
assay, such as the Buehler assay, the GPMT, or the murine LLNA (FDA
CDER, 2002). While the GPMT and murine LLNA were the only assays
reported in 24 reviews, only two of these reviews were for drugs in-
tended for topical administration (Supplementary File, “Summary”
sheet).
These observations suggest a need for FDA to communicate clearly
Fig. 4. Local tolerance studies in vivo and in vitro or ex vivo.
7
Regulatory Toxicology and Pharmacology 114 (2020) 104666
to sponsors that the evaluation of local tolerance by routes not intended
for human administration and the evaluation of skin sensitization for
drugs not intended for topical administration are not recommended. In
addition, FDA should consider whether its recommendation that the eye
irritation/corrosion potential of reformulated topical drug products be
evaluated by appropriate in vitro or ex vivo methods can be extended to
other classes of drugs.
While the significant acceptance of in vitro and ex vivo methods for
the evaluation of eye and skin irritation/corrosion is encouraging, it is
disappointing that similar progress is not evident in the evaluation of
skin sensitization, especially considering the increasing acceptance of
such methods in other sectors and the length of time for which NAMs
have been available. Kleinstreuer et al. (2018a,b) used murine LLNA
and human skin sensitization data to evaluate the performance of six
defined approaches, comprising eight non-animal testing strategies, for
both hazard and potency characterization. Multiple non-animal testing
strategies incorporating in vitro, in chemico, and in silico inputs de-
monstrated equivalent or superior performance to the LLNA when
compared to both animal and human data for skin sensitization
(Kleinstreuer et al., 2018b). In April 2018, EPA released a draft interim
science policy, Use of Alternative Approaches for Skin Sensitization as a
Replacement for Laboratory Animal Testing (EPA, 2018). This draft policy
builds upon substantial work done by the Organization for Economic
Cooperation and Development (OECD) to establish the adverse outcome
pathway (AOP) for skin sensitization and to develop guidelines for
several methods that assess the ability of chemicals to activate the first
three key events in this AOP (OECD, 2018a; OECD, 2018b; OECD,
2019b). Under this interim policy, EPA will accept submissions in
which two defined approaches combining these methods are used. We
recommend that FDA develop general guidance for the evaluation of
skin sensitization and consider whether a similar approach can be ap-
plied to the testing of new drugs.
As noted in sections 3.1-3.3, multiple single dose studies were re-
ported in 49 reviews, multiple local tolerance studies in 15, and mul-
tiple skin sensitization studies in nine. In many cases, these studies
differed in the test article, route of administration, species of animal, or
experimental protocol used (Supplementary File, “Multiples” sheet). In
several cases, reporting was inadequate to determine how studies dif-
fered. By beginning with problem formulation and proceeding through
a decision-making framework that accounts for all available informa-
tion and considers how NAMs can address information needs at each
step, Integrated Approaches to Testing and Assessment (IATA) can help
sponsors avoid the conduct of such similar studies. In particular, in-
formation derived from read-across, quantitative structure–activity re-
lationship models, and in vitro or ex vivo methods can supplement
available information, minimizing the conduct of in vivo studies.
