Transcript Genotoxicity

Prof. David Kirkland
Kirkland Consulting
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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
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In such cases, unnecessary animal use occurs
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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
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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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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
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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.
Chemical
Lowest +ve concs.
Tert-butylhydroquinone
Lowest +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
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 >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 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)