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ATLA 32, 437459, 2004

437

Strategies to Replace In Vivo Acute Systemic Toxicity


Testing
The Report and Recommendations of ECVAM Workshop 501,2
Alessandra Gennari,3 Christine van den Berghe,4 Silvia Casati,3 Jos Castell,5 Cecilia
Clemedson,6 Sandra Coecke,3 Alessandra Colombo,7 Rodger Curren,8 Gianni Dal Negro,9 Alan
Goldberg,10 Catherine Gosmore,11 Thomas Hartung,3 Ingrid Langezaal,12 Iglika Lessigiarska,12
Wilfred Maas,13 Inge Mangelsdorf,14 Ralph Parchment,15 Pilar Prieto,3 Juan Riego Sintes,12
Michael Ryan,16 Gabriele Schmuck,17 Katherine Stitzel,18 William Stokes,19 Joan-Albert
Vericat20 and Laura Gribaldo3
3ECVAM,

Institute for Health & Consumer Protection, European Commission Joint Research Centre, Ispra,
Italy; 4LOral Research, Aulnay sous Bois, France; 5Departamento de Bioqumica Facultad Medicina/Centro
de Investigacin Hospital Universitario La Fe, Valencia, Spain; 6Expertrdet AB, Sundbyberg, Sweden;
7Direzione Salute, Sicurezza e Ambiente Sicurezza Prodotti Polimeri Europa, San Donato Milanese, Italy;
8Institute for In Vitro Sciences, Gaithersburg, MD, USA; 9DVM-Medicines Safety Evaluation,
GlaxoSmithKline Medicines Research Centre, Safety, Verona, Italy; 10Center for Alternatives to Animal
Testing, Johns Hopkins University, Baltimore, MD, USA; 11Health and Safety Executive, Bootle, Merseyside,
UK; 12European Chemicals Bureau, Institute for Health & Consumer Protection, European Commission Joint
Research Centre, Ispra, Italy; 13Physiological Sciences Department, TNO Chemistry, Zeist, The Netherlands;
14Fraunhofer, Department of Chemical Risk Assessment, Institute of Toxicology and Experimental Medicine,
Hannover, Germany; 15SciTech Development, Grosse Pointe, MI, USA; 16Department of Pharmacology,
Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin, Ireland;
17Bayer, Project Managment AH PH-PD Toxicology, Germany; 18West Chester, OH, USA; 19National
Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods, National
Institute of Environmental Health Sciences, Research Triangle Park, NC, USA; NeuroPharma, Madrid, Spain
Address for correspondence: L. Gribaldo, ECVAM, Institute for Health & Consumer Protection, European Commission
Joint Research Centre, 21020 Ispra (VA), Italy.
E-mail: laura.gribaldo@jrc.it

Preface
This is the report of the fiftieth of a series of workshops organised by the European Centre for the Validation of Alternative Methods (ECVAM). ECVAMs
main goal, as defined in 1993 by its Scientific
Advisory Committee, is to promote the scientific and
regulatory acceptance of alternative methods which
are of importance to the biosciences and which
reduce, refine or replace the use of laboratory animals. One of the first priorities set by ECVAM was
the implementation of procedures that would enable
it to become well informed about the state-of-the-art
of non-animal test development and validation, and
the potential for the possible incorporation of alternative tests into regulatory procedures.
It was decided that this priority would be best
achieved by the organisation of ECVAM workshops

on specific topics, at which small groups of invited


experts would review the current status of various
types of in vitro tests and their potential uses, and
make recommendations about the best ways forward (1).
This ECVAM workshop on strategies to replace
in vivo acute systemic toxicity testing was held at
ECVAM on 1518 September 2003, under the
chairmanship of Laura Gribaldo. The participants
represented academia, national organisations,
international regulatory bodies and industry. The
aim of the workshop was to review the state-of-theart of in vitro methods for estimating acute systemic
toxicity, and to develop research, development, and
validation strategies necessary for the replacement of
in vivo testing, with a focus on oral acute toxicity. The
report describes the strategy proposed by the participants for using in vitro methods, and provides rec-

Address for reprints: ECVAM, Institute for Health & Consumer Protection, European Commission Joint Research Centre,
21020 Ispra (VA), Italy.
1ECVAM The European Centre for the Validation of Alternative Methods. 2This document represents the agreed report
of the participants as individual scientists.

438

ommendations for the test method development


and validation activities necessary to support such
a strategy that might replace the use of animals for
the acute toxicity testing of chemicals.

Introduction
Acute systemic toxicity testing involves an assessment of the general toxic effects of a single dose of
a chemical or product, or, in some cases, the effects
of multiple doses given within 24 hours, and that
occur during a subsequent 21-day observation
period. The test material can be administered by
various routes (orally, by inhalation, intravenously,
or transdermally). Acute systemic tests include
lethal dose tests, such as the classical and modified
LD50 tests (2, 3), the Acute Toxic Class (ATC)
method (46), up-and-down methods (7, 8) and nonlethal tests (for example, the Fixed Dose Procedure
[FDP; 9, 10]).
A major use of acute systemic toxicity data is for
the classification and labelling of chemicals in relation to their manufacture, transport and use, but
the data are also used for defining modes of action,
setting dose levels for other tests, and providing
medical advice in cases of poisoning (based on the
toxic effects observed).
The use of lethal dose tests has been widely criticised, not least by toxicologists themselves, and
much effort has been put into the development of
alternative methods which could reduce or replace
the numbers of animals required (8, 11).
Many studies have shown very good correlations
between in vitro basal cytotoxicity data obtained
with undifferentiated cell lines and LD50 data (for
example, 1217). Good correlations have also been
shown between in vitro cytotoxicity data (IC50 values) and human lethal blood concentrations (as in
the Multicentre Evaluation of In Vitro Cytotoxicity
[MEIC] study [12]). However, acute systemic toxicity can be caused by a variety of mechanisms, many
of which would not be detected by a single cytotoxicity test, so more-sophisticated approaches are also
necessary. An objective reappraisal of what has
been achieved with regard to the development of in
vitro tests for cytotoxicity is required.
Numerous tests for basal cytotoxicity have been
developed, but insufficient attention has been paid
to how the data they provide can be applied, particularly as a means of making decisions or predictions.
In 1999, an ECVAM Task Force on Integrated
Testing Strategies was established, with the aim of
making proposals with regard to the design and
implementation of integrated testing strategies
(18). The first step in such an approach is usually to
determine the chemical functionality of a substance, on the basis of its structure and physicochemical properties (quantitative structureactivity

A. Gennari et al.

relationship [QSAR] techniques). Then, the biokinetic and dynamic behaviours of the compound are
assessed, and the various elements are integrated to
predict local or systemic toxicity.
An international workshop was organised in 2000
by the Interagency Coordinating Committee on the
Validation of Alternative Methods (ICCVAM), during which the current status of in vitro methods for
assessing acute toxicity was reviewed (19). From
this review, a process was devised to offer realistic
short-term and long-term goals for further refinement and replacement of the animal studies, in particular for acute oral toxicity (the same principles
could then be used for acute dermal and inhalation
toxicity). Although no standardised in vitro cytotoxicity assays, with optimised protocols and prediction models, have yet been formally validated, it
appears from the number of studies showing positive correlations between cytotoxicity results in
vitro and acute toxic effects in vivo, that the application of such in vitro methods does have potential.
The workshop concluded that the replacement of
animal tests by in vitro assays would require much
more time to implement. It further concluded that
the development and validation of in vitro methods
should concentrate on the prediction of human,
rather than rodent, acute toxicity.
Moreover, cytotoxicity assays would be likely only
to provide estimations of the inherent LD50 of a
test material, while other in vitro assays would be
needed to indicate how this potential toxicity would
be affected by the absorption, distribution, metabolism and excretion (ADME) of the compound.
Finally, in vitro testing strategies will need to integrate biokinetic factors, which affect the interactions between cells and test materials.

