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Prof. David Kirkland Kirkland Consulting. For many, the frequency of misleading positive results in vitro is unacceptable Where follow-up testing in vivo.

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Presentation on theme: "Prof. David Kirkland Kirkland Consulting. For many, the frequency of misleading positive results in vitro is unacceptable Where follow-up testing in vivo."— Presentation transcript:

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 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 mM Ethyl acrylate+ve in MLA at 20 µg/ml (0.2 mM) Eugenol+ve in MLA at 120 nl/ml (approx 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 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 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)


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