5 TH INTERNATIONAL WORKSHOP
ON GENOTOXICITY TESTING
Basel, August 17-19, 2009
1

Chair
Co-Chair
Sheila Galloway
Elisabeth Lorge
Rapporteur Marilyn Aardema
Working group members:
David Eastmond, Mick Fellows, Bob Heflich,
David Kirkland, Dan Levy, Anthony Lynch,
Daniel Marzin, Takeshi Morita, Maik
Schuler, Günter Speit
2

Appropriate measures of cytotoxicity for in vitro cytogenetic assays
(chromosome aberrations and micronucleus)
• Requirements: Balance
• Excessive toxicity could give false negative because cells are delayed
• Excessive toxicity can give positive results that only occur associated with toxicity, not at lower doses
• Metaphase assays
– Cells must reach metaphase: Allow time for cell cycle delay
• Micronucleus assays
– Nucleus must divide for production of micronuclei
3

What are appropriate cytotoxicity measures for in vitro cytogenetics assays?
Measure
Mitotic Index
Term
(cf control)
RMI
Chrom
Abs
Micronucleus

Evidence of proliferation
Yes
Binucleate cell index with cytocholasin B
Population doubling
Increase in cell counts
Cell counts
CBPI/
RI
RPD
RICC
RCC







Yes
Yes
Yes
NO
4

Example of need for measure based on proliferation
Culture cell number at treatment start
(million)
Ending cell number
Control 0.5
1.0
Relative population doubling/ increase in cell count
-
Relative cell counts
-
Treated 0.5
0.5
0 50% **
**Relative cell counts underestimate toxicity;
NO NET CELL GROWTH
5

Appropriate measures of cytotoxicity for in vitro cytogenetic assays
(chromosome aberrations and micronucleus)
Data were reviewed from in vitro micronucleus collaborative studies that assessed;
– Multiple toxicity measures concurrently
– Multiple cell types
– 14 clastogens and aneugens of various chemical classes/mechanisms, including weak genotoxins
• Studies followed draft OECD guideline (TG487) for in vitro micronucleus assay
• The question: What are the preferred cytotoxicity measures?
6

Cytotoxicity measures for chromosome aberration and micronucleus assays in vitro
• Known Positive Chemicals:
– Cell type and toxicity measure affected concentrations selected
– Despite this, use of all the measures allowed detection of all the positive chemicals tested
(both weak and potent chemicals) in the TG487 validation exercise in all the cell types
– RICC; RPD; CBPI/RI; RCC
• Group preference for measures based on proliferation during the treatment (RICC, RPD,
CBPI/RI)
– Ongoing data collection/review on “misleading positives” (positives associated with toxicity, i.e., positive results with non-mutagenic/non carcinogens) may support dropping RCC
7

Cyto B has advantage that cells selected for scoring have undergone nuclear division
• This study reaffirmed that when increases in MN in mono-nucleated cells are noted they must be scored
• Caution with Cyto B*:
– Cells with asymmetrical multinucleated morphology complicate scoring and indicate need to repeat assay without cyto B
• e.g., seen with phenolphthalein; also known with aneugens such as taxol, colchicine, vincristine
• Recommendation that if equivocal results are found in the above two scenarios, a repeat test without Cyto B is needed
* Also note that cyto B can increase toxicity of test chemical 8

• In this dataset, all chemicals were detected without exceeding 50% toxicity, (50% reduction in growth), but some were quite close to 50%
– The group agreed that the target range in the draft
OECD in vitro micronucleus guideline is appropriate:
55 % +/- 5 %
• Dose spacing
– Good study design may require closely spaced concentrations (much closer than half-log dilutions) particularly for aneugens, nucleoside analogues and others with steep toxicity dose relations
9

Appropriate top concentration for in vitro cytogenetic assays
• Is the change from 10 to 1 mM that has been recommended for pharmaceuticals acceptable for testing for all types of product development?
• If not, can we identify exceptions where higher/lower top concentrations than 1 mM might be recommended?
• Chemical class
• Type/use of product?
10

Information on top concentration
• ECVAM analysis of rodent carcinogens from Gold database
– Least effective concentration for positive results in in vitro mammalian cell assays; correlation with Ames test and rodent carcinogenicity results
– Analysis of the compounds positive only between 1 and 10 mM
• For Ames test negative compounds: (about 20)
• Are they “compounds of concern”, in vivo genotoxins/genotoxic carcinogens?
• Pharmaceutical industry rationale for 1 mM limit
– Industry survey found most drug candidates positive above 1 mM had evidence for lack of in vivo relevance (weight of evidence, in vivo negative genotoxicity)
– Biological relevance (e.g., K m for metabolic enzymes)
– Consideration of human exposure levels to pharmaceuticals
11

• Additional data considered:
– Data from Japan on high production volume chemicals:
• in vitro chromosome aberration data from 1994 –
2006 (later assays followed OECD guideline)
• 45% of 249 chemicals positive
– 37 Ames negative, pos >1 to 10 mM
– 20 had high toxicity, altered pH, neg in vivo (all of 5 tested), polyploidy only
– 17 of the 113 positives remain
– NIHS (Japan) support for 1 mM limit
12

Comments from working group 1a on top concentration limit
Consensus that there are reasons to reduce the 10 mM limit
•Based on data examined, for screening the majority of chemicals, 1 mM is appropriate.
•Discussion of possible exceptions……
13

Examples discussed of chemicals detected at >1 mM
•Some were recognized to reflect
• Inadequate metabolism; e.g., Cyp2E1 required
• Volatility; e.g., low molecular weight chemicals
• These illustrate need for improved test design, not high test concentration limit
• Specific chemical classes e.g., aldehydes; others?
• Complex mixtures: mM not practical
•Test to limit of cytotoxicity, max solubility etc
• Discussion incomplete……….
14

Overall summary for in vitro chromosome aberration and micronucleus assays
Toxicity:
• Observations:
– All the measures allowed detection of all the positive chemicals tested in the micronucleus assay: RICC;
RPD; CBPI/RI; RCC
– In this dataset, all chemicals were detected without exceeding 50% toxicity, (50% reduction in growth), but some were quite close to 50%
• Recommendations :
– Group preference for measures based on proliferation during the treatment (RICC, RPD,
CBPI/RI) for aberration and micronucleus assays
– No re-examination of the OECD limit for micronucleus testing (55 +/- 5 %) was recommended
15

Overall summary for in vitro chromosome aberration and micronucleus assays
Concentration:
• Observation:
– In the data examined, the majority of chemicals were detected at

1 mM
• Recommendation:
– There are reasons to reduce 10 mM limit
– While a 1 mM limit can be considered, exceptions need to be defined
16