Estimating Acute Toxicity Estimate (ATE)
to animal welfare legislation in Europe, it is illegal for contract testing organisations to administer a chemical to an animal
where it is known to cause distress. Dose levels for testing must be set at levels that do not cause distress
to animals; it is obviously impossible to know what level of stress the animals are under throughout the whole procedures,
but testing facilities have a legal duty to minimise this.
are harmed and the outcome of many animal studies will include death and severe clinical signs, especially to the limited
numbers of animals used in preliminary or screening studies. However, performing LD50 studies
has been outlawed in Europe for many years; by definition, to find a dose or concentration causing an estimated 50% mortality,
it is necessary to deliberately administer dose levels above thresholds reasonably expected to cause mortality and other severe
In Europe, the end-point for short-term single dose studies is the ‘discriminating dose’.
This is the highest concentration administered that has not caused significant effects to the animals.
In repeat-dose studies, this is known as the No Observed Adverse Effect Level (NOAEL).
LD50 is still permitted in parts of the World with less regard to animal welfare and to allow harmonisation under
GHS, CLP describes the ‘Acute Toxicity Estimate’ as a way of converting discriminating dose to the equivalent
of an LD50 for purposes of classification. This mechanism for conversion was also in place under
earlier EU Directives.
The use of the Acute Toxicity Estimate for classification is described in Annex I of CLP, Table 3.1.1.
For example, Table 3.1.1 suggests that if the Acute Toxicity (Range) Estimate is 300 – 2000 mg/kg, then Category
4 applies. This means that the discriminating dose is > 300 mg/kg and LD50 is < 2000 mg/kg.
For mixtures, it get more confusing, especially if only the Categories for classification are provided and it is
not known whether this is based on a ‘point’ ATE (eg LD50) or ‘range’ ATE (discriminating
dose). Table 3.1.2 tries to remedy this and if the Category is known, then a fixed ATE can
be used for calculation.
For example, a Category 4 acute oral substance with ATE (range) 300
– 2000 mg/kg can be assumed to have a ‘point’ ATE of 500 mg/kg. Therefore, if diluted
to < 25%, the calculated ATE based on simple dilution is 2000 mg/kg and is therefore outside the 300 – 2000 range
for classification as Cat 4. Note that this would then fall into Cat 5, if being used (early indications
suggest that Cat 5 will be used by many international suppliers, even though not part of European CLP).
simple process of dilution to remove or reduce hazard needs to be considered with caution and interaction between components
must be understood or estimated on a case-by-case basis.
affecting toxicity of mixtures
Simple dilution and additivity of effects will
often be valid, especially if mixing in water or with truly inert materials. However, even non-hazardous
materials can interact with other components and mixtures can be more or less hazardous than estimates.
Some key properties to be aware of when predicting if simple dilution or addition is appropriate are outlined below
and in most cases are the result of enhancing a substance through formulation to change the physico-chemical properties:
Change in physical form
(eg solid dissolved in liquid)
· Surface tension reduction (use of surfactants as wetting and penetration
· Solvents (increased fat solubility and penetration)
Emulsions (fatty materials
dispersed finely in water)
There are many cases of two or more substances not individually classified
as hazardous being mixed together to form a hazardous mixture; in the US, it is normal to test mixtures and not the single
components and this is perhaps a better option for good assessment, although it will require the use of more laboratory animals
as each new formulation would need testing.
In-vitro skin and eye irritation can be performed
on mixtures in Europe, but this does not always pick up synergistic or antagonistic effects seen in biological systems.
However, as indicated above, testing mixtures on animals that contain substances known to be hazardous and likely to
harm the animal is not permitted in Europe.
STOTs / CMRs etc
Dilution factors for acute toxicity, corrosivity
and irritation and aquatic toxicity are easy to justify and the effects are generally reduced on dilution.
For substances that do not have clearly defined no-effect levels (for example, mutagens, carcinogens, sensitisers
etc), then it is a bit of a guess to determine the concentration of no concern or establish a classification limit.
In practice, it is necessary to establish threshold limits to allow
minor traces of substances of concern to be ignored. These are defined in Annex I of CLP and two limits
are described; one is the “cut-off value” (i.e. the limit of concern) to identify the component and declare as
part of the content (whether impurity or deliberately present) and the second is the “concentration limit” (the
higher limit above which classification is triggered).