In their article following a 2017 workshop, Towards Global
Elimination of the Acute Toxicity Six-Pack, Prior et al. (2019) state that
these tests are primarily conducted for the classification and labelling of
industrial chemicals and agrochemicals. They note that they are largely
banned for testing of cosmetics and rarely required for testing of
pharmaceuticals (Prior et al., 2019). Likewise, in their report on the
status of acute systemic toxicity testing requirements by U.S. regulatory
agencies, Strickland et al. (2018) omitted FDA CDER, noting that it does
not request nonclinical acute systemic toxicity data (Strickland et al.,
2018). These authors state that FDA CDER's 1996 guidance document,
Single Dose Acute Toxicity Testing for Pharmaceuticals, is outdated and no
longer used by FDA, noting that any acute toxicity information needs
are satisfied by repeated-dose toxicity studies, and single dose acute
toxicity studies may not even be reviewed by CDER. While our results
confirm that single dose acute toxicity studies are sometimes not re-
viewed by CDER, even if acute systemic toxicity data are not requested,
sponsors nevertheless frequently submit such data. Regarding whether
FDA CDER's 1996 guidance is no longer used, we note that it is still
accessible, and we found no indication that it is not to be used. Further,
in its 2005 guidance, Nonclinical Studies for the Safety Evaluation of
J. Manuppello, et al.
Pharmaceutical Excipients, FDA CDER and FDA CBER cite this 1996
guidance to support their recommendation that acute toxicology studies
be performed in both a rodent species and a nonrodent species (FDA
CDER and FDA CBER, 2005). Finally, many reviews included a “Phar-
macology/Toxicology Filing Checklist” (e.g., FDA, 2015) in which one
of the questions asked is: “Are all required … and requested IND studies
… completed and submitted (carcinogenicity, mutagenicity, ter-
atogenicity, effects on fertility, juvenile studies, acute and repeat dose
adult animal studies, animal ADME studies, safety pharmacology, etc.)?”
[Emphasis added] This would seem to indicate that acute systemic
toxicity studies are required or requested in at least some cases.
5. Conclusions
Using publicly-available information, we tallied the single dose
acute toxicity, local tolerance, and skin sensitization studies that were
submitted by sponsors in approved, original NDAs, as reported by FDA,
from 2015 through 2018. We found that sponsors submit such studies
frequently, challenging the perception that they are generally not re-
quired or conducted for pharmaceuticals. We found little association
between the information reported by FDA in its reviews of these studies
and the information identified as useful by FDA in its guidance to in-
dustry. Sponsors frequently conducted these studies by routes other
than those intended for human administration and in more than one
species of animal. For single dose acute toxicity studies, we accessed
two conflicting guidance documents, and our results suggest that many
sponsors are still following the older guidance. While we found sig-
nificant use of in vitro and ex vivo methods for the evaluation of skin and
eye irritation/corrosion, the use of NAMs for the evaluation of skin
sensitization was not reported, despite their increasing acceptance by
regulators in other sectors.
In order for FDA to reduce the use of animals in the assessment of
new drugs, our analysis suggests a need for FDA to update its guidance
for industry such that its recommendations both reduce the number of
studies conducted by sponsors and identify appropriate NAMs that can
replace animal studies. In particular, FDA CDER should revise its 1996
single dose acute toxicity guidance to be consistent with the more re-
cent ICH guidance or retire this guidance. In addition, FDA should
emphasize that the evaluation of local tolerance and skin sensitization
by routes not intended for human administration is not recommended.
In order for FDA to measure progress toward this goal, it must improve
and standardize its reporting of animal use, ideally extending that re-
porting beyond approved NDAs to include INDs. So doing would allow
more new drugs to be evaluated across a broader range of potential
effects in less time while using fewer animals and reducing costs.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
Funding for the work reported in this article was provided by the
Caroll Petrie Foundation.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.yrtph.2020.104666.
References
Burt, T., Vuong, L.T., Baker, E., Young, G.C., McCartt, A.D., Bergstrom, M., Sugiyama, Y.,
Combes, R., 2018. Phase 0, including microdosing approaches: applying the Three Rs
8
Regulatory Toxicology and Pharmacology 114 (2020) 104666
and increasing the efficiency of human drug development. Altern Lab Anim 46,
335–346. https://doi.org/10.1177/026119291804600603.
Chapman, K., Creton, S., Kupferschmidt, H., Bond, G.R., Wilks, M.F., Robinson, S., 2010.
The value of acute toxicity studies to support the clinical management of overdose
and poisoning: a cross-discipline consensus. Regul. Toxicol. Pharmacol. 58, 354–359.
https://doi.org/10.1016/j.yrtph.2010.07.003.
EPA, 2002. Health effects test guidelines OPPTS 870.1100 acute oral toxicity. https://
www.regulations.gov/contentStreamer?documentId=EPA-HQ-OPPT-2009-0156-
0003&contentType=pdf, Accessed date: 15 January 2020.
