The 10 mM Debate

1. The 10 mM Debate Prof. David Kirkland Kirkland Consulting

2. Why are we debating this issue? • 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. Why this debate? • 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 Pfuhler’s 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. Review of published data • 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. 7/19 Misleading positives at <1 mM +ve MLA = >GEF; +ve CA = >5% cells with abs

6. 12/19 Misleading positives at >1 mM