Labels and safety data sheets need to identify any dangerous chemicals
present within prescribed concentration limits. For substances, this is a simple requirement to provide
the chemical name of the substance itself if classified, but for mixtures containing diluted substances, limits are set at
which the content needs to be declared.
There are therefore two concentration limits to
consider; one is the concentration that will result in classification and the other is the generic cut-off value; in CLP,
this is described in Annex I. Table 1.1 is particularly useful for short term toxicity, irritant or environmental
toxicity. For CMRs, STOTs and sensitisers, the limit for classification is also thecut-off value (typically
0.1 % or 1%)
In the generic guide below, the % for ‘concern’ (taken
in part from Table 1.1 in Annex I) and gives the concentration limit that dictates whether the component needs identifying
on the label and SDS and if it is ‘contributing to potential hazard’. If these limits are exceeded,
an SDS will need to be provided on request, even if the concentrations are below thresholds for classification
Category of Danger
Acute toxicity 1 – 3
1 or 2
Skin corrosion 1
or respiratory sensitiser
CMR category 1a, 1b, 2
Aquatic chronic 1
Aquatic chronic 2
are difficult and the No Observed Adverse Effect Levels must be considered in relation to classification limits
The CLP legal text does note that these are generic,
where no other factors are known that could impact on the safety of these limits.
If a substance is classified Aquatic Acute 1 or Aquatic Chronic 1with
acute EC50 < 1mg/l, a multiplication factor is added to aid classification of diluted mixtures.
Very simply, if the EC50 is 0.01 – 0.1 % the M factor is 10 and the level of concern is 10 times lower
(ie. limit of concern becomes 0.01%). If the actual EC50 and no-effect concentrations are known
for a substance and the mixture is a simple dilution in water, M factors are not required and direct effect of dilution can
be determined from real test data.
factors for classification in CLP
For each classification end point described in CLP, limits for classification
are defined. In some cases, such as acute toxicity or irritation, limits can be set based on estimated
effects of dilution, but for other endpoints such as flammability and aspiration toxicity (viscosity), there is often little
option than to assess the properties of the mixture.
The table below simplifies some
of the key classification endpoints described in the CLP Regulation. However, at the most basic level,
consider these as dilution in water or inert material where there is no real chance of interaction between the components.
If mixing with other hazardous substances or substances that could change biological properties, extra care is needed.
Category of Danger
Concentration limit for classification
Acute oral toxicity 1*
Acute oral toxicity 2*
Acute oral toxicity 3*
Acute oral toxicity 4*
(but needs SDS on request at 1%)
Skin corrosion 1
5% (becomes Cat 2 skin)
Skin corrosion 1
(becomes Cat 1 eye)
1% (no classification)
Skin irritation 2
Skin or respiratory sensitiser
1% (but needs SDS on request at 0.1%)
CM Cat 1a, 1b
R Cat 1a and 1b
0.3% (but needs SDS on request at 0.1%)
CM Cat 2
(but needs SDS on request at 0.1%)
3% (but needs SDS on request at 0.1%)
Aquatic acute 1
Aquatic chronic 2
Aquatic chronic 3
Aquatic chronic 4
* Based on ATE
point estimate in Table 3.1.2,
** Note that if below limit of concern of 0.1% for Cat 1, the legal text implies that substances can be ignored
unless it is known to be of concern. Rather vague !
*** Consider on case-by-case, especially if potential vPvB or PBT.
**** Note M factor
that the text of the CLP Regulation covers this in detail and this is a summary of limited endpoints.
It is not possible
to apply simple factors to physico-chemical properties to estimate mixtures and as testing does not involve animals, there
is no legislation to prevent testing of mixtures, indeed, CLP obliges testing for physical hazard endpoints if no suitable
data is available on which to base classification.
Obviously, if none of the components have hazardous
properties, these can be ignored (for example, if none are considered oxidising, the mixture will not have oxidising properties).
Many endpoints can be estimated; diluting ethanol to 4% in water is likely to make the mixture non-flammable (eg beer).
Some properties such as vapour pressure and partition coefficient may not necessarily be removed on mixing and estimation
of interaction is difficult.
some help is provided with dilution factors and lower limits of concern, there is a need for expert judgment in classifying
mixtures and many software tools need to be used with caution.