Acute Toxicity

     1. ECVAM validated test methods

     2. Test methods under validation by ECVAM

     3. Development and optimisation of alternative methods

     4. Workshop reports and relevant publications





Acute systemic toxicity testing involves an assessment of the general toxic effects of a single dose or multiple doses of a chemical or product, within 24 hours by a particular route (oral, dermal, inhalation), and that occur during a subsequent 21-day observation period.

Acute toxicity data are common requirements under many regulatory frameworks to provide classification and labelling warning or the possible consequence of exposure to a chemical.


Substances that require classification and labelling include industrial chemicals (REACH Annex VII and VIII), biocides (REGULATION (EU) No 528/2012), pesticides (link to REGULATION (EC) No 1107/2009), cosmetic ingredients (SCCS notes of guidance, 2010). Acute toxicity requirement for pharmaceuticals is now largely historic (ICH M3(R2) guideline).


Test method


(EC) No 440/2008


Test Method

OECD Test Guideline


in vitro/in vivo

Acute Oral Toxicity - Fixed Dose Procedure

B.1 bis

TG 420

Evident toxicity

in vivo

Acute Oral toxicity - Acute Toxic Class Method

B.1 tris

TG 423


in vivo

Acute Oral Toxicity: Up-and-Down Procedure


TG 425


in vivo

Acute Dermal Toxicity


TG 402


in vivo

Acute Inhalation Toxicity


TG 403


in vivo

Acute Inhalation Toxicity - Acute Toxic Class Method


TG 436


in vivo



Method Deletion

  • Acute Oral Toxicity test, Lethal Dose (LD50)
    Date of the ESAC opinion: 21 March 2000
    Remarks: The ESAC statement reflects the concern about the delay in the process of deletion of the Guideline 401.
    Status: The method has been deleted in 2001 from both the Annex V of Council Directive 67/548/EEC (Method B.1.), as well as from the OECD Test Guidelines (TG 401).

    Related Documents:



Alternative test methods and approaches


1. ECVAM validated test methods

1.1 The Colony Forming Unit-Granulocyte/Macrophage (CFU-GM) Assay for predicting acute neutropenia in humans

1.2. The neutral red uptake in vitro cytotoxicity tests to estimate starting doses for acute oral systemic toxicity tests

The international The NICEATM/ECVAM In Vitro Cytotoxicity Validation Study generated in vitro cytotoxicity data to predict rodent in vivo LD50 values and starting doses for acute oral systemic toxicity test methods. The in vitro tests evaluated used rodent (mouse fibroblast [3T3]) and human (normal human epidermal keratinocyte [NHK]) cells. The study involved 72 reference chemicals. The peer review panel of the NICEATM/ECVAM validation study concluded that the NRU basal cytotoxicity assay may be useful in a weight-of-evidence approach to determine the starting dose for acute oral in vivo toxicity protocols. The results of this study also showed that the overall accuracy of the NRU based cytotoxicity assay in BALB/3T3 cells and NHK for correctly predicting each of the GHS acute oral toxicity classification categories was low (31% and 29%, respectively).

Available here:


1.3 The 3T3 Neutral Red Uptake (3T3 NRU) Cytotoxicity assay for the identification of substances not requiring classification for acute oral toxicity (LD50 > 2000 mg/kg b.w.) according to the EU CLP system


This ECVAM follow-up study aimed to evaluate the capacity of the 3T3 NRU cytotoxicity assay to identify specifically substances considered "negative" in terms of acute systemic oral toxicity. These are substances not requiring classification for acute oral toxicity. According to the legal provisions in the EU laid out in the EU Classification, Labelling and Packaging (CLP) Regulation (EU 2008) which implements the UN Globally Harmonised System (GHS), all substances with LD50 values greater than 2000 mg per kg of body weight fall into this category "non-classified".

The study was completed in 2011 and the ECVAM Scientific Advisory Committee (ESAC), at its meeting on 22 March 2011, provided the scientific advice on the study in the form of an ESAC Opinion. EURL-ECVAM recommendation has been drafted and underwent public consultation.

All relevant documents are available in the EURL ECVAM Recommendations section.


2. Test methods under validation

Currently there are no test methods under validation.

3. Development and optimisation and alternative methods

EU FP6 ACuteTox project Optimisation and pre-validation of an in vitro test strategy for predicting human acute toxicity (EU contract no.LSHB-CT-2004-512051)

The ACuteTox project represented the first attempt to create an integrated testing strategy based solely on in vitro and in silico methods, with the ultimate purpose of replacing animal testing for predicting human acute oral systemic toxicity and classification of chemicals into the different EU Classification, Labelling and Packaging (CLP) and GHS toxicity classes. The main objectives of the project included the compilation, evaluation and generation of high quality in vitro and in vivo data for comparative analyses and the identification of factors that influence the correlation between in vitro (concentration) and in vivo (dose) toxicity, particularly taking into consideration biokinetics, metabolism and organ toxicity (liver, central nervous system, kidney). Moreover, innovative tools (e.g. cytomics) and new cellular systems for anticipating animal and human toxicity were explored. Ultimately, the goal was to design a simple, robust and reliable in vitro test strategy amendable for robotic testing, associated with the prediction models for acute oral toxicity.


EURL ECVAM was involved in the Steering Committee and the Management Board of the project and was also leading activities in several work packages, such as the compilation of in vivo data, setting up the project database as a useful tool for a quality controlled transfer and organisation of large in vitro and in vivo toxicological data sets, and the pre-validation of the testing strategy. A special issue in Toxicology In Vitro is in press and includes most of the results obtained during the last year of the project (see relevant publication section).



4. Workshop reports and relevant publications