1. Prof. David Kirkland Kirkland Consulting

2. For many, the frequency of misleading positive results in vitro is unacceptable Where follow-up testing in vivo is not permitted (e.g. cosmetics ingredients in EU) new products may be abandoned and businesses suffer For other chemicals in vivo follow-up may be triggered by in vitro +ve when not otherwise required Low tonnage industrial chemicals Food flavours and additives In such cases, unnecessary animal use occurs

3. ECVAM workshop (Kirkland et al, 2007) identified several design issues of mammalian cell tests thought to contribute to high level of misleading positives – including top conc.; extent & measures of cytotoxicity Others will be discussed in Stefan Pfuhlers group Proposed revisions to ICH S2 guideline for pharmaceuticals suggested lowering top conc. for non-toxic drugs to 1 mM (Lutz Müller will present) So are there data to suggest a reduction in the top conc. for non-pharmaceutical chemicals can also be considered?

4. ECVAM commissioned Jim and Liz Parry to review published literature At what concs. do carcinogens that are –ve in Ames test give detectable +ve responses in mammalian cells? Raffaella Corvi will discuss this review I will discuss some of the key chemicals from this review From the 19 chemicals identified (Kirkland et al, 2008) as giving misleading +ve results in mammalian cells, 12 are only +ve above 1 mM, and 11 are only +ve above 2 mM These would be avoided if lower top conc.

5. ChemicalLowest +ve concs. Tert-butylhydroquinoneLowest +ve conc. in CA test not identified, but <100 µg/ml, i.e. <1 mM 1,3-dihydroxybenzene+ve in CA test 20-80 µg/ml, i.e. from 0.18 mM upwards Curcumin+ve for MN at 10 µM Propyl gallate+ve in MLA at 1 µg/ml and in CA at 16 µg/ml, i.e. at 0.005-0.075 mM Ethyl acrylate+ve in MLA at 20 µg/ml (0.2 mM) Eugenol+ve in MLA at 120 nl/ml (approx. 0.73 mM) although only +ve for CA at 300 µg/ml (1.83 mM) 2,4-dichlorophenol+ve in MLA at 40 µg/ml (0.25 mM) and in CA at 0.6 mM +ve MLA = >GEF; +ve CA = >5% cells with abs

6. ChemicalLowest +ve concs. D,L-menthol+ve in CA at 1.6-1.9 mM Phthallic anyhydride+ve in CA at 10 mM o-Anthranilic acid+ve in MLA at 886 µg/ml (6.47 mM) 2-ethyl-1,3-hexanediol+ve in CA at 4000 µg/ml (27.4 mM) Ethionamide+ve in MLA at 500 µg/ml (3 mM) and in CA at 5-8 mM Benzyl alcohol+ve in MLA and CA at 30-40 mM Urea+ve in CA at >10 mM Sodium saccharin+ve in CA at 8000 µg/ml (33.2 mM) p-Nitrophenol=ve in CA at 1500 µg/ml (10.8 mM) Sodium xylene sulfonateEquivocal in MLA at 4000 µg/ml (19.1 mM) Isobutyraldehyde+ve in MLA at 250 µg/ml (3.47 mM) and in CA at 500 µg/ml (6.94 mM) SulfisoxazoleWeak +ve in MLA at 1000 µg/ml (3.75 mM)