Current Regulatory Testing Requirements


for Acute Systemic Toxicity
LD50 values and the regulation of chemical
products
Historically, acute toxicity has been quantified from
a compounds potency in a lethality assay, using a
route of administration judged to be most relevant
to any human exposures that might occur when it is
handled or used (oral, dermal, inhalation). Less
often, a compounds lethality can be measured by
using intravenous (i.v.) administration to circumvent problems due to low absorption from the gut,
and/or extensive hepatic metabolism prior to
accessing the systemic circulation. The acute systemic toxicity of a chemical is defined as the dose
that is lethal to 50% of the treated animals, and this
dose is named the lethal dose 50 (LD50). LD50 values are widely used by government regulatory
authorities to place chemicals in hazard categories,

ECVAM Workshop 50: strategies to replace in vivo acute systemic toxicity testing

which dictate appropriate hazard warning labels,


permissible uses and exposure limits, and requirements for transport, handling and disposal under
existing laws and regulations. Acute systemic toxicity studies to determine the LD50 are a regulatory
requirement for all newly developed industrial
chemicals, and other chemical products with potential for human exposure. The hazard classification a
chemical receives is based on its potency in the test,
i.e. its LD50 value. Alternative testing designs have
been proposed to refine or replace the acute lethality test in animals (20), but all of these methods are
fundamentally a way of estimating the LD50 with
sufficient accuracy for regulatory use to protect
human health and the environment. It is important
to recognise the importance of the LD50 in chemical regulations worldwide, not only because there is
considerable background knowledge about its accuracy, meaning, strengths and weaknesses, but also
because legal statutes are built on its historic use.
Thus, validation and rapid regulatory acceptance of
alternative tests for evaluating acute systemic toxicity, including in vitro tests such as the ones discussed at this workshop, will depend on proven
capability of the alternative test to accurately predict the acute toxicity hazard category. The alternative way, for example, to build a regulatory and
legal framework around an in vitro lethality value
or alternative in vivo endpoint that cannot be
related to an in vivo LD50 value, would undoubtedly take much longer and require regulatory bodies to build an entirely new knowledge and
experience base around the new endpoint.
Therefore, this workshop focused on development
of an in vitro alternative to the in vivo lethality test
that would still provide an accurate estimate of
acute toxicity hazard according to current and
internationally harmonised classification schemes.
After validating an in vitro test that predicted
existing LD50 values in the diverse universe of regulated chemicals with sufficient accuracy to support
decision making in the regulatory community, the
validated test would be used to evaluate existing
chemical inventories that may still need acute toxicity testing, as well as new chemical compounds
planned for market introduction.
EU requirements
A strong regulatory impulse in the European Union
(EU) to find alternatives to animal tests comes from
the need for increased assessment of the so-called
industrial chemicals. Currently, the data requirements for industrial chemicals in the EU are laid
down, respectively, in Directive 67/548/EEC (21),
and its amendments, for the so-called new chemical
substances, and in Regulation 793/93 (22) for existing chemical substances. Existing substances are
chemical substances that existed in the European

439

market before 18 September 1981, whereas new


substances are the ones that were introduced after
that date.
Other regulations such as the Directive on Plant
Protection Products (91/414/EEC [23]) and the
Biocides Directive (98/8/EC [24]), also require the
hazard and risk assessment of substances to be
used for these purposes.
New substances
Currently, for any new chemical substance put on
the EU market, the manufacturer or importer is
required under Directive 67/548/EEC (21) to prepare a notification dossier containing, in addition to
details of the notifier and identity of the substance,
and its production process and production volume,
data on its physicochemical properties and results
from a number of toxicological and ecotoxicological
tests. These include tests on the acute systemic toxicity of the substance in rodents. The data requirements already begin at a production level of
10kg/year per manufacturer, with further testing
required as the production volume reaches higher
tonnage thresholds (Table 1). Since 1981, more
than 3600 new chemical substances have been notified following this harmonised scheme. The
Competent Authorities in the Member States are
responsible for assessing the risk of new chemical
substances according to Directive 92/32/EEC (25).
All data on the notification of new substances in
the EU are kept in the New Chemicals Database
(NCD) that is hosted by the European Chemicals
Bureau (ECB) of the European Commission (see
http://ecb.jrc.it).
Data should be obtained by applying the standardised tests laid down in Annex V of Directive

Table 1: Requirements for acute systemic


toxicity data for new chemical
substances according to Annex VII
of Directive 67/548/EEC
Production
level

Information required

10kg

Acute toxicity: one route (normally oral,


gases and volatile liquids by inhalation)

100kg

Acute toxicity: one route (normally oral,


gases and volatile liquids by inhalation)

1 tonne
and above

Acute toxicity: two routes (oral + one


other, gases by inhalation only);
assessment of toxicokinetics based on
available data

A. Gennari et al.

440

67/548/EEC (21; see http://ecb.jrc.it/testingmethods/). The approved methods in Annex V for


the oral route are the method B.1bis Acute Oral
Toxicity: Fixed Dose Procedure (equivalent to
OECD Test Guideline [TG] 421), and the method
B.1tris Acute Oral Toxicity: Acute Toxic Class
Method (equivalent to OECD TG 423). In addition,
results from the Up-and-Down Procedure (OECD
TG 425) may also be accepted. The classical LD50
method B.1 for acute oral toxicity was deleted from
Annex V in 2001 (as OECD TG 401), so it cannot be
conducted any more, but results from existing studies are still acceptable. For the dermal route, the
approved method is B.3 Acute Dermal Toxicity
(OECD TG 402) and for the respiratory route, B.2
Acute Inhalation Toxicity (OECD TG 403).
Existing substances
The European Inventory of Existing Chemical
Substances contains the list of the approximately
100,000 chemical substances that existed on the
European market before 18 September 1981. It is
estimated that about 30,000 of these chemicals are
actually being produced in the EU today (26).
Regulation 793/93 (22) establishes the need to
evaluate the existing chemical substances for their
possible risks for both human health and the environment, within a framework subsequently established in Regulation 1488/94 (27). This obligation is
on the Competent Authorities, based on data provided by the producers/importers. Because of the
large number of substances involved, a prioritisation exercise was required. So far, four priority lists
have been drawn up by the Commission in consultation with the EU Member States. Substances on
priority lists must undergo an in-depth risk assessment covering the risks posed by the priority chemical to humans and the environment (see
http://ecb.jrc.it/existing-chemicals).
Currently, around 140 priority substances have
undergone, or are undergoing, in-depth risk assessment. Around 65 assessments have been finalised,
and 34 have been published. The producer/importer
is obliged to provide data on acute systemic toxicity
prior to conducting the risk assessment. Available
data are used if they exist, and when new data generation is necessary, the use of the standardised
methods already mentioned is usually required. A
derogation from testing may be allowed, if it can be
established that the information is unnecessary for
risk assessment or is (technically) impossible to
obtain.
Classification and labelling
The EU legislation also requires an evaluation of
the hazard of a substance or preparation (classifica-

tion) and a communication of that hazard via a


label put on its package (labelling). This evaluation
must be made for any substance or preparation
manufactured within or imported into the EU and
placed on the EU market, and results in classification as dangerous for one or several endpoints concerning physical and chemical properties, and
health or environmental effects. All marketed substances and preparations must be classified and
labelled, irrespective of the quantity placed on the
market. This is performed according to the criteria
established in one of the annexes to the legislation,
and on the basis of existing information or new data
generated, normally by using the methods available
in Annex V, as already mentioned.
The Technical Guidance Document
The current guidance on conducting risk assessments for both new and existing chemical substances is implemented in the detailed Technical
Guidance Document on risk assessment for new
and existing substances and biocidal products (28).
This document establishes that, before any testing
for acute systemic toxicity is performed, it is necessary to gather and assess all the available information. Only when this information is not sufficient
for a proper assessment of the chemical, and if no
other means of obtaining the information are adequate, should testing with animals be carried out. If
the substance is indicated as corrosive, the need for
further testing should only be considered for noncorrosive concentrations.
Future chemicals legislation
Following the publication in 2001 by the European
Commission of a White Paper entitled Strategy for
a Future Chemicals Policy (26), in May 2003, DG
Environment and DG Enterprise made publicly
available on the Internet, a consultation document
on a new system for industrial chemicals, called the
Registration, Evaluation and Authorisation of
Chemicals (REACH) system, as a result of which
(and subsequent to this workshop), revised proposals have now been published (29).
In these proposals, the tonnage thresholds for
testing for acute toxicity are higher than those in
the current scheme for new chemical substances
(Table 2). This would mean a reduced need for animal testing. On the other hand, the REACH system
includes both new and existing chemical substances
within the same framework. Although there are
indications that data on acute systemic toxicity for
many existing chemical substances may already be
available (30, 31), some additional testing may be
needed.

ECVAM Workshop 50: strategies to replace in vivo acute systemic toxicity testing

Table 2: Requirements for acute systemic


toxicity data for new (and existing)
chemical substances according to
draft Internet REACH consultation
document
Production
level

Information required

10kg

None

100kg

None

1 tonne

None

10 tonnes
and above

Acute toxicity: two routes (oral + one


other, gases by inhalation only);
assessment of toxicokinetics based on
available data

441

product or substance with an estimated LD50 of


less than 5000mg/kg.
The US Department of Transportation also
requires that the hazard potential for all substances
transported interstate must be properly determined
and labelled (34). These requirements are harmonised internationally via the United Nations
(UN) Committee on the Transport of Dangerous
Goods.
The internationally harmonised acute
systemic toxicity testing requirements

REACH = Registration, Evaluation and Authorisation


of Chemicals.

