Documente Academic
Documente Profesional
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437
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
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.
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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.
ECVAM Workshop 50: strategies to replace in vivo acute systemic toxicity testing
439
Information required
10kg
100kg
1 tonne
and above
A. Gennari et al.
440
ECVAM Workshop 50: strategies to replace in vivo acute systemic toxicity testing
Information required
10kg
None
100kg
None
1 tonne
None
10 tonnes
and above
441
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
III
> 500 to 5000mg/kg
> 2 to 20mg/l
> 2000 to 20,000mg/kg
IV
> 5000mg/kg
> 20mg/l
> 20,000mg/kg
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Table 4: Labelling requirements for precautionary statements for substances with acute
toxicity hazards
Toxicity
category
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
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
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.
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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-
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Source
Notes
2030
10,600
1000s
4000
10,000
(100 into
humans)
1000s
Food
contaminants
5000
High production volume chemicals databases
(USA and EU)
Confidential information
reference 65.
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Advisory group
Regulators/industry
In vivo
database
Classes/
families
Databases
Data mining
Reproducibility
Interspecies predictivity
Human predictivity
Modelling
446
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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
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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
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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
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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)
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Susceptible function
Kidney
Transport, filtration
Liver
Metabolic transformation:
xenobiotics
nutrients
protein regulation and excretion
Neurotransmission,
structural integrity
Cardiovascular system
Lung/respiratory system
Membrane integrity,
gas exchange
Blood
Oxygen transport,
white cell production
Gastrointestinal tract
Transport:
hydration
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).
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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
Blood-forming capacity
mRNA production
Fluorescein diacetate
Intracellular structure
Cell communication
intracellular signalling
cellcell interaction (dieldrin, botulinum toxin,
acetylcholinesterase inhibitor)
[Ca2+] measurement
Gap-junction assays
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.
5.
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454
7.
8.
9.
10.
11.
12.
13.
14.
15.
Cytotoxicity studies
16.
17.
18.
19.
20.
21.
22.
ECVAM Workshop 50: strategies to replace in vivo acute systemic toxicity testing
Organ-specific toxicity
23.
30.
31.
25.
26.
27.
28.
29.
Pharmaceutical chemicals, especially anticancer drugs that are routinely and ethically
tested in animals and humans at toxic doses,
455
Read-across
32.
33.
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35.
36.
37.
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.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
ECVAM Workshop 50: strategies to replace in vivo acute systemic toxicity testing
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
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44.
45.
46.
47.
48.
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