The 10 mM Debate

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The 10 mM Debate

• Prof. David Kirkland
• Kirkland Consulting

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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

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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 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?

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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.

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7/19 Misleading positives at lt1 mM
Chemical Lowest ve concs.
Tert-butylhydroquinone Lowest ve conc. in CA test not identified, but lt100 µg/ml, i.e. lt1 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 gtGEF ve CA gt5 cells with abs
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12/19 Misleading positives at gt1 mM
Chemical Lowest 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 gt10 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 sulfonate Equivocal 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)
Sulfisoxazole Weak ve in MLA at 1000 µg/ml (3.75 mM)