US requirements
US government agencies require determination of
safety and hazard for a wide range of substances
and products. Acute systemic toxicity is one component of the basic testing requirements. As with
other regulatory authorities, acute toxicity testing
data is used to classify the hazard category of the
substance and, if it is considered to be hazardous,
appropriate hazard labelling is required to inform
consumers and workers of the potential hazard, and
of precautions that should be taken to avoid injury
or illness. Table 3 provides the four current toxicity
classification categories used by the US Environmental Protection Agency (EPA) for pesticides
and chemicals (32). As previously noted, classification is based on the estimated rat LD50 values.
Table 4 provides the precautionary labelling
requirements based on the four toxicity categories
(33). Precautionary labels must be placed on any

An internationally harmonised classification system has recently been developed and recommended
by the UN (35). This new system provides five hazard classification categories based on the estimated
rat LD50 values (Table 5).
Since this hazard classification system will be
adopted globally during the next few years, it is recommended that any validation efforts for acute toxicity methods should evaluate the predictive
performance of the methods for correctly estimating all of the Globally Harmonized System (GHS)
hazard categories.
National policies and laws impacting acute
oral toxicity
The Seventh Amendment to the EU Cosmetics
Directive prohibits the use of animals for the safety
testing of finished cosmetic products, and bans the
marketing of any cosmetic product, if animals are
used to test the safety of the product. This requirement forces cosmetic companies to use other nonanimal approaches to estimate whether the
accidental ingestion of a cosmetic product, typically
by unsuspecting children, might cause serious toxic
or lethal effects. Fortunately, most cosmetic ingredients have been tested in animals for their acute
oral toxicity, and companies are able to use this
information to estimate the acute oral toxicity
potential of a product or formulation containing

Table 3: US Environmental Protection Agency hazard classification categories for acute oral
toxicity

Hazard
indicators
Oral LD50
Inhalation LC50
Dermal LD50

Toxicity category
I
50mg/kg
0.2mg/l
200mg/kg

II
> 50 to 500mg/kg
> 0.2 to 2mg/l
> 200 to 2000mg/kg

LC50 = 50% lethal blood concentration; LD50 = lethal dose 50.

III
> 500 to 5000mg/kg
> 2 to 20mg/l
> 2000 to 20,000mg/kg

IV
> 5000mg/kg
> 20mg/l
> 20,000mg/kg

A. Gennari et al.

442

Table 4: Labelling requirements for precautionary statements for substances with acute
toxicity hazards

Toxicity
category

Precautionary statements by toxicity category


Oral, inhalation or dermal toxicity

Fatal (poisonous) if swallowed (inhaled or absorbed through the skin). Do not breathe vapour (dust or
spray mist). Do not get in eyes, or on skin or clothing. (Front panel statement of practical treatment
required.)

II

May be fatal if swallowed (inhaled or absorbed through the skin). Do not breathe vapours (dust or spray
mist). Do not get in eyes, or on skin or clothing. (Approximate first aid statement required.)

III

Harmful if swallowed (inhaled or absorbed through the skin). Avoid breathing vapours (dust or spray
mist). Avoid contact with skin (eyes or clothing). (Approximate first aid statement required.)

IV

(No precautionary statement required.)

Table 5: Acute toxicity hazard categories and (approximate) LD50/LC50 values defining the
respective Globally Harmonized System categories
Category
Exposure route
Oral (mg/kg body weight)
Dermal (mg/kg body weight)
Gases (ppmV)a,b
Vapours (mg/l)b,c,d
Dusts and mists (mg/l)b,f
aGas

5
50
100
0.5
0.05

50
200
500
2.0
0.5

3
300
1000
2500
10
1.0

2000
2000
5000
20
5

5000
e
e
e
e

concentrations are expressed in parts per million per volume (ppmV).

bInhalation

cut-off values are based on a 4-hour exposure test. Existing inhalation toxicity data, generated according to
a 1-hour exposure protocol, should be converted by dividing by a factor of two for gases and vapours and a factor of
four for dusts and mists.
cIt is recognised that saturated vapour concentration can be used as an additional element by some regulatory systems,
to provide for specific health and safety protection (for example, United Nations Recommendations for the Transport of
Dangerous Goods).
dFor

some chemicals, the test atmosphere will not be merely a vapour, but will consist of a mixture of liquid and vapour
phases. For other chemicals, the test atmosphere may consist of a vapour which is close to the gaseous phase. In these
latter cases, classification should be based on ppmV, as follows: Category 1 (100ppmV), Category 2 (500ppmV),
Category 3 (2500ppmV), Category 4 (5000ppmV). Work in the OECD Test Guidelines Programme should be undertaken
to better define the terms dusts, mists and vapours in relation to inhalation toxicity testing.

eCriteria for Category 5 are intended to permit the identification of substances which are of relatively low acute toxic
hazard, but which may present a danger to vulnerable populations under certain circumstances. These substances are
anticipated to have an oral or dermal lethal dose 50 [LD50] value in the range 20005000mg/kg body weight, with
equivalent doses for inhalation. The specific criteria for Category 5 are: i) the substance is classified in this category if
reliable evidence is already available that indicates the LD50 (or 50% lethal blood concentration [LC50]) to be in the
range of Category 5 values, or other animal studies or toxic effects in humans indicate a concern for human health of
an acute nature; and ii) the substance is classified in this category, through extrapolation, estimation or measurement
of data, if assignment to a more hazardous category is not warranted and: reliable information is available indicating
significant toxic effects in humans; or any mortality is observed when tested up to Category 4 values by the oral,
inhalation or dermal routes; or where expert judgement confirms significant clinical signs or toxicity, when tested up to
Category 4 values, except for diarrhoea, piloerection or an ungroomed appearance, or where expert judgement confirms
reliable information indicating the potential for significant acute effects from other animal studies.
fThe values for dusts and mists should be reviewed, to adapt to any future changes to OECD Test Guidelines with
respect to technical limitations in generating, maintaining, and measuring dust and mist concentrations in respirable
form.

ECVAM Workshop 50: strategies to replace in vivo acute systemic toxicity testing

many ingredients. However, such estimates do not


take into account the possibility that the ingredients could interact to produce new chemical entities
that might have toxic effects, or that the presence of
other chemicals might increase the bioavailability
of one or more toxic ingredients. Thus, there is a
need for in vitro methods that can help increase the
accuracy of the estimates of the toxicity of formulations.
The Cosmetics Directive further mandates that
cosmetic ingredients may not be tested for acute
oral toxicity after 2009. Thus, as new cosmetic
ingredients are introduced after this date, it will be
imperative to have accurate non-animal estimates
of their acute oral toxicities, in order to adequately
protect the health of consumers and the general
public.

The Necessity for High Quality In Vivo


LD50 Reference Data
Validation studies for alternative tests usually
determine the accuracy of the proposed test method
by comparing its predictive performance against
data from a reference method that is currently
accepted and used by regulatory authorities to
classify hazard and evaluate risks to the public. If
the alternative test is as accurate as, or more accurate than, the reference test method, it can be considered a potential substitute or replacement. It is
generally accepted that only high quality in vivo
data should be used as the reference standard, but
the workshop participants noted that the quality of
the in vivo reference data, including the accuracy of
the LD50 values, has never been formally evaluated.
LD50 values are derived from studies largely conducted in the private sector, although, infrequently,
such data may be generated by government laboratories. For new commercial chemicals and products,
manufacturing companies conduct internal toxicology studies according to their own Standard
Operating Procedures (SOPs), in compliance with
regulatory requirements. Sometimes data must be
submitted to regulatory bodies as part of a product
registration application, but there is often no
requirement that data must actually be submitted
(for example, for consumer products in the USA).
When data are submitted, this information is often
considered proprietary to the company, and is
therefore not usually released into public databases. In cases where a particular chemical is identified as a public health concern, the US Federal
authorities may undertake independent testing of
toxicity, although this rarely involves the determination of acute oral toxicity. LD50 values for a
smaller number of compounds are published as
results of research studies, or confirmatory studies
for lethality data sets. Thus, LD50 values are

443

derived from a large number of individual laboratories and organisations, from tests conducted for a
variety of reasons, and much of this information
may be filed in confidential product registration
applications.
A number of databases have been generated,
which could be considered as possible sources of
LD50 values for validation studies on alternative
methods (Table 6). These databases have been evaluated in terms of their purpose, the origin of the
data they contain, their policies for dealing with
conflicts between, or wide variations in, LD50 values from multiple studies, and the breadth of the
chemical compounds represented in the data. The
goals of this review were to collect a summary of
resources from which LD50 values could be
obtained for a wide range of chemical products
(such as insecticides, pharmaceuticals and industrial chemicals), to assess the adequacy of these
existing data for establishing reference LD50 values
for use in validation studies, and to develop an
objective process for selecting particular LD50 values to include in learning (for QSAR models) and
validation sets.
The database used for validating the ATC method
has detailed rat toxicity data, but the number of
compounds is too small to capture the required
variety of chemicals.
The International Uniform Chemical Information
Database is valuable, because it contains data on high
production volume chemicals. Although older substance entries are accompanied by endpoint values, a
range estimate of the LD50 value is now only available for more-recent chemical substance entries, evaluated by using the ATC method or the FDP.
The Registry of Toxic Effects of Chemical Substances (RTECS), formerly from the US National
Institute for Occupational Safety and Health, but
recently transferred to MDL Information Systems
(Elsevier MDL, San Leandro, CA, USA), is a frequently used database of LD50 values and other
toxicological information. LD50 values from this
database were correlated with in vitro IC50 values
in the review by Halle (36). The RTECS does not
require standardised methodology or supporting
data for determining LD50 values, and the lethal
dose is taken as the most potent value reported in
the literature at the time of the update. An important question to consider is whether or not a validation study can update its in vivo data set with
RTECS data entered after the validation study
begins. A predictive in vitro test should not have to
fail validation because of inaccurate in vivo data.
The ECB maintains an active database of new
chemical substances that have been notified prior to
marketing (the NCD). This database contains a
large number of chemicals with relatively recent,
and possibly more precise, toxicology data (of high
quality, because they were derived by applying
Good Laboratory Practice [GLP]), and would there-