EPA, 2018. Interim science policy: use of alternative approaches for skin sensitization as a
replacement for laboratory animal testing. https://www.regulations.gov/
contentStreamer?documentId=EPA-HQ-OPP-2016-0093-0090&contentType=pdf.
EPA, 2020. CompTox chemicals dashboard (predictions). https://comptox.epa.gov/
dashboard/predictions/index, Accessed date: 27 January 2020.
FDA, 1988. LD50 test policy. Fed. Regist. 53, 39650–39651.
FDA, 2015. Orkambi (lumacaftor/ivacaftor) oral tablet. https://www.accessdata.fda.gov/
drugsatfda_docs/nda/2015/0206038Orig1s000PharmR.pdf, Accessed date: 6
February 2020.
FDA, 2017. FDA's predictive toxicology Roadmap. https://www.fda.gov/media/109634/
download, Accessed date: 15 January 2020.
FDA, 2020a. Drugs@FDA: FDA-approved drugs. https://www.accessdata.fda.gov/scripts/
cder/daf/, Accessed date: 15 January 2020.
FDA, 2020b. Search for FDA guidance documents. https://www.fda.gov/regulatory-
information/search-fda-guidance-documents, Accessed date: 15 January 2020.
FDA, 2020c. Research list of guidance documents. https://www.fda.gov/drugs/
guidances-drugs/research-list-guidance-documents, Accessed date: 15 January 2020.
FDA, 2020d. FDA-TRACK: agency-wide program performance. https://www.accessdata.
fda.gov/scripts/fdatrack/view/track.cfm?program=cber&status=public&id=
CBER-All-IND-and-IDEs-recieved-and-actions&fy=All, Accessed date: 15 January
2020.
FDA CDER, 1996. Guidance for industry. Single dose acute toxicity testing for pharma-
ceuticals. https://www.fda.gov/media/72288/download, Accessed date: 14 January
2020.
FDA CDER, 2002. Guidance for industry. Immunotoxicology evaluation of investigational
new drugs. https://www.fda.gov/media/72228/download, Accessed date: 15
January 2020.
FDA CDER, 2015. Nonclinical safety evaluation of reformulated drug products and pro-
ducts intended for administration by an alternate route. Guidance for industry and
review staff. https://www.fda.gov/files/drugs/published/Nonclinical-Safety-
Evaluation-of-Reformulated-Drug-Products-and-Products-Intended-for-
Administration-by-an-Alternate-Route.pdf, Accessed date: 15 January 2020.
FDA CDER, FDA CBER, 2005. Nonclinical studies for the safety evaluation of pharma-
ceutical Excipients. https://www.fda.gov/media/72260/download, Accessed date:
15 January 2020.
FDA CDER, CBER, 2010. M3(R2) nonclinical safety studies for the conduct of human
clinical trials and marketing authorization for pharmaceuticals. https://www.fda.
gov/media/71542/download, Accessed date: 15 January 2020.
Forsby, A., Bal-Price, A.K., Camins, A., Coecke, S., Fabre, N., Gustafsson, H., Honegger, P.,
Kinsner-Ovaskainen, A., Pallas, M., Rimbau, V., Rodríguez-Farré, E., Suñol, C.,
Vericat, J.A., Zurich, M.G., 2009. Neuronal in vitro models for the estimation of acute
systemic toxicity. Toxicol. Vitro 23, 1564–1569. https://doi.org/10.1016/j.tiv.2009.
07.017.
ICCVAM, 2018. A strategic Roadmap for establishing new approaches to evaluate the
safety of chemicals and medical products in the United States. https://ntp.niehs.nih.
gov/iccvam/docs/roadmap/iccvam_strategicroadmap_january2018_document_508.
pdf, Accessed date: 15 January 2020.
