E L S E V I E R Mutation Research 308 (1994) 243-244

Fundamental and Molecular Mechanisms of Mutagenesis

Series: Current Issues in Mutagenesis and Carcinogenesis, No. 50

Regulatory requirement for three dose levels and proof of tissue localisation in rodent genetic toxicity assays - a call

for supporting data

John Ashby * Zeneca Central Toxicology Laboratory, Alderley Park, Macclesfield SKIO 477, UK

Accepted 27 April 1994

Key words: OECD mutagenicity guideline; Call for data

The current OECD mutagenicity guideline re- view is presently at the stage of discussion. One of the current proposals is the use of three dose levels in rodent genetic toxicity tests (e.g. mouse bone marrow micronucleus (MN) and rat liver UDS tests). A related issue under discussion in some regulatory circles is the requirement for proof that the test chemical has ' reached' a tissue in order that a negative result can be considered to be of any value.

The above two protocol requirements will in- volve a substantial increase in the amount of work and the number of animals needed to pro- duce an assay response that is acceptable to regu- latory authorities. It is therefore appropriate that the data supporting these extra requirements are made available for scrutiny and comment. I am not aware of data supporting the need for the use of three dose levels, and I do not know how to prove that a chemical has reached a tissue, short of the conduct of a full pharmacodynamic analy-

* Corresponding author.

sis. Further, if these two new requirements are defensible we should cease publication in the literature of negative rodent genetic toxicity data derived in their absence. To achieve this, journal editors will need to be aware of the supporting data bases in order to justify their rejection slips.

1. Regarding the use of three dose levels

If the maximum tolerated dose (MTD) of a chemical gives a negative assay response with no concomitant evidence of target tissue toxicity ( P E / N E ratio depression in the marrow or pyk- notic liver cells), what is the evidence that a positive effect will be seen at lower dose levels? If a negative assay response and toxicity are ob- served at the MTD, the present use of two dose levels should cover this situation. We have en- countered two situations (vincristine and etopo- side) where a positive dose-response relationship was observed in the mouse bone marrow MN assay, and where the magnitude of the response decreased at higher dose levels concomitant with

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244 J. Ashby/Mutation Research 308 (1994) 243-244

a decrease in the P E / N E ratio, but we have not encountered data to support the routine use of three dose levels in either the MN or the liver UDS assays. It would be interesting to review the level of evidence supporting the need for even two dose levels, but that is not being contested here.

bioassays could be reduced to an analytical en- deavour. Of course, consideration of the possible local genotoxicity (skin, stomach, etc.) of unstable direct-acting electrophiles that are systemically non-genotoxic, is a separate topic. Chemical structure is the major alert to such situations, and each instance must be individually addressed.

2. Regarding proof that a chemical has reached the target tissue

Liver toxicity and a depression in the marrow P E / N E ratio provide possible proof that a chem- ical (or its metabolites) have reached the target tissue. If a negative assay response is observed at the MTD in the absence of such indications of localisation, that provides data indicating either the absence of absorpt ion/systemic circulation of the chemical, or its genetic inactivity. The fact that a chemical is not absorbed is valuable toxico- logical information for hazard estimation, but how is that to be proved unequivocally? Will determi- nation of a blood level for the chemical, or its metabolites (which?), suffice to prove the in- evitable circulatory exposure of the bone marrow and liver? If so, what minimum detection level will be set as acceptable? Detection of levels as low as pg /m l blood could probably be achieved for most chemicals, but the cost required to es- tablish, individually, such analytical methods would be prohibitive. Alternatively, is proof of urinary excretion of radiolabelled material suffi- cient? Again, though, this would involve individ- ual radiolabel custom synthesis. Alternatively, all rodent tests could be conducted using intra- venous injection. The end of this particular road is to regard all in vitro genotoxins that are ab- sorbed into the blood, at any concentration, as de facto mammalian mutagens. In this way, rodent genotoxicity and even rodent carcinogenicity

3. Regarding the real problem

A negative result observed for a chemical in a rodent genetic toxicity assay can mean one of four things. First, that it is absorbed and is sys- temically genetically inactive. Second, that it is not absorbed. These two conclusions indicate, equally, the absence of a systemic genetic hazard. The third possibility is that it is absorbed and is genetically active in a different tissue - thus the advice offered in some regulatory guidelines to refer to a second tissue when faced with inactivity in the first. The fourth possibility is that the agent is absorbed and produces a genetic response in the tissue under study, but of a magnitude below the statistical power of the assay to resolve. The solution to this potential problem is to maximise the resolving power of the assay. Neither the use of a third (lower) dose level, nor the study of tissue localisation, will contribute to resolution of this problem.

The use of rodent genotoxicity assays remains essentially empirical, and this increases the need for confirmed precedents when suggesting the use of additional assays, or when recommending major protocol enhancements to existing assays. In the absence of confirmed precedents, an al- most endless list of variables can be speculated upon to meet all possible contingencies (species, strain, route, vehicle, number and frequency of administrations, genetic endpoint, etc.). Thus the title of this communication.