A. Gennari et al.

444

Table 6: Sources of LD50 values


Number of
substances

Source

Notes

2030

Acute Toxic Class validation

Individual rat data

10,600

International Uniform Chemical


Information Database

Endpoint (newer entries)


or range (older entries)

1000s

Registry of Toxic Effects of Chemical


Substances

Based on published studies, dynamic values; most


toxic value listed as the LD50

4000

European Chemicals Bureau


New Chemicals Database

Confidential information; requires


authorised access

10,000
(100 into
humans)

Cancer drugs before 1985 (National


Institutes of Health/National Cancer
Institute)

Mouse and rat LD10 and LD50 values (probit). Oral


versus intravenous values for bioavailability. Not in
electronic format

1000s

US Environmental Protection Agency


pesticide registry

Pesticide LD50 values; confidential information

US Food and Drug Administration/Martindalea

Drug LD50 values; confidential information

European Chemical Industry Council, Centre


for Food Safety and Nutrition (US Food
and Drug Administration)

Highly toxic, but perhaps structurally


irrelevant

Food
contaminants

5000
High production volume chemicals databases
(USA and EU)

Confidential information

LD50 = lethal dose 50.


aSee

reference 65.

fore be a valuable resource for a validation study on


an in vitro method. However, the confidential
nature of the data, most of which would represent a
competitive advantage to the submitting company,
will limit access to them. On the other hand, under
right-to-know and consumer protection laws, the
hazard classification of all of these products
becomes public information at the time of marketing approval. Since this classification is presumably
based on LD50 values from acute systemic toxicity
testing, it would seem straightforward for the ECB,
or the applicant companies, to make lists of new
approved chemicals available with their corresponding LD50 values. Such information may become
available in the EU under the legislation to create
the REACH system.
The US National Cancer Institute (NCI) has historic rat and mouse toxicity data on thousands of
compounds that were developed as anticancer
agents. Although current toxicology paradigms
emphasise maximum tolerated doses in preclinical
species, older toxicology studies always included
dose-ranging studies to identify the lethal dose in

mice of both sexes, and in many cases, in rats as well,


by using several routes of exposure. The LD50 values
were very accurately estimated by probit analysis.
This information is of substantial value for evaluating how well animal values predict human safety, as
hundreds of these compounds were advanced to clinical trials, in which the detailed human toxicology
profile and maximum tolerated dose were ethically
determined, sometimes with accompanying blood
levels (37). The database is also valuable because it
contains compounds representing a wide range of
potencies, including compounds that exhibited
potency differences in the rat and the mouse, or even
in different inbred mouse strains, which would be a
fair challenge to an in vitro assay that predicts in
vivo toxicity data. The NCI data are not yet converted into an electronic format, but the data are
accessible from archive sites, and can usually be
released, because many compounds are off patent,
are no longer used clinically, or are sponsored by the
NCI itself.
Both the US EPA pesticides database and the
Food and Drug Administration (FDA) drug data-

ECVAM Workshop 50: strategies to replace in vivo acute systemic toxicity testing

base contain LD50 data of good quality. However,


most of these data will not be publicly accessible,
because of confidentiality restrictions by sponsors.
The Center for Food Safety and Nutrition of the
US FDA maintains toxicity information about food
contaminants. This is a non-proprietary source of
information about chemical toxicants, and toxicity
data in animals. However, most of these compounds
are highly potent natural products (such as shellfish toxins or mycotoxins) that may be too unique to
be considered as learning or validation compounds
for the development of predictive in vitro tests for
chemical safety.
The general consensus was that no single database is an ideal source of in vivo data to be the gold
standard or in vivo benchmark for validation testing. This is not surprising, since these databases
exist for purposes other than the support of in vitro
validation studies. Existing databases will have to
be mined for the most accurate information, and for
compounds that will maximise the chances for successful validation. Among the several sources of
multiple LD50 values for the same compounds, perhaps the best source would be that for the data used
for the validation studies on the three replacement
methods for LD50 determination, adopted by the
OECD during the 1990s. For two of these methods,
many laboratories repeated the LD50 tests with the
same compounds. The EU database on existing
chemical data is another source of LD50 data from
multiple studies, although it is uncertain whether
all the studies have been conducted with the same
chemical.
In addition, the workshop participants strongly
supported the idea of regulatory recommendations
that industries complete an in vitro cytotoxicity
assay conducted according to one of the existing validation study protocols, before conducting acute
toxicity tests in animals. The EPA already recommends that organisations consider using in vitro
basal cytotoxicity data to estimate starting doses for
in vivo studies, and that both the in vitro and in
vivo data should be submitted to the National
Toxicology Program Interagency Center for the
Evaluation of Alternative Toxicological Methods
(NICEATM), to assist in evaluating the usefulness
of the in vitro methods. The in vitro assay could be
conducted internally or outsourced to certified testing laboratories that can demonstrate SOP performance with reference standards that is on a par
with the validation study laboratories. The in vitro
and in vivo data, together with a sample of the compound, could be submitted to ECVAM, NICEATM,
ICCVAM, and/or other validation organisations
involved in the validation of in vitro methods. The
data could then be used to establish a repository of
chemicals with high quality LD50 and IC50 data,
which could be used in the future to quickly test the
validity of new or revised in vitro methods. For this
plan to be successful, the regulations would need to

445

explicitly define the in vitro testing as research, so


that the in vitro data produced would be submitted
but not used in the regulatory assessment of hazard
class by the Competent Authorities, until such time
as the in vitro test or tests had been formally validated and accepted as an alternative to the acute
systemic toxicity test in animals. In return,
ECVAM, and other validation organisations, would
be required to exercise due diligence in the use of
these data to expedite the introduction of a validated alternative, and to rapidly complete an internal study to determine whether the in vitro data
could replace the in vivo data as an anchoring data
set in read-across analysis at the regulatory agencies.
Several important aspects of mining chemical
toxicity databases to select chemicals for learning
and validation sets were identified during the workshop (Figure 1). First and foremost is the need for
an objective process to evaluate and select only
chemicals with useful in vivo data. In the context
of in vitro validation studies, useful data means that
the data should be of high quality, publicly accessible, reportable, and complete. High quality in vivo
data should include explicit details about the testing methodology used to determine the LD50 values, including sex, strain and age of the animals.
Inaccurate and highly variable in vivo data could
cause the validation failure of an accurate in vitro
test. Zbinden & Flury-Roversi (38) reported that so
many testing variables affect the LD50 that it cannot be considered a biological constant. This conclusion is underscored by the fact that, even with
standardised husbandry and dose administration,
oral LD50 values can vary by more than 10-fold
across individual studies (39). Some concern was
expressed, since this selection criterion might
exclude highly toxic compounds from the testing
repository, because low toxicity compounds in vitro

Figure 1: Database selection for chemicals

Advisory group
Regulators/industry

In vivo
database

Classes/
families

Databases

Data mining
Reproducibility
Interspecies predictivity
Human predictivity
Modelling

Chemical prioritisation and selection

446

show the largest variance in multiple in vivo LD50


studies (36). Highly potent compounds may exhibit
steep doseresponse curves in vivo that are difficult
to titrate, because small variations in bioavailability
dramatically influence toxicity.
There are considerable differences of opinion
about using pharmaceutical compounds in the validation of in vitro tests. A related issue is whether
highly toxic natural products, such as food contaminants, should be included in learning and validation sets. On the one hand, many pharmaceutical
compounds are designed to interact with specific
targets in certain organs, so biological effects are to
be expected. In contrast, many industrial chemicals
are designed for certain reactions, or to interact
with non-human biological systems, so adverse
biological effects in man are not expected.
Pharmaceutical compounds undergo more-accurate, quantitative toxicology conducted for market
registration, and the chemicals are formulated to
achieve consistent dosing. Anticancer drugs are
particularly useful, because dose-dependent systemic toxicity following a single exposure is characterised in detail in the rodent, the dog and the
human. A statistical evaluation of LD50 values for
pharmaceuticals versus non-pharmaceuticals
should answer a question raised at the workshop:
are LD50 values in pharmaceutical databases more
precise than LD50 values in chemical databases? If
the answer to this question is yes, it will be important to think through the implications of emphasising the use of pharmaceutical databases for
validating a test that ultimately will be applied to a
diverse set of chemicals, in which approximately
75% are not hazardous at all.
It is also generally accepted that the acute toxicities of pharmaceuticals usually result from organspecific toxicity, whereas the acute systemic toxicity
of other chemicals is often a result of general or
basal toxicity. Most likely, this distinction is an
artificial one that arises because, unlike drug toxicity, general chemical toxicity is not investigated in
detail at the organ, cell and molecular levels. Acute
death from a single exposure to a non-pharmaceutical chemical is most likely to be due to complex
physiological changes induced by adverse chemical
interactions with the biological system, which are
similar to the toxic effects of drugs; however, the
resources and the justification for understanding
these physiological changes do not exist in the
chemical industry. If the lethality of bioactive pharmaceuticals can be predicted accurately by an in
vitro test, it seems logical to expect that the same
test would be just as accurate for predicting the
absence of biological activity of a larger number of
chemical substances. It was noted that about 50% of
bioactivated chemicals, insecticides and neurotoxins were false negatives in the in vitroin vivo correlation registry published by Halle (36), because
the in vitro test underestimated the LD50 value.

