ACuteTox News

The ACuteTox prevalidation finalized

During the Prevalidation phase, the selected test methods, presented in Table 1 below, were challenged under blind conditions with a new set of 32 test compounds. The ultimate aim was to evaluate how well different combination of the selected methods where able to classify the chemicals into the official acute oral toxicity categories (EU CLP and GHS). For this purpose the Classification and Regression Trees (CART), and the Random Forest model were used.

The strategy that uses the Random Forest model including 9 endpoints (CFU-GM/human cord blood cells assay; 3T3/NRU assay; Primary rat brain aggregates including the following 3 endpoints: lowest gene expression, HSP-32 expression, NF-H expression; SH-SY5Y cell line/Lowest EC value obtained in cytomic panel; HepG2 cell line/MMP; rat hepatocytes/MTT assay; whole blood/IL-1 release), gave the best correct classification rate (69.26%) and resulted in only 2 compounds with underestimated toxicity (Brucine, Paraquat), as compared to the official acute toxicity classification.

However, the results of the classification analysis showed that the incorporation of additional endpoints did not improve significantly the outcome of the 3T3/NRU cytotoxicity assay alone, and only compounds with oral LD50 > 2000 mg/kg (e.g. unclassified chemicals according to EU CLP classification system) are best classified.

These results lead us to question the scientific motivation for the current classification systems for acute oral toxicity and to suggest the revision of the GHS/EU CLP systems.


The ACuteTox partner Dr. Richard Clothier, receives the Bjorn Ekwall Memorial Award 2010 See In vitro news

 

The best performing in vitro tests for estimation of acute oral systemic toxicity have been selected for pre-validation

During 2009, a statistical analysis of all cytotoxicity data and organ-specific toxicity data was performed. The multivariate CART analysis revealed which in vitro assays were the best performing ones according to their reproducibility and reliability and most importantly, according to their potential to classify chemicals into the official acute oral toxicity classes (GHS and EU). The best performing assays (Table 1) will be challenged in the pre-validation study planned to take place during January-May 2010 with a new set of 33 coded chemicals. The final tiered testing strategy will be revealed after the retrospective analysis of its capacity to predict the official acute oral toxicity classes (GHS and EU). The impact of kinetic factors such as volume of distribution, protein binding, clearance, and oral absorption, together with estimated passage over the blood-brain barrier, will also be integrated in the final prediction model.

Table 1. The best performing in vitro assays, which were selected as candidates for the final tiered testing strategy, will be further evaluated in the pre-validation phase.

 

Selected assay

Target (workpackage involved)

The neutral red uptake assay using  the3T3 fibroblast cell line (3T3/NRU)

General basal cytotoxicity (WP2)

The cytokine release assay using human whole blood (IL-1, IL-6, TNF-alpha)

Haemotoxicity (WP 4)

Cell differentiation in human cord blood-derived cells (CBC/CFU-GM)

Haemotoxicity (WP 4)

Gene expression (GFAP, HSP-32, MBP and NF-H) in primary rat brain aggregate cultures

Neurotoxicity (WP 7.1)

Uridine incorporation measuring the total mRNA synthesis in primary rat brain aggregate cultures

Neurotoxicity (WP 7.1)

Cytomic panel measuring oxidative stress (intracellular peroxidative activity, intracellular levels of superoxide anion, oxidized DNA base 8-oxoguanine) in HepG2, SH-SY5Y and A.704 cells

New endpoints (WP 4)

Cytomic panel for cytotoxicity screening (intracellular Ca2+ levels, mitochondrial membrane potential, plasma membrane potential) in HepG2, SH-SY5Y and A.704 cells

New endpoints (WP 4)

The MTT assay using primary rat hepatocytes

Metabolism (WP 6)

Kinetic parameters: volume of distribution, protein binding, clearance, and oral absorption (Caco-2 cells) for the estimation of the oral dose from the effective concentration observed in vitro

Biokinetics (WP5)

The estimation of compound passage through the blood-brain barrier using neuronal networks (for neurotoxicity assays)

Biokinetics (WP5)