1. Note lack of cytotoxicity
The in vitro chromosome aberration assay is very sensitive to chemical mutagens
The chromosome aberration assay is a sensitive test for real DNA damaging agents. We spend a lot of time working with compounds that are negative in other genetox tests but positive in the in vitro chromosome aberration assay, commonly at high doses or associated with cytotoxicity, but we should not forget that the assay does its job of screening out the real “bad actors” . It is notable that I have listed the substantial amount of aberration induction at doses that have only a minor effect on cell growth at the time of sampling for aberration analysis, the day after treatment. If assayed later, by colony forming ability for example, there would be marked cytotoxicity, but there is little immediate toxicity during the time of the aberration assay, with these true clastogens.
Note lack of cytotoxicity

2. Chromosome aberrations in vitro in CHO cells
With S-9
Without S-9
In contrast to the potent DNA damaging agents on the first slide, here is the first example of a drug, an angiotensin II receptor antagonist, that we developed using the strategy of examining the relationship of aberration to toxicity and to an indirect mechanism, DNA synthesis inhibitions. Note the high dose levels, closely spaced and the sudden increase in aberrations from control levels at 2.5 mM to significant increases at 3 mM. Note that these increases occurred as we approached 50% cytotoxicity (cell counts at the time of sampling for aberrations). At that time we were also measuring at mitotic index after treatment and again at the time of sapling for aberrations, and we saw great mitotic suppression followed by recovery. That was one of the things that led us to follow up on our experience with inhibitors of DNA synthesis that caused aberrations.
Numbers on top of bars are cell counts as % control.

3. Negative genotoxicity assays of Angiotensin II receptor antagonist and analogs
Microbial mutagenicity in Salmonella and E.Coli
Alkaline elution (DNA strand breaks) in rat hepatocytes
Mammalian cell mutagenicity; hprt in V79 cells
In vivo:
Chromosome abs in M & F mice (1500 mg/kg)
Alkaline elution in F rat liver (1978 mg/kg)
Additional negative tests on analogs:
DNA adducts in calf thymus DNA
‘phage DNA fragmentation
unscheduled DNA synthesis
All the other evidence was that the Angiotensin II receptor antagonist was not a DNA damaging compound. DNA strand breaks were induced at very toxic doses in hepatocytes, giving us another clue that aberration induction might be associated with cytotoxicity.

4. Inhibition of DNA synthesis by AIIRAs
Inhibition of DNA synthesis measured by uptake of bromodeoxyuridine in pulse for the last 30 minutes of a 3-hour treatment. The horizontal axis shows DNA content from G1 on the left through S phase to G2/M on the right. The height of the horseshoe shows the amount of BrdUrd uptake; this vertical axis is a log scale. You can see dramatic suppression of DNA synthesis by Angiotensin II receptor antagonist and two analogs, in the dose range that induced chromosome aberrations (thick arrows). Classical DNA damaging agents such as those in the first slide cause little or no suppression of DNA synthesis in this assay.

5. Significance of aberrations associated with cytotoxicity
Potential threshold
no structural alert
no mutation
no DNA binding
no aberrations in vivo
likely secondary to toxicity, not relevant at human exposure levels
In vitro positive at µg/ml
In vivo: mouse exposure at 500 mg/kg/d x8
C Max 74 µg/ml, AUC 90 µg/ml.hr
Human 40mg oral:
C Max 0.2 µg/ml, AUC 0.4 µg/ml.hr

6. Mechanisms of chromosome aberrations
Aberrations result from DNA strand breaks
Strand breaks induced by e.g.,
ionizing radiation
processing of lesions/adducts/abnormal bases
but also by
inhibition of DNA synthesis
inhibition of topoisomerases
Cytotoxicity
Mis-incorporation, altered bases
nucleoside analogs; ribonucleotide reductase inhibitors; folate antagonists
DNA synthesis inhibition; chain termination; mis-pairs, gaps;

7. Mechanisms of genotoxicity that may have threshold
Disruption of cell division
Disruption of chromosome segregation
Inhibition of DNA synthesis
Inhibition of topoisomerases
Nucleotide pool imbalance
Overloading of oxidative defence mechanisms
Metabolic overload (phase II enzymes etc)
Ion chelation; disturbance of metal homeostasis
Extremes of pH/osmolality
Cytotoxicity
(adapted from Henderson, Albertini & Aardema, Mutat Res 464, , 2000)

8. Possible mechanisms for chromosome aberration induction by nucleoside analogs
DNA synthesis inhibition
incorporation
chain termination
attempted repair of mispaired bases
pool imbalance
polymerase errors- attempted repair
Nucleoside analogs are a good example of how on one level one can treat compounds as a class (for example long treatments may be required to detect these in chromosome aberration assays) but for safety evaluation the individual characteristics of each analog should be examined.