A. Gennari et al.

Because a validated test will eventually be used


prospectively with novel chemical structures, learning and validation sets of chemicals should contain
sufficient chemical diversity to represent a wide
variety of structures, applications, hazard classifications and possible mechanisms of action. Acute
toxicity testing is required for drugs, pesticides and
biocides, and food contaminants, so some bioactive
agents should be included, as well as some chemicals that are not metabolised (renal clearance only).
In addition, all hazard classes of chemicals should
be represented (GHS Categories 15), taking into
account diverse physicochemical properties.
An unresolved issue at this time is whether to
include chemicals with low bioavailability, i.e.
chemicals with significantly higher LD50 values
after oral than after i.v. exposure. In the Halle registry of IC50LD50 correlations, i.v. LD50 values
correlated better with in vitro IC50 values than did
oral LD50 values (36). However, restricting learning and validation sets to chemicals for which i.v.
LD50 values exist significantly reduces the available number of compounds. Low oral bioavailability
can result from poor absorption in the gastrointestinal tract, or extensive first-pass metabolism by
the liver, or combinations thereof. In vitro alternatives to animals that provide estimates of these
processes were presented at the workshop. While it
might be more fruitful to develop a validated in
vitro test to predict the LD50 of chemicals following
i.v. exposure, without the complications of bioavailability, regulatory testing requirements and the
protection of public health will continue to require
estimates of oral acute toxicity. However, the predicted oral LD50 values might be able to be estimated by considering bioavailability, as estimated
with additional in vitro testing. It seems unreasonable to ask that a simple in vitro cytotoxicity test, to
which the compound is added directly to the cell
culture medium, should model a complex interplay
between cellular toxicity and chemical permeation
through tissue barriers and elimination from the
body. A multistep testing strategy seems appropriate, in which the first step uses the strengths of the
in vitro cytotoxicity test to predict the rat LD50
from a direct, i.v. exposure. Then, this predicted
LD50 is adjusted for inter-species differences in susceptibility between rat and man, based on specialised in vitro tests, such as testing on
bone-marrow progenitors (3841), and evaluating
bioavailability by using in vitro tests for drug solubility, permeation of the gut mucosa, and first-pass
metabolism.
There was general concern that correlating LD50
values and IC50 values was not appropriate, because
dose (i.e. LD50 measurement) is related to concentration (i.e. IC50 measurement) by absorption and
consequent disposition in the body, which is not a
component of simple in vitro tests for cytotoxicity. It
seems more scientifically sound to compare IC50 val-

ECVAM Workshop 50: strategies to replace in vivo acute systemic toxicity testing

ues with blood levels at the LD50 dose, as in the


MEIC study, and then extrapolate blood levels to
dose based on the in vitro and in silico modelling of
pharmacokinetics and bioavailability. Although i.v.
and oral values may be similar for many chemicals,
i.v. LD50 values of 5100% of oral LD50 values have
been reported; this 20-fold difference in LD50 value
is larger than the 1-log prediction interval from the
actual LD50 that is the goal of published in vitro
models (36). Regulatory officials are most concerned
about distinguishing non-toxic from slightly toxic
chemicals, so the workshop participants considered
that the in vitro tests must have a narrower prediction interval than 1-log. Category 1 and Category 2
hazards are separated by only a 6-fold difference in
LD50 value (50mg/kg versus 300mg/kg), and the distinction between not hazardous and Category 4 is
only 2.5-fold (2000mg/kg versus > 5000mg/kg). To
be accepted by the regulatory community, the in
vitro test must be able to reliably distinguish chemicals in these adjacent hazard categories, and the best
way to achieve a more-precise prediction range of the
LD50 value is to adjust the predicted LD50 value for
bioavailability estimates in separate testing components.

State-of-the-art of the In Vitro Methods


Over the last 50 years, research has been conducted worldwide to evaluate the potential use of
in vitro cell systems for predicting acute toxic
effects in vivo. A large number of in vitroin vivo
correlation studies have shown that rodent LD50
values and human lethal concentrations can be
derived, with a sufficient level of precision, from in
vitro IC50 values. The report of the International
Workshop on In Vitro Methods for Assessing Acute
Systemic Toxicity (19), organised by ICCVAM and
NICEATM in 2000, is a comprehensive review of
such studies. The participants in the ECVAM
workshop acknowledged the contribution of these
studies in enhancing the comprehension of the
relationship between in vitro basal cytotoxicity
and in vivo systemic toxicity. Furthermore, conclusions and recommendations were provided for
test systems that would be necessary to predict
target organ toxicity, and relevant toxicokinetic
parameters such as ADME. The purpose of this
workshop was to build on the progress of the ICCVAM workshop, and to identify efforts needed to
advance the development and validation of in vitro
methods that could accurately predict acute systemic toxicity.
The MEIC and EDIT programmes
The MEIC programme was a 7-year study coordinated by the Scandinavian Society for Cell Tox-

447

icology. Fifty-nine laboratories worldwide volunteered to test 50 reference chemicals selected by the
Swedish Poison Information Centre, according to
their in-house protocols. Each chemical was backed
by relevant data on human toxicity and kinetics
(MEIC Monograph [MEMO] programme) to permit
the derivation of approximate 50% lethal blood concentration (LC50) curves over time to be used for
comparison with in vitro data. The main conclusion
of the MEIC study was that certain human basal
cytotoxicity tests correlate with the LC50 values
calculated from patients with intentional or accidental drug and chemical overdoses. Furthermore,
the prediction increased considerably when known
toxicokinetic data (i.e. passage across the
bloodbrain barrier) were used to correct the in
vitro data. It was further recognised that other
important toxic mechanisms exist, which could only
be measured by using supplementary in vitro toxicity tests. Subsequently, the managers of the MEIC
study started the Evaluation-guided Development
of New In Vitro Test Batteries (EDIT) programme
to evaluate tests relevant to toxicokinetics and
organ-specific toxicity, eventually to be incorporated into a test battery for the prediction of human
acute systemic toxicity (42).
The results of the MEIC programme have been
published as a series of eight papers in ATLA
(4350).
The Registry of Cytotoxicity
The Registry of Cytotoxicity (RC) is a database of
acute oral LD50 data from rats and mice (taken
from the RTECS) and the geometric means of the
IC50 values (IC50x; in mmol/l medium), taken from
in vitro cytotoxicity assays available in the literature, for 347 chemicals (36). The data included in
the RC were collected according to defined acceptance criteria, and have been used to derive a linear
prediction model by plotting pairs of the log-transformed IC50x values and oral rodent LD50 values
expressed in mmol/kg. An empirical prediction
interval of log5 was defined, based on information
on the required and expected precision of the
rodent oral LD50 data (51). The analysis of both
positive and negative outliers has shown that this
might be partly attributed to the large variation in
the LD50 data reported in the literature.
Furthermore, analysis of the negative outliers
showed that they are characterised by specific systemic in vivo effects. Although the RC prediction
model was developed with in vitro data obtained
from non-validated tests, it has demonstrated a
good correlation between basal cytotoxicity and
rodent oral systemic LD50 values. It has been suggested that this relationship is sufficiently strong
for in vitro data to be considered for estimating
starting doses for in vivo acute lethality assays, to

448

further reduce the use of animals in this type of


test.
The ECVAMNICEATM validation study on
two in vitro basal cytotoxicity tests
During the International Workshop on In Vitro
Methods for Assessing Acute Systemic Toxicity
(19), recommendations were made on the need to
evaluate two sufficiently developed and standardised basal cytotoxicity assays for their ability to predict rodent LD50 values according to the RC
regression model, in order to improve the dose
selection for in vivo studies, and to predict human
lethal concentrations (52). In 2002, NICEATM and
ECVAM designed and started a joint validation
study involving two US and one European laboratory. Seventy-two chemicals, which comprise the
majority of the 50 MEIC chemicals and chemicals
nominated by various regulatory authorities, were
selected according to the availability of acute oral
rodent and human toxicity data and the need to
represent a wide range of toxicities. These chemicals are being tested in a mouse immortalised cell
line (BALB/c 3T3) and in normal human keratinocytes by using the neutral red uptake cytotoxicity assay (53). The primary study objectives are: a)
to classify the chemicals according to the GHS categories, as well as to identify non-toxic chemicals
(i.e. chemicals with an oral LD50 above
5000mg/kg); b) to evaluate the predictability of the
starting dose in in vivo testing by using reliable in
vitro data, and to determine the reduction in the
number of animals that would be obtained by using
information from an in vitro assay prior to performing an in vivo study; and c) to evaluate the correlation with human lethal concentrations. This
study will furthermore provide a high quality in
vitro database and selected high quality in vivo
LD50 values for the 72 chemicals, for future use in
the development, refinement and validation of
other methods that could be part of a new testing
strategy for assessing the acute systemic toxicity of
chemicals.