ICH, 2009. Guidance on nonclinical safety studies for the conduct of human clinical trials
and marketing authorization for pharmaceuticals M3(R2). https://database.ich.org/
sites/default/files/M3_R2__Guideline.pdf, Accessed date: 15 January 2020.
Jang, K.J., Otieno, M.A., Ronxhi, J., Lim, H.K., Ewart, L., Kodella, K.R., Petropolis, D.B.,
Kulkarni, G., Rubins, J.E., Conegliano, D., Nawroth, J., Simic, D., Lam, W., Singer, M.,
Barale, E., Singh, B., Sonee, M., Streeter, A.J., Manthey, C., Jones, B., Srivastava, A.,
Andersson, L.C., Williams, D., Park, H., Barrile, R., Sliz, J., Herland, A., Haney, S.,
Karalis, K., Ingber, D.E., Hamilton, G.A., 2019. Reproducing human and cross-species
drug toxicities using a Liver-Chip. Sci. Transl. Med. 11https://doi.org/10.1126/
scitranslmed.aax5516. eaax5516.
Kleinstreuer, N.C., Hoffmann, S., Alépée, N., Allen, D., Ashikaga, T., Casey, W., Clouet, E.,
Cluzel, M., Desprez, B., Gellatly, N., Göbel, C., Kern, P.S., Klaric, M., Kühnl, J.,
Martinozzi-Teissier, S., Mewes, K., Miyazawa, M., Strickland, J., van Vliet, E., Zang,
Q., Petersohn, D., 2018. Non-animal methods to predict skin sensitization (II): an
assessment of defined approaches. Crit. Rev. Toxicol. 48, 359–374. https://doi.org/
10.1080/10408444.2018.1429386.
Kleinstreuer, NC, Karmaus, A, Mansouri, K, Allen, DG, Fitzpatrick, JM, Patlewicz, G.,
2018. Predictive models for acute oral systemic toxicity: a workshop to bridge the gap
from research to regulation. Comput. Toxicol. 8, 21–24. https://doi.org/10.1016/j.
comtox.2018.08.002.
Kneuer, C., Lakoma, C., Honscha, W., 2007. Prediction of acute toxicity in HPCT-1E3
hepatocytoma cells with liver-like transport activities. Altern Lab Anim 35, 411–420.
https://doi.org/10.1177/026119290703500410.
Kurokawa, Y.K., Shang, M.R., Yin, R.T., George, S.C., 2018. Modeling trastuzumab-re-
lated cardiotoxicity in vitro using human stem cell-derived cardiomyocytes. Toxicol.
Lett. 285, 74–80. https://doi.org/10.1016/j.toxlet.2018.01.001.
Mannerström, M., Toimela, T., Sarkanen, J.R., Heinonen, T., 2017. Human BJ fibroblasts
is an alternative to mouse BALB/c 3T3 cells in in vitro neutral red uptake assay. Basic
Clin. Pharmacol. Toxicol. 121 (Suppl. 3), 109–115. https://doi.org/10.1111/bcpt.
12790.
J. Manuppello, et al.
OECD, 2014. Test No. 431: in Vitro Skin Corrosion: Reconstructed Human Epidermis
(Rhe) Test Method. OECD Guidelines for the Testing of Chemicals, Section 4. OECD
Publishing, Paris. https://doi.org/10.1787/9789264224193-en.
OECD, 2017. Test No. 437: Bovine Corneal Opacity and Permeability Test Method for
Identifying I) Chemicals Inducing Serious Eye Damage and Ii) Chemicals Not
Requiring Classification for Eye Irritation or Serious Eye Damage. OECD Guidelines
for the Testing of Chemicals, Section 4. OECD Publishing, Paris. https://doi.org/10.
1787/9789264203846-en.
OECD, 2018a. Test No. 442D: in Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test
Method. OECD Guidelines for the Testing of Chemicals, Section 4. OECD Publishing,
Paris. https://doi.org/10.1787/9789264229822-en.
