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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.

Regulatory context

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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 (Regulation (EC) No 1107/2009), cosmetic ingredients (SCCS notes of guidance 2016, SCCS/1564/15). Acute toxicity requirement for pharmaceuticals is now largely historic (ICH M3(R2) guideline).

The following in vivo methods have received regulatory approval:

  • Acute oral toxicity - Fixed Dose Procedure: OECD test guideline 420
  • Acute oral toxicity - acute toxic class method: OECD test guideline 423
  • Acute oral toxicity - up-and-down procedure: OECD test guideline 425
  • Acute dermal toxicity: OECD test guideline 402
  • Acute inhalation toxicity: OECD test guideline 403
  • Acute inhalation toxicity - acute toxic class method: OECD test guideline 436
  • Acute inhalation toxicity - fixed concentration procedure: OECD test guideline 433

The European Chemicals Agency (ECHA) has revised its guidance document on the conduct of acute systemic toxicity studies and has incorporated a Weight-of-Evidence (WoE) approach that gives the possibility to adapt the Annex VIII standard information requirement for an oral acute toxicity study in certain cases and instead use data from an oral repeated-dose toxicity study together with other pieces of information, such as results from in vitro cytotoxicity testing, physico-chemical properties, structural analysis and toxicokinetic assessment (ECHA, 2016).
The WoE adaptation proposed primarily applies to low toxicity substances (i.e. LD50 > 2000 mg/kg).

An OECD acute toxicity waiver guidance document has been published recently (OECD GD 237) and includes, among other criteria, the possibility to waive the acute oral toxicity study based on the results of an alternative test or test battery, if the LD50 is predicted to be greater than 2000 mg/kg.

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Method deletion

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Acute Oral Toxicity test, Lethal Dose (LD50).

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).

Remarks: The

ESAC Statement on the acute oral toxicity test
English
(16.49 KB - PDF)
Prenesi

reflects the concern about the delay in the process of deletion of the OECD Guideline 401.

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EURL ECVAM validated test methods

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

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

The international NICEATM/ECVAM validation study of the Neutral Red Uptake (NRU) basal cytotoxicity assay 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) and human (normal human epidermal keratinocyte) cells. The study involved 72 reference chemicals.

The peer review panel of the NICEATM/ECVAM joint 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:

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.

More information and all relevant documents are available here.[/collapse]

Test methods under validation

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Currently there are no test methods under validation.[/collapse]

Development and optimisation and alternative methods

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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 Global Harmonise System (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 kinetics, 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 summary of the main achievements and recommendations is available here. In addition, a special issue was published in 2013 in Toxicology In Vitro (Volume 27 (4):1347-1424) and includes most of the results obtained during the last year of the project.[/collapse]