9. Genetic Toxicology of Antivirals/Nucleosides
Note that even those that are positive in the micronucleus assay are not necessarily rodent carcinogens.
All are negative in AMES (no data on adenosine)
Wutzler and Thust Antiviral Research. 49, 55-74; Phillips et al, Env Molec Mutagen 18, , 2001

10. Risk evaluation for nucleoside analog
Specificity for viral vs mammalian enzyme
Is it incorporated into DNA? RNA?
If incorporated is it pro-mutagenic? (AZT)
If not incorporated: High dose effect.
Possible mechanisms
pool imbalance (try to restore)
DNA synthesis inhibition (demonstrate)
Genotoxicity may have threshold
in vivo cytogenetic assay negative
good safety margin (exposure)
May not be genotoxic risk to humans.
Nucleoside analogs that are incorporated and mutagenic like AZT are a potential risk even at low doses. (Mutations have been demonstrated in lymphocytes of people taking AZT and it is reportedly a transplacental carcinogen.) Those that are not incorporated may induce chromosome aberrations through pool imbalance and or DNA synthesis inhibition; those are amenable to experimental investigation. For example, one can reverse the effects of pool imbalance by supplying appropriate nucleosides.

11. ? Indirect DNA Synthesis
Strategy for Genotoxicity Follow-Up Testing for Unique in vitro Positive
In vitro Chromosome Aberrations +
Mechanistic Studies
Second tier
follow-up
? Indirect DNA Synthesis
Inhibition
Direct DNA Damage DNA adducts, or
neg Ames & Alk elut
Acute Mouse Bone Marrow Micronucleus
Human (TK6) Cell Mutation -
Measure Exposure
2nd in vivo
test if
exposure appropriate
yes
+ = No Go - -
Good Margin
This is the scheme we use to follow up a unique in vitro chromosome aberration positive results. Although a potential risk of genotoxicity may well be acceptable in a risk benefit assessment for treating disease, it is often preferable to do the first clinical trials in normal volunteers. Here there is no benefit associated with any risk, so we have to evaluate especially carefully any signals of genotoxicity for their potential relevance to people. Understanding as much as possible about the reason for in vitro genotoxicity helps determine whether it is likely to be relevant in vivo, and precludes doing complex or long term in vivo studies in animals. We do further studies to rule out potential for DNA damage (given a negative Ames test) such as a DNA strand break assay (we use the alkaline elution assay in rat hepatocytes) and/or a DNA adduct assay, usually 32P post-labeling. We also look for any evidence for an indirect mechanism, such as inhibition of DNA synthesis. We have demonstrated this effect with many non-mutagenic compounds that induce aberrations at high doses. The significance is that this mechanism will not operate at lower doses. Additional weight of evidence (as indicated in the ICH guidelines) may be obtained by an in vitro mammalian cell mutation assay (other than the mouse lymphoma cell assay), such as the tk locus mutation assay in human TK6 cells. The in vivo micronucleus test is done (one administration), and exposure is measured under the same conditions in mice. Finally, a second in vivo test has to be considered, as recommended in the ICH guidelines. There may be no value to a second in vivo test in liver, e.g., if the in vitro alkaline elution assay is negative and the in vitro chromosome aberration positive is not dependent on S-9 metabolic activation. However, in some cases one can achieve higher exposure in the liver in vivo than was achieved in vitro, and here an in vivo alkaline elution assay in liver is useful.
OK for single or multiple dose trials in normal volunteers.

12. Risk evaluation when mechanism is indirect
Chromosome aberrations may be induced only above a threshold dose.
Therefore if:
in vivo cytogenetic assay negative
second in vivo assay negative
good safety margin (exposure)
May not be genotoxic risk to humans.