administration should be the same as that planned
in humans, whereas the animal studies should
involve higher doses and longer durations of expo-
sure than those planned clinically. The type and
duration of specific studies, and those that are
needed relative to different stages of clinical
development, were mentioned previously (Federal
Register, 25 November 1997). Protocols must
specify the number of animals per group, numbers
of groups and experimental procedures to be
carried out, and standard versions of these have
been available for some time. In general, for initial
repeated-dose studies, protocols require the use of
three dose groups plus a control, and a minimum of
10 rodents and 3 non-rodents per sex per group.
Doses must be selected that will allow for the
identification of toxic effects at the highest dose
as well as at a no-effect level at the middle or lowest
dose.
Usual experimental procedures include the
determination of body weights and food con-
sumption on at least a weekly basis, evaluation of
hematology and blood chemistry parameters
during the treatment period, ophthalmoscopic
examinations, the recording of macroscopic exam-
inations at necropsy and the determination of organ
weights. A complete histopathological examina-
tion of tissues from animals is required. In rodent
studies, this can take the form of examination of
all high-dose and control animals and the
examination of target organs at the two lower
doses. In non-rodent studies, it is typical to exam-
ine tissues from all animals in the study.
It is crucial that plasma concentrations of drug
are measured in these studies to allow for determi-
nation of effects on the basis of exposure. Frequ-
ently this is a more appropriate measure of
comparing effects in animals and man, as rates of
absorption, distribution and excretion can vary
extensively between these species. This aspect,
now commonly referred to as ‘toxicokinetics’,
has been outlined in an ICH guideline (Federal
Register, 1 March 1995). This guideline specifies
minimum requirements in terms of number of time
points examined, number of animals per time
point, and the requirements for calculation of
various pharmacokinetic parameters such as
Cmax, AUC and so on. Thesewill become important
for comparison with human data as it becomes
available later.Mutagenicity studies
Mutagenicity studies are highly specialized but, in
general, include studies of genetic mutation, clas-
togenesis and nuclear maturation. There are multi-
ple hereditary components in both somatic and
germinal cells that may be affected by drugs. Dur-
ing 1970s, it was thought naı ̈vely that these studies
may be replacements for the long and costly carci-
nogenicitystudies thatare requiredfor many drugs.
Although this goal was never realized, mutageni-
city studies nonetheless provide useful indications
of the ability of a drug to alter genetic material,
which may later be manifested in studies of carci-
nogenic or teratogenic effects (Kowalski, 2001).
Genotoxicity studies are relatively inexpensive and
may also serve, early in the drug development
process, to assure drug developers and regulators
that no obvious risk of such adverse effects exists,
albeit knowing that more definitive studies to eval-
uate teratogenic and carcinogenic effects will not
come until later.
An exhaustive review of various components of
a mutagenicity evaluation will not be attempted in
this chapter. Multiple guidelines are available.
Those issued by the ICH includegeneral guidelines
(Federal Register, 24 April 1996) and specifics
related to the core battery of studies required
(Federal Register, 3 April 1997). Tennantet al.
(1986) have summarized the correlation between
the results of a battery of mutagenicity assays and
the probability of the material producing a positive
carcinogenic response in long-term rodent studies.
Obviously, mutagenicity studies cannot predict
nongenetic carcinogenicity or teratogenicity (e.g.
estragen-induced breast tumors in rodents).
Positive results in one or more mutagenicity
assays do not necessarily translate into human
risks. Mechanistic studies may show that such
responses would not occur in the human cell
population or that the concentrations at which
positive responses occurred may far exceed any
concentration of drug that may occur in the
clinical setting. Many drugs are in the market68 CH6 NONCLINICAL TOXICOLOGY