The Integration of Biokinetics and Basal


Cytotoxicity to Predict Acute Toxicity
A large number of in vitro test methods exist for
determining the basal cytotoxicity of chemicals.
These methods use various types of mammalian
cells that are exposed to a concentration range of
the test compound for certain periods of time. Thus,
the in vitro models are used to determine a critical
concentration of a test compound at a specific target site by direct exposure of the cells to the test
compound. In the in vivo situation, however, a
number of toxicokinetic factors, such as ADME,

A. Gennari et al.

also play an important role in determining the critical concentration at the target site. Due to the complex pathways that lead from external exposure to
the local susceptible target organ concentration, a
direct extrapolation of in vitro data to an acute toxic
dose in vivo is very difficult, if not impossible. In
order to develop a scientifically sound alternative
approach to predict acute toxicity in humans,
knowledge of the kinetics of a test compound should
be combined with in vitro toxicity data.
Such an approach should aim at predicting the
toxic dose for a chemical compound by integrating
data obtained from a number of relatively simple in
vitro tests. The concentration at which a toxic effect
is observed in vitro can be compared with the critical concentration at the target sites, which induces
acute toxicity in vivo. Since the concentration at the
target site is related to the concentration in the
plasma, plasma levels can be used in making in
vivoin vitro comparisons (20). In some cases, however, these concentrations are not identical, due to
processes that may actively increase or decrease
concentrations in the target tissue. Therefore, one
relevant issue would be whether plasma levels could
be accurately predicted by using in vitro data.
There are a number of aspects that determine the
bioavailability of a chemical compound after oral
exposure. Solubility and/or binding in the gastrointestinal tract and intestinal mobility largely
determine the free fraction of the compound available for uptake. A number of models exist for studying these processes in vitro; for example, those used
to study the release of specific compounds from various matrices, such as food matrices (5456). An
important parameter in determining plasma levels
is the permeability of the intestinal wall to the
chemical compound. The systemic toxicological risk
will be largely reduced, when the uptake into the
body is negligible. Apart from QSAR models that
have been developed to describe intestinal absorption, a number of cell-based and tissue-based in
vitro gastrointestinal barrier models exist, which
can be used to determine intestinal permeability
(for example, TC7 cells, HT29 cells and Caco-2
cells). For example, several groups have described
the relationship between human oral absorption
and the permeability coefficient of a test compound
determined in Caco-2 cells (57, 58), which has been
used to predict intestinal uptake. Some of these in
vitro barrier models also permit the study of the
effects of intestinal membrane transporters and
metabolism on bioavailability.
When a chemical enters the systemic circulation,
hepatic first-pass metabolism may noticeably influence its concentration in the blood. The (timerelated) blood concentration of a potentially toxic
chemical may be considerably reduced when
hepatic first-pass metabolism is high and the compound is rapidly excreted in the urine or bile. Liver
subcellular fractions (such as microsomes) are, for

ECVAM Workshop 50: strategies to replace in vivo acute systemic toxicity testing

example, used to estimate hepatic clearance. It


should be noted that, in acute toxicity, instances
exist in which hepatic first-pass metabolism actually causes the production of metabolites with
higher intrinsic toxicity than the parent compound
(for example, paracetamol).
After passage through the liver and entry into the
systemic circulation, the plasma levels of a compound are largely influenced by its tissue distribution, which is predominantly determined by its
partition between tissue and plasma. At this stage,
additional kinetic parameters of active transport
mechanisms into or out of the target organ can also
be considered. Finally, binding to plasma proteins
may significantly lower the free bioavailable fraction in the plasma (59, 60).
In order to study the combined effects of a large
number of kinetic parameters, data can be combined in in silico models (6164). With respect to
acute toxicity, these models may help in extrapolating the in vitro critical concentration to the in vivo
critical dose. Preferably, these biokinetic models
should be generic models, in order to be applicable
to a large number of chemicals. In this respect,
attempts can be made to simplify the model by considering the actual relevance of a number of the
aspects mentioned above. For example, acute toxicity will primarily occur under conditions of overloading. Under these conditions, it can be
considered that certain aspects, such as the release
of a chemical compound from a particular matrix or
the involvement of intestinal membrane transporters and metabolism, may be of lesser importance in determining the bioavailable fraction. In
addition to the oral route, the dermal and inhalation routes are important routes-of-entry in acute
toxicity, and kinetic parameters which describe
uptake via these routes (for example, absorption
rate through the skin and the airblood partition
coefficient) can relatively easily be incorporated
into these models.
In conclusion, the integration of in vitro kinetic
models, in silico kinetic computer modelling and in
vitro target organ toxicity assays, makes the comparison of in vitro and in vivo data more meaningful.

Read-across and New Substances


The use of toxicological read-across data in the notification for new substances is an area to which the
EU has been committed over recent years, primarily and most importantly for animal welfare reasons. For certain new substances which require
notification, toxicological data are already available
on structurally related analogues. In these circumstances, and where scientifically justified, reading
information across to the new substance is clearly a
desirable route to notification, not least as a means

449

of reducing animal testing. A number of factors are


considered, including the purity/impurity profiles of
the two chemicals, together with an assessment of
the quality of the available data. Once a read-across
approach has been agreed, full physicochemical
testing is conducted, alongside an acute oral toxicity test and the Ames genotoxicity test, which are
used to underpin the validity of the approach.
These tests give an indication of the likely toxicokinetics and, together with information on the general toxicity profile of the chemical, provide
reassurance that the two compounds are indeed
sufficiently similar and that further toxicity testing
(skin and eye irritation, skin sensitisation, repeated
dose toxicity) is not required. If different results are
obtained for the new chemical and its analogue, further testing is required, to address the particular
concerns.
This approach achieves a reduction in the use of
animals for regulatory testing, but it is not able to
function as a complete replacement, since some animal testing is still required. The ultimate aim
would be for an in vitro alternative for the acute
toxicity endpoint to be validated and to replace the
animal test, thereby enabling a complete in vitro
read-across package to be used for notification purposes. For the present, however, the workshop participants recommended that in vitro cytotoxicity
data should be included, wherever possible, in the
read-across data set, to complement the in vivo
results and to begin to build a knowledge base on in
vitroin vivo relationships for use in future validation studies. However, there is no legislation to
enforce this recommendation, so it would require
cooperation and participation of industry. A potential problem with this approach is one of confidentiality. Although industry may well be willing to
help support the development of alternative methods, the data on these compounds are commercially
sensitive, so it is necessary for approaches to be
investigated to resolve any such concerns.

The Development of a Database on Acute


Toxicity
In order to make available more useful data on
acute toxicity, for analysing the outcome of in vitro
test validation studies, a new database on acute toxicity data should be developed and made accessible
to the scientific and regulatory community. To
allow comparison with the in vitro data and to facilitate development of alternative tests, the toxicity
database should contain, as a minimum, the compounds from the MEIC study and from the Halle
registry (36), and those tested in vitro in the
ECVAMNICEATM validation study. This database should include acute toxicity data from animal
studies, human acute toxicity data, and physicochemical data (such as vapour pressure, pKa,

A. Gennari et al.

450

n-octanolwater partition coefficient and water solubility), as well as the results from in vitro testing
programmes for acute toxicity.
One major purpose of the database will be to
derive general principles for acute toxicity studies
in animals, including: the identification of structural alerts for compounds with high or low acute
toxicity; the analysis of poisoning symptoms (which
poisoning symptoms, and of what severity, are
related to death, and what are specific/non-specific
poisoning symptoms); the analysis of necropsy findings (distinguishing between specific and nonspecific findings, identifying indicators of overload); the analysis of interstudy variability, including differences between strains, species and sexes;
and the analysis of human predictivity.
The database should also be used for the analysis
of in vitro data, including: the comparison of test
systems (with respect to identifying suitable endpoints and cell systems); and the analysis of the
reproducibility of test results.
Furthermore, in vivo and in vitro data should be
compared, including: the calculation of regression;
the analysis of outliers in regression analysis; the
influence of physicochemical data (as indicators of
absorption or accumulation) on regression; the
identification of compounds tested in in vitro studies which should also be tested in in vivo studies;
and the development of rules by which test systems
or combinations of cytotoxicity and other test systems could replace acute toxicity studies in animals.
Based on the experience gained with the repeated
dose toxicity database, developed by Fraunhofer
(Institute of Toxicology and Experimental
Medicine, Hannover, Germany) on behalf of the
European Chemical Industry Council, the following
information from acute in vivo toxicity studies
should be included in the database:

species;
strain;
sex;
age;
weight;
test protocol/guideline;
use (or not) of GLP;
score (to be developed) for quality of study;
study deficiencies;
dose groups;
number of animals per dose group;
clinical signs;
clinical measurements (for example, heart rate,
blood pressure);
necropsy findings;
LD50 value; and
time to death.
Glossaries should be developed (for example, for
clinical signs and necropsy findings), to allow questions to be asked of the database with consistent

terminology. Guidance for constructing these glossaries and codifying toxicity can be found in the systematic classification of clinical toxicity caused by
acute exposure to anticancer drugs, which has been
developed by the NCI (http://ctep.cancer.gov/forms/
CTCAEv3.pdf). These Common Terminology
Criteria (CTC) were developed to achieve uniform
reporting of toxicity data.
Most acute toxicity studies for common compounds were not conducted according to standardised protocols, as they are rather old. Nevertheless,
older studies provide useful data. Therefore, it is
important also to include non-standardised studies
in the database, although any protocol deficiencies
should be recorded. As mentioned above, a score for
the quality of the database should be included in
the database.
Cytotoxicity data should also be entered in the
database, including, for example:

cell line;
dose groups;
number of replicates per dose;
solvent and solvent maximum concentration;
exposure duration/observation period;
testing endpoint (for example, protein content,
ATP content, morphological changes); and
IC50 value.