OECD, 2018b. Test No. 442E: in Vitro Skin Sensitisation: in Vitro Skin Sensitisation
Assays Addressing the Key Event on Activation of Dendritic Cells on the Adverse
Outcome Pathway for Skin Sensitisation. OECD Guidelines for the Testing of
Chemicals, Section 4. OECD Publishing, Paris. https://doi.org/10.1787/
9789264264359-en.
OECD, 2019a. Test No. 492: Reconstructed Human Cornea-like Epithelium (RhCE) Test
Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye
Irritation or Serious Eye Damage. OECD Guidelines for the Testing of Chemicals,
Section 4. OECD Publishing, Paris. https://doi.org/10.1787/9789264242548-en.
OECD, 2019b. Test No. 442C: in Chemico Skin Sensitisation: Assays Addressing the
Adverse Outcome Pathway Key Event on Covalent Binding to Proteins. OECD
Guidelines for the Testing of Chemicals, Section 4. OECD Publishing, Paris. https://
doi.org/10.1787/9789264229709-en.
Passini, E., Britton, O.J., Lu, H.R., Rohrbacher, J., Hermans, A.N., Gallacher, D.J., Greig,
R.J.H., Bueno-Orovio, A., Rodriguez, B., 2017. Human in silico drug trials demon-
strate higher accuracy than animal models in predicting clinical pro-arrhythmic
Regulatory Toxicology and Pharmacology 114 (2020) 104666
cardiotoxicity. Front. Physiol. 8, 668. https://doi.org/10.3389/fphys.2017.00668.
Prieto, P., Cole, T., Curren, R., Gibson, R.M., Liebsch, M., Raabe, H., Tuomainen, A.M.,
Whelan, M., Kinsner-Ovaskainen, A., 2013. Assessment of the predictive capacity of
the 3T3 Neutral Red Uptake cytotoxicity test method to identify substances not
classified for acute oral toxicity (LD50 > 2000 mg/kg): results of an ECVAM vali-
dation study. Regul. Toxicol. Pharmacol. 65, 344–365. https://doi.org/10.1016/j.
yrtph.2012.11.013.
Prior, H., Casey, W., Kimber, I., Whelan, M.4, Sewell, F., 2019. Reflections on the progress
towards non-animal methods for acute toxicity testing of chemicals. Regul. Toxicol.
Pharmacol. 102, 30–33. https://doi.org/10.1016/j.yrtph.2018.12.008.
Robinson, S., Delongeas, J.L., Donald, E., Dreher, D., Festag, M., Kervyn, S., Lampo, A.,
Nahas, K., Nogues, V., Ockert, D., Quinn, K., Old, S., Pickersgill, N., Somers, K., Stark,
C., Stei, P., Waterson, L., Chapman, K., 2008. A European pharmaceutical company
initiative challenging the regulatory requirement for acute toxicity studies in phar-
maceutical drug development. Regul. Toxicol. Pharmacol. 50, 345–352. https://doi.
org/10.1016/j.yrtph.2007.11.009.
Sirenko, O., Grimm, F.A., Ryan, K.R., Iwata, Y., Chiu, W.A., Parham, F., Wignall, J.A.,
Anson, B., Cromwell, E.F., Behl, M., Rusyn, I., Tice, R.R., 2017. In vitro cardiotoxicity
assessment of environmental chemicals using an organotypic human induced plur-
ipotent stem cell-derived model. Toxicol. Appl. Pharmacol. 322, 60–74. https://doi.
org/10.1016/j.taap.2017.02.020.
Strickland, J., Clippinger, A.J., Brown, J., Allen, D., Jacobs, A., Matheson, J., Lowit, A.,
Reinke, E.N., Johnson, M.S., Quinn Jr., M.J., Mattie, D., Fitzpatrick, S.C., Ahir, S.,
Kleinstreuer, N., Casey, W., 2018. Status of acute systemic toxicity testing require-
ments and data uses by U.S. regulatory agencies. Regul. Toxicol. Pharmacol. 94,
183–196. https://doi.org/10.1016/j.yrtph.2018.01.022.