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Section D
In Vitro Toxicity Tests: Genotoxicity
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In Vivo Toxicological Process
3
In Vitro Toxicological Process
4
In Vitro Tests
! Genotoxicity
- Structure/activity
- Mutagenicity (formation of mutations)
- Chromosomal damage
- DNA damage
! Cellular toxicity
- Various endpoints
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Genotoxicity/Mutagenicity
! Mutagens exist as—or are metabolically converted to—
reactive electrophiles
- Genotoxic DNA reactive metabolites
- Mutagens can “initiate” the carcinogenic process
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S/A Analysis for Genotoxicity/Mutagenicity
Potential
! Structure activity (S/A) relationships
- Prediction of potential genotoxicity/ mutagenicity
based on chemical structure
- Identifies potentially reactive sites on the chemical OR
- Sites on the chemical that can be biotransformed to
reactive sites
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S/A Analysis for Genotoxicity/Mutagenicity
Potential
! By their very nature,
one would expect S/A
relationships to be
overly sensitive
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In Vitro Tests for Mutagenicity
! Short-term cell-based tests for genotoxic/mutagenic
carcinogen identification
- Gene mutation, DNA damage assays
! ! Bacterial-Ames assay (histidine reversion assay
in Salmonella)
Various mammalian cell culture systems
(mutations, damage, DNA, chromosomes)
- Mammalian cell transformation assays
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In Vitro Cell-Based Tests for Mutagenicity in
Bacterial Cells
! Ames assay
- Depends on the ability of mutagenic chemicals to
cause mutations in mutant strains of Salmonella
typhimurium
- Basis of assay
! Mutation in gene encoding an enzyme in the
histidine biosynthesis pathway
- Bacterial cells require histidine for growth;
cannot grow in absence
! Assay detects reversion to histidine independence
caused by mutation
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Ames Assay
Salmonella that do not
synthesize histidine
Chemicals
Histidine-free media
Microsomal S9
preparation
Chemical is converted by
microsomal protein to a
reactive metabolism
Mutations can occur in bacterial genome. Bacteria that can
synthesize histidine form colonies in histidine-free media.
Source: adapted by CTLT from Casarett & Doull. (1996). Chapter 8.
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Ames Assay
Control (spontaneous
mutations)
Direct-acting
mutagen
Mutagen requiring
metabolic activation
–
S9
+S
9
Source: adapted by CTLT from Casarett & Doull. (1996). Chapter 8.
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Ames Assay: Spot Test
A. Spontaneous
B. Furylfuraminde
C. Aflatoxin B1
D. 2-Aminofluorene
Source: adapted by CTLT from McCann and Ames. (1977).
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Ames Assay Data
Ames Assay Data
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Ames Assay Data
The Range of Mutagenic Potency in the Salmonella/Microsome Test
1,2 Epoxybutane
0.006
1
Benzyl chloride
0.02
3
Methyl methanesulfonate
0.63
105
2-Naphthylamine
8.5
1,400
2-Acetyleaminoflourene
108
18,000
7,057
1,200,000
20,800
3,500,000
Aflatoxin B1
Furylfuramide
Source: adapted by CTLT from McCann et al. (1975); and McCann and Ames. (1977).
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Correlation of Mutagenicity and Carcinogenicity
! Conclusions
- Positive: Ames strongly suggests that compound will
be carcinogenic in rats and/or mice
- Negative: Ames does not imply that compound will not
be carcinogenic in rats and/or mice (Zeiger, 1987)
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