Assessment of the predictive capacity of the 3T3 Neutral Red Uptake cytotoxicity test method to identify substances not classified for acute oral toxicity (LD 50 > 2000 mg/kg): Results of an ECVAM validation study Pilar Prieto a,⇑ , Thomas Cole a , Rodger Curren b , Rosemary M. Gibson c , Manfred Liebsch d , Hans Raabe b , Anita M. Tuomainen a , Maurice Whelan a , Agnieszka Kinsner-Ovaskainen a a Institute for Health and Consumer Protection, European Commission Joint Research Centre, 21027 Ispra, Italy b Institute for In Vitro Sciences, Inc., 30 W. Watkins Mill Road, Suite 100 Gaithersburg, MD 20878, USA c Health and Safety Laboratory, Harpur Hill, Buxton, SK17 9JN, UK d Federal Institute of Risk Assessment (BfR) Centre for Alternative Methods to Animal Experiments, Berlin (ZEBET), Berlin, Germany article info Article history: Available online 13 December 2012 Keywords: Acute oral toxicity Validation Cytotoxicity BALB/3T3 Neutral Red Uptake assay CLP Industrial chemicals abstract Assessing chemicals for acute oral toxicity is a standard information requirement of regulatory testing. However, animal testing is now prohibited in the cosmetics sector in Europe, and strongly discouraged for industrial chemicals. Building on the results of a previous international validation study, a follow up study was organised to assess if the 3T3 Neutral Red Uptake cytotoxicity assay could identify sub- stances not requiring classification as acute oral toxicants under the EU regulations. Fifty-six coded industrial chemicals were tested in three laboratories, each using one of the following protocols: the pre- viously validated protocol, an abbreviated version of the protocol and the protocol adapted for an auto- mation platform. Predictions were very similar among the three laboratories. The assay exhibited high sensitivity (92–96%) but relatively low specificity (40–44%). Three chemicals were under predicted. Assuming that most industrial chemicals are not likely to be acutely toxic, this test method could prove a valuable component of an integrated testing strategy, a read-across argument, or weight-of-evidence approach to identify non toxic chemicals (LD 50 > 2000 mg/kg). However, it is likely to under predict chemicals acting via specific mechanisms of action not captured by the 3T3 test system, or which first require biotransformation in vivo. Ó 2012 Elsevier Inc. All rights reserved. 1. Introduction Acute oral toxicity studies are performed mainly for classifica- tion and labelling in order to assign substances their potential haz- ard categories and estimate the dose required to cause toxicity (Creton et al., 2010; Seidle et al., 2010). In the EU, the Regulation on Classification, Labelling and Packaging (CLP) (European Com- mission, 2008) of substances and mixtures entered into force in 2009 to align the previous EU legislation Dangerous Substances Directive (Directive 67/548/EEC) and the Dangerous Preparations Directive (Directive 1999/45/EC), with the Globally Harmonised System of Classification and Labelling (GHS) developed under the auspices of the United Nations (UN, 2011), in order to increase con- sistency among diverse frameworks used in different jurisdictions and industrial sectors. Formerly, Annex I of Directive 67/548/EEC provided an official EC inventory of classified substances. The same inventory is currently available as Annex VI to Regulation 1272/ 2008, now transposed as a GHS version, where previous warning terms and risk (R) phrases have been replaced by updated classifi- cation categories and hazard (H) statements. According to the CLP Regulation, chemicals are allocated in one of four toxicity catego- ries based on their acute oral toxicity properties: category 1 (LD 50 6 5 mg/kg b.w.), category 2 (5 < LD 50 6 50 mg/kg b.w.), category 3 (50 < LD 50 6 300 mg/kg b.w.), and category 4 (300 < LD 50 6 2000 mg/kg b.w.). Under this EU CLP scheme, the limit dose above which chemicals are not required to have a hazard label for acute oral toxicity is 2000 mg/kg b.w. According to the cosmetics regulation (European Commission, 2009) it is prohibited in the EU to market cosmetic products and their ingredients that have been tested on animals for most of the human health effects, including acute toxicity. This imposes a great need for the cosmetic industry to have alternative approaches avail- able for safety testing of ingredients of consumer products. All the accepted methods for determining acute oral toxicity are based on in vivo experiments that estimate the LD 50 value (i.e. the single dose of a substance that can be expected to cause death in 0273-2300/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.yrtph.2012.11.013 ⇑ Corresponding author. Address: Institute for Health and Consumer Protection (IHCP), The European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM), European Commission Joint Research Centre, TP 580, Via Fermi 2749, 21027 Ispra, VA, Italy. Fax: +39 0332 785336. E-mail address: maria.prieto-pilar@jrc.ec.europa.eu (P. Prieto). Regulatory Toxicology and Pharmacology 65 (2013) 344–365 Contents lists available at SciVerse ScienceDirect Regulatory Toxicology and Pharmacology journal homepage: www.elsevier.com/locate/yrtph