Organ-specific Effects
In vitro methods are used by both the pharmaceutical and chemical industries, as part of a testing
strategy, to decide whether a new chemical entity
could be developed as a consumer product. Once a
compound has been selected for product development, regulatory testing is initiated. Most of the
compounds that pass these in vitro development
and safety screens eventually lead to new products.
The workshop participants considered this to be
proof of the principle that in vitro and short-term
tests alone, without animal studies, can be used to
make correct decisions about the risk of chemical
toxicity, and thus should be adequate for acute toxicity testing, if properly developed and validated.
If multiple, but specific, adverse organ-specific
effects combine to cause acute systemic toxicity, as
occurs with some pharmaceuticals, a panel of in
vitro tests that each predicts specific, acute organ
toxicity might yield more-accurate predictions than
a cytotoxicity test based on a single cell line from
one species. In fact, many in vitro tests for adverse
organ effects are ideally suited to acute toxicity testing, where exposure to the toxicant is shorter than
the life-span of the cell cultures. Once a relationship
between an effect on the functional endpoint in the
in vitro assay and a clinical manifestation of organ
toxicity can be established with pharmaceutical
compounds, it should be possible to identify the

ECVAM Workshop 50: strategies to replace in vivo acute systemic toxicity testing

acute systemic toxicity and hazard categories of


non-pharmaceutical chemicals.

Dual Use of Acute Toxicity Data and New


Strategies for In Vitro Testing
Acute toxicity data can be used for two specific purposes: for labelling and classification (for example,
based on an LD50 value), and to provide data on
loss of function or functional integrity (for example,
based on the effects of poisoning). In the first case,
cellular failure or death might be closely related to
the LD50 value, whereas, in the second, a morespecific loss of function would be expected to bring
about organ system failure. However, it is important to note that organ system failure can be due
either to the death of cells in the affected organ, or
to a loss of function in the surviving cells of that
organ, resulting in cellular death or loss of function
in other organs.
If accurate in vitro data for predicting acute toxicity are to be collected, it is important to have
some prior information about the test compound.
Whatever information is available should be used
in defining specific assays that are most relevant
for predicting the toxicity of the particular chemical or group of chemicals. Ideally, ADME should
be known prior to choosing the tests. ADME predictions may indicate that basal cytotoxicity testing without adjustment for biokinetics will be
highly inaccurate for a certain chemical, which
would redirect the testing strategy toward combined assays for toxicity and ADME, or complementary, more-specialised assays for organ
function.

451

Cellular Failure or Death Mechanisms for


LD50 Classification
For evaluating chemical effects on specific endpoints of toxicity that are generally applicable to
many cell types, the workshop participants developed a list of potential susceptible targets that are
common to many cell types and could serve as in
vitro endpoints for acute toxicity (Table 7). These
include the production of reactive oxygen species
(ROS), energy production and metabolism, membrane structure or function, protein turnover, gene
regulation, and cell communication. Identified in
Table 8 are agents known to affect these systems,
which can cause death. The participants suggested
that assays that would measure these particular
endpoints might be very accurate tools for predicting lethality and LD50 values, either individually or
as a battery of tests (which, it is anticipated, is more
likely to be appropriate).

Organ-specific Toxicity Mechanisms for


LD50 Classification
Functional aspects of specific organs are a potential
area of interest for modelling acute toxicological
consequences and for predicting acute lethality.
Assays of acute organ dysfunction may be very
important for assessing the acute systemic toxicity
of chemicals that are false negatives in basal cytotoxicity assays. An alternative to basal cytotoxicity
assays would be to assay for critical clinical
adverse effects on organ systems that result in
acute toxicity (described at the workshop as what
the pathologist would see histologically, as well as

Table 7: List of mechanisms common to many cell types and responsible for cellular failure
or death
Reactive oxygen species formation (paraquat, CCl4)
Energy production and metabolism (3-nitropropionic acid, fluoroacetic acid, 3-acetylpyridine, 6-aminonicotinamide)
Mitochondrial function (CN-, uncouplers, ATPases)
Glycolysis (acrylamide, n-hexane)
Membrane structure or function (CCl4, peptide toxins)
Protein turnover (cycloheximide, proteosome inhibitors)
Gene regulation (tetrachlorodibenzo-p-dioxin)
Cell communication

intracellular signalling
cellcell interaction (dieldrin, botulinum toxin, acetylcholinesterase inhibitor)

A. Gennari et al.

452

Table 8: Organ-specific functions that are commonly compromised in organ failure


Organ

Susceptible function

Examples of toxic agents

Kidney

Transport, filtration

Direct action: cephalosporins, aminoglycosides,


platin analogues

Liver

Metabolic transformation:
xenobiotics
nutrients
protein regulation and excretion

Direct action: spicamycin


Metabolised: chloroform, aflatoxin, paracetamol

Central nervous system

Neurotransmission,
structural integrity

Direct action: organotins, organic solvents


Metabolised: organophosphates

Cardiovascular system

Electrical conduction, contraction

Direct action: digoxins, terfenadine


Metabolised: doxorubicin

Lung/respiratory system

Membrane integrity,
gas exchange

Direct action: NO2, CoCl2, CdO2


Metabolised: paraquat

Blood

Oxygen transport,
white cell production

Direct action: mycotoxins


Metabolised: aromatic amines

Gastrointestinal tract

Transport:
hydration

Direct action: antibiotics, cholera toxin

physiologically). In fact, modelling critical clinical


adverse effects is a way of identifying what alterations in the function of a particular organ system
result in acute toxicity at the clinical level. The
results of this discussion are presented in Table 8.
The main organ systems known to be susceptible
to acute toxicity resulting in lethality were identified as the kidney, liver, central nervous system,
cardiovascular system, lung/respiratory system,
blood, and the gastrointestinal tract. In Table 8, the
susceptible function for each organ is identified,
and examples are given of compounds that affect
one or more of these susceptible systems, divided
into those that act directly and those that require
metabolic activation. The table is meant to be illustrative, rather than all-inclusive, and it is certain
that many other compounds could also serve as
examples.
Although the text of the table is largely selfexplanatory, the following two cases will serve to
further illustrate its value. Firstly, the two main
functions of the kidney are filtration and transport.
These systems, when inhibited, can result in either
loss of function or, if sufficiently severe, death
within the parameters of acute toxicity.
A second example is the blood, or, more specifically, blood cell production by the haematopoietic
bone-marrow. It is possible to monitor the ability to
produce differentiated myeloid cells in vitro by
using the colony-forming unit-granulocyte/
macrophage (CFU-GM) assay. In clinical studies, it

has been observed that significant inhibition (probably in the 90% range) can increase the risk of septicaemia. ECVAM sponsored a successful validation
study on the use of this assay for predicting the
human dose that causes severe neutropenia, which
defines the maximum tolerated dose in humans in
the absence of toxicity in other organs (3941).

Relationships Between Cellular Failure or


Death and Organ-specific Toxicity
When the susceptible functions of various organ systems are considered, there are several physiological
processes common to many, if not all, of the organ
systems listed in Table 8: transportation of molecules, membrane integrity and secretion of molecules
(such as enzymes and proteins). Thus, it became clear
that cellular failure or death can result in organ system failure and vice versa. An attempt has been made
to identify these common physiological processes and
the potential assay systems for quantifying specific
endpoints related to each of them (Table 9).
The specific systems identified by two asterisks in
Table 9 can be considered to be fundamental cellular processes, and a discussion ensued on whether
or not these could represent a potential general
model of acute cytotoxicity. This might be called the
second generation approach, to develop alternative tests that could predict all aspects of acute toxicity, providing for both classification and

ECVAM Workshop 50: strategies to replace in vivo acute systemic toxicity testing

453

Table 9: Potential models for acute toxicity testing


Susceptible function

Potential assay system

A transport model
membrane functions (CCl4, peptide toxins)**
fluids, electrolytes, molecules, nutrients

Caco-2 cells
Reconstituted renal tubule model

A secretion model**
enzyme, protein, hormones, neurotransmitters

Neural transmission
Neuroendocrine cell lines
Cytokine patterns for whole blood

Energy production and metabolism**


mitochondrial** (CN-, uncouplers, ATPases)
glycolysis** (acrylamide, n-hexane)

ATP chemiluminescence assay

Blood-forming capacity

ECVAM-validated colony-forming unitgranulocyte/macrophage assay

Protein turnover (cycloheximide, proteosome inhibitors)

Rate of protein synthesis

Gene expression (tetrachlorodibenzo-p-dioxin)

mRNA production

Reactive oxygen species (paraquat, CCl4)

Fluorescein diacetate

Intracellular structure
Cell communication
intracellular signalling
cellcell interaction (dieldrin, botulinum toxin,
acetylcholinesterase inhibitor)

[Ca2+] measurement
Gap-junction assays

**These systems are considered to be fundamental cellular processes.

predicting functional consequences for specific


organ(s). It should be noted that this would be a
dynamic battery of tests, tailored to test the individual compound with a particular level of knowledge. For example, if read-across identified a new
chemical as a potential neurotoxin, the secretion
model might focus on the nervous system. If readacross indicated a more generalised pattern of toxicity, a secretion model such as cytokine pattern
release in whole blood might be an appropriate
model. The same rationale holds for membrane
transport and other physiological processes that are
the targets of toxicants. There are several common
functions in many different organ systems.

holders. In particular, DG Enterprise and the


US Federal Agencies should encourage industry to submit all toxicological information (in
vitro and in vivo) for any of the 72 materials
that are currently being used to evaluate the
cytotoxicity hypothesis.
2.

The use of these chemicals in additional types


of in vitro tests should be encouraged. A chemical repository for these chemicals should be
established at ECVAM for distribution within
the scientific community.

3.

Until interspecies differences are fully investigated and understood, both human and rodent
cells should continue to be considered for use
in in vitro cytotoxicity studies.

4.

Absorption by the required routes of administration should be estimated by using in vitro


or in silico systems.

5.

All the technical limitations of in vitro tests


should be identified and addressed (solubility

Conclusions and Recommendations


General
1.

The creation of alternatives to animal testing


should be a joint effort among many stake-

A. Gennari et al.

454

and volatility problems, endpoint limitations


for some materials).
6.

Methods to estimate dermal and inhalation


LD50 values should be developed. For example, an in vitro dermal absorption assay should
be conducted before a dermal toxicity assay. If
there was no absorption, only a limit dose test
might be considered necessary.

7.

Research should be conducted to determine


whether acute toxicity from slightly toxic
chemicals occurs when physiological processes
(such as metabolism) are overloaded.

8.

Organ-specific tests should be developed, based


on critical clinical adverse effects. Assay systems based on susceptible functions are identified in Table 9, for example, a transport system
that could be relevant to the kidney, gastrointestinal tract and bloodbrain barrier.
Robotic technology is now available for measuring transepithelial resistance. With modifications to this system, it could also be possible to
measure ROS and energy metabolism.

9.

Human-based systems for the detection of


metabolism-mediated bioactivation and detoxification should be further developed and
prevalidated.

10.

Knowledge-based alert systems should be


developed and used to evaluate and identify
possible relationships between physicochemical properties and biological activities.

11.

Absorption models (intestinal: Caco-2 cells;


skin penetration; and, if deemed necessary,
bloodbrain barrier) should be standardised,
validated and integrated with the basal cytotoxicity approach.

12.

13.

14.

Simplified physiologically based biokinetic


(PBBK) computer models to predict maximum
blood level and duration of exposure, based on
partition coefficient and in vitro clearance,
should be used to estimate chemical disposition in the body.
Basic metabolic systems (for example, engineered Hep G2 cells) should be used to
increase the accuracy of acute toxicity predictions.
High priority should be given to the establishment of an in vivoin vitro database (including
all toxicological endpoints). The quality of the
in vivo data should be described, quantified
and ranked. Study details and results should
be documented.

15.

The LD50 values of new chemicals should be


made publicly available at the time of marketing approval. Since a new products hazard
classification is based on its LD50 value, and
since the hazard classification of commercial
products is public information, the LD50 value
should be made public at the same time,
including species and route of administration.
Companies should be encouraged to make raw
data available to databases that will facilitate
the development of in vitro and in silico predictive test systems.

Cytotoxicity studies
16.

An appropriate in vitro gut absorption model


should be used to translate from circulating
blood levels or effective dose (actual dose
absorbed) to exposure dose.

17.

The in vitro tests should be evaluated for their


utility for predicting the most relevant in vivo
endpoint, namely, the LC50 value the blood
concentration that is found at the LD50 dose.
The LC50 value can be converted into an
LD50 dose by using pharmacokinetic and
bioavailability models.

18.

The current, well-managed cytotoxicity assay


study (the ECVAMNICEATM validation
study) should be completed, in order to provide a high quality in vitro database for the
two assays, and to facilitate the development
and validation of the additional methods
needed to increase the accuracy of the cytotoxicity predictions.

19.

Cytotoxicity studies should be evaluated with


reference to the GHS categories.

20.

Outliers should be further evaluated by using


toxicological knowledge and by using QSAR
and/or functional assays (to improve the prediction).

21.

If a cytotoxicity study demonstrates that the


method is sufficiently predictive, the cytotoxicity assay and the appropriate regression
model should be used to select a starting dose
for an acute oral toxicity test.

22.

A cytotoxicity assay should be conducted


according to one of the existing validation
study protocols before conducting the animal
test. The in vivo LD50 values and the in vitro
IC50 values should be submitted to
ECVAM/ICCVAM, and should be made publicly available, if appropriate. Alternatively,
the test compounds should be sent to ECVAM

ECVAM Workshop 50: strategies to replace in vivo acute systemic toxicity testing

for in-house in vitro testing. These data will


assist in assessment of the predictivity of the
test.

provide high quality LD50 data, because of the


detailed nature of preclinical and clinical toxicology studies which are conducted. These
chemicals play an important role in the validation of alternative tests for acute systemic
toxicity, but care should be exercised to
include a prevalence of these chemicals in testing sets that approximates their prevalence in
the commercial pipeline.

Organ-specific toxicity
23.

A test battery should be developed to include


the three assays identified, namely, ROS production, energy metabolism, and cytokine
secretion profile in blood. Initially, data
should be collected in a single laboratory or in
one laboratory for each endpoint, then the
performance of the assays should be evaluated
against known standard toxicants. This
should be required before validation studies
are initiated.

30.

The regulatory authorities are particularly


concerned about distinguishing between nontoxic chemicals and slightly toxic chemicals.
GHS Category 1 and Category 2 hazards are
separated by only a 6-fold dose range (50mg/kg
versus 300mg/kg), and the distinction between
not hazardous and Category 4 is only 2.5-fold
(2000mg/kg versus > 5000mg/kg). It will be
important to evaluate the precision of the
LD50 values in the databases, in order to find
out whether the data can support such distinctions. The in vitro test must also be able to
reliably distinguish between chemicals in adjacent classifications, and the workshop participants consider that the in vitro tests need a
narrower prediction interval than 1-log. A
narrower prediction range for the LD50 values
may be achievable by adjusting the predicted
LD50 value with bioavailability estimates
from additional tests.

31.

An objective, uniform process of selecting test


chemicals for learning and validation sets
should be used, that emphasises the need for
chemicals with the highest quality in vivo
data.

In vivo data/chemical selection


24.

Although several databases contain rat LD50


values, no single, currently available database
is an ideal source of in vivo data or is adequate
to serve as the in vivo benchmark for validation testing. Therefore, the existing databases
must be mined for chemicals with the most
accurate information for use as test articles
and to maximise the chances of success for validation studies. The basis for acceptance or
rejection of chemicals for studies should be
clearly documented.

25.

The accuracy of the in vivo data (LD50 values)


should be evaluated. A definition is needed of
how the reliability of the LD50 data should be
judged.

26.

Chemicals exhibiting significant inter-species


differences in potency will be outliers in the in
vitroin vivo correlation. There can be several
causes of inter-species differences, and specialised in vitro tests for chemical toxicity to
normal cells (for example, the validated CFUGM assay) can be used to translate across
species.

27.

All classes of chemicals should be represented


(GHS Categories 15), taking into account
diverse physicochemical properties.

28.

Acute toxicity testing is required for drugs,


pesticides and biocides, so appropriate examples of these types of chemicals/products
should be included in the learning and validation sets for testing.

29.

Pharmaceutical chemicals, especially anticancer drugs that are routinely and ethically
tested in animals and humans at toxic doses,

455

Read-across
32.

Currently, two assays (toxin acute oral test


and a genotoxicity test) are used to underpin
the validity of the read-across (i.e. if similar
results are obtained, it is assumed that the
substances are indeed similar). It is recommended that in vitro cytotoxicity data should
be included in the anchoring set, to complement the in vivo data. In the future, it should
be considered whether in vitro data alone
would be sufficient to anchor the data. The
workshop participants recommend that cytotoxicity data should also be submitted with a
read-across package, to complement the current data set. The ultimate aim would be for in
vitro data to fully support the read-across,
along with the genotoxicity test data (i.e. no in
vivo test would be necessary).

33.

Policies should be developed to resolve any


confidentiality concerns that limit the contri-

A. Gennari et al.

456

bution and significance of industry data to validation efforts.


34.

35.

36.

37.

Criteria used for the inclusion and quality


estimation of toxicity data should accompany
any sets of animal or human data used in the
validation process. This includes data sets that
are used as the basis for any computational
methods.
Consideration should be given to the inclusion
of in vitro measurements along with physicochemical parameters.
QSAR systems may be useful for suggesting
the appropriate cell or tissue biomarkers (perhaps measuring functional activity) to be used
to supplement the basal cytotoxicity information for specific chemicals.
The full data set should always be considered
with read-across (i.e. toxicity data, physicochemical data, toxicokinetics, impurity profiles, etc., so that a fully informed decision can
be made on the validity of the read-across).

Concluding Remarks
The main outcome of the workshop is the suggestion that an integrated testing strategy be developed for the replacement of in vivo acute toxicity
testing, based on the use of physicochemical data, in
vitroin vivo data, computational methods, basal
cytotoxicity assays, and complementary assays (for
metabolism, transport, kinetics and target organ
toxicity). This represents the important issues that
contribute to acute systemic toxicity and hazard
classification. When integrated, the information
will be the basis for a good prediction model to estimate LD50 values that could progress to the validation process.

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