PRINCIPLES