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Abstracts / Toxic

mportance of understanding the maturation of enzymeystems involved in the processes of metabolic activationn the liver of young rats. Although the consensus of theroup was that the more information with regard to theetabolic capabilities of young rats would be useful, the

ublished literature shows that young rats have sufficientetabolic capacity for the purposes of this assay. The use

f young rats as a model for detecting MN induction inhe liver offers a good alternative methodology to the usef partial hepatectomy or mitogenic stimulation. Addi-ional data obtained from colon and skin MN modelsave integrated the databases, enhancing confidence inhe utility of these models.

A fourth topic discussed by the working group washe regulatory acceptance of the single-dose-level assay.here was no consensus regarding the acceptability ofsingle dose level protocol when dose-limiting toxicityccurs. The use of a single dose level can lead to prob-ems in data interpretation or to the loss of animals dueo unexpected toxicity, making it necessary to repeat thetudy with additional doses. A limit test at a single doseevel is currently accepted when toxicity is not dose-imiting.

eference

ayashi, M., MacGregor, J.T., Gatehouse, D.G., Adler, I.-D., Blakey,D.H., Dertinger, S.D., Krishna, G., Morita, T., Russo, A., Sutou,S., 2000. Environ. Mol. Mutagen. 35 (3), 234–252.

oi:10.1016/j.tox.2006.05.053

ourth international workgroup on genotoxicity test-ng: Results of the Comet assay workgroup

rian Burlinson

Huntingdon Life Sciences, UK

-mail address: burlinsb@ukorg.huntingdon.com

As part of the ongoing IWGT initiative, a series oforkshops were held as part of the ICEM/EMS joint

onference in San Francisco, September 2005. The mainurpose of this workshop was to enhance the ability ofhe Comet assay to be used for regulatory decision mak-ng with the primary focus being the rodent alkaline (pH13) Comet assay as a replacement/alternative for the inivo unscheduled DNA synthesis (UDS) assay. Topicsnd conclusions were:

. Number of dose levelsIn situations where evident toxicity is not present,

i.e. maximum dose level of 2 g/kg is reached, can the

6 (2006) 12–77 35

limit dose only be tested? The group decision wasno, because downturns in response exist (bell-shapeddose-response curve). Also, positive responses atmultiple dose levels reinforce the biological relevanceof the result.

2. Does the method used to process tissues, i.e. isolationof nuclei or whole cells, make a difference?

The consensus was that there were not enoughdata to decide although data that were presented sug-gested that the issue was unlikely to be a problem.However, any international validation study shouldconsider both processing methods for different tissuesusing reference chemicals with diverse mechanismsof action and covering a range of potencies.

3. Do we need to include measures of cytotoxicity?The consensus was “yes” and suggested methods

included dye exclusion tests for membrane integrityand/or metabolic competency. Determining the fre-quency of “dead” cells (hedgehogs or ghost cells)by the neutral diffusion assay was also suggested asa useful measure of cytotoxicity. The final methodwas using histopathology to evaluate levels of necro-sis and apoptosis when results were positive. It waspointed out that there was a need to standardise waysto present histopathological findings.

4. Image analysis (IA) or manual scoringThe consensus was that IA is preferred but not

required. Hedgehogs (ghost or dead cells) should beexcluded from IA data collection although determin-ing their frequency may be useful for data interpre-tation. If IA is used then percentage of tail DNAappeared to be the most linearly related to dose andthe easiest to compare across studies and thereforeshould always be presented. Data on the distributionof migration among cells should also be presented.

5. Need to develop and include historical negative/positive control data

The minimal number of studies needed was notdefined but should be enough studies to demonstratethe stability of the negative/positive controls. Crite-ria for determining the acceptability of new studies,based on historical control data, should be devel-oped for each tissue by each lab. There was somediscussion on the background responses for nega-tive controls and there was a consensus that negativecontrols should exhibit measurable DNA migration.A suggestion was made that to detect crosslinkingagents a mean value of around 10–20% tail DNA is

needed.

6. Minimal reporting standardsIt was agreed that to ensure that all studies can

be independently evaluated a minimum reporting

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36 Abstracts / Toxic

standard for regulatory submissions and publicationswill be developed. This standard will be consistentwith OECD in vivo genetic toxicology test methodguidelines. Previous publications have covered someaspects of protocol design and reporting (Hartmannet al., 2003; Wiklund and Agurell, 2003).

References

Hartmann, A.A., Agurell, E., Beevers, C., Brendler-Schwaab, S.,Burlinson, B., Clay, P., Collins, A., Smith, A., Speit, G., Thybaud,V., Tice, R.R., 2003. Mutagenesis 18, 45–51.

Wiklund, S.J., Agurell, E., 2003. Mutagenesis 18, 167–175.

doi:10.1016/j.tox.2006.05.054

Oral AbstractshOGG1 is specific for detecting 8-oxoguanine in themodified comet assay

Catherine C. Smith, Mike O’Donovan, Elizabeth Mar-tin

Genetic Toxicology, Safety Assessment, AstraZeneca,Mereside, Alderley Park, Cheshire SK10 4TG, UK

E-mail address: Catherine.Smith@astrazeneca.com(C.C. Smith)

The specificity and sensitivity of the comet assaycan be enhanced by incubating lysed cells with lesion-specific endonucleases that recognise certain damagedbases. The European Standards Committee on Oxida-tive DNA Damage (ESCODD) recommended the useof formamidopyrimidine DNA-glycosylase (FPG) in thecomet assay to measure levels of oxidative damage,in particular 8-oxoguanine (Gedik and Collins, 2005).In the present study, the use of FPG was compared

with endonuclease III (ENDOIII) and human 8-hydroxy-guanine DNA-glycosylase (hOGG1) for detecting differ-ent types of DNA lesions.

Table 1The effect of FPG, ENDOIII and hOGG1 on TI following treatment with D(23 �mol/L) and EMS (2 mmol/L)

Treatment % Tail intensity

No enzyme +FPG

DMSO (6%) 3.7 ± 1.3 6.6Gamma (10 Gy) 15.0 ± 4.3 28.0KBrO3 (2.5 mmol/L) 2.1 ± 0.5 91.3MMS (23 �mol/L) 4.5 ± 2.5 94.0ENU (2 mmol/L) 14.6 ± 3.2 62.6

Mean ± S.D. from at least three independent experiments; *p < 0.001, one-sid

26 (2006) 12–77

Mouse lymphoma L5178Y tk+/− cells were treatedwith dimethylsulphoxide (DMSO) as a standard sol-vent; agents known to induce oxidative damage: gammairradiation and potassium bromate (KBrO3) and well-established alkylating agents: methyl methansulphonate(MMS) and ethylnitrosourea (ENU).

No increase in DNA break sites was seen withFPG, ENDOIII or hOGG1 following treatment with1–6% DMSO (Table 1). With gamma irradiation(1–10 Gy), there was a significant increase in breakswith all three enzymes (Table 1). Treatment with KBrO3(0.25–2.5 mmol/L), showed similar increases in breakswith both FPG and hOGG1 but increases in breaks withENDOIII were only seen at the highest concentration2.5 mmol/L (Table 1). Following treatment with MMSand ENU, there were significant increases in breaks withFPG and ENDOIII but, in contrast, there was no addi-tional damage with hOGG1 (Table 1).

The data indicate that FPG, ENDOIII and hOGG1are all able to detect oxidative DNA damage. However,FPG and ENDOIII also detect high levels of damage toDNA following alkylation and, therefore, are not spe-cific for oxidative DNA damage alone. hOGG1 does notdetect alkylation damage and, therefore, appears to bemore specific for oxidative DNA damage, in particular8-oxoguanine.

Acknowledgements

We would like to thank Andrew Collins for his kinddonation of the FPG and ENDOIII enzymes and DebbieGodwin and Karen Oldman (AstraZeneca) for perform-ing statistical analyses.

References

MSO (6%), gamma irradiation (10 Gy), KBrO3 (2.5 mmol/L), MMS

+ENDOIII +hOGG1

± 1.7 5.0 ± 1.2 3.9 ± 4.8± 7.1* 34.3 ± 7.2* 19.6 ± 4.3*

± 5.0* 42.1 ± 3.7* 89.8 ± 6.0*

± 2.3* 47.9 ± 11.7* 4.9 ± 1.2± 4.9* 61.8 ± 3.6* 19.2 ± 5.8

ed ANOVA.

Gedik, C.M., Collins, A., ESCODD (European Standards Committeeon Oxidative DNA Damage), 2005. FASEB J. 19, 82–84.

doi:10.1016/j.tox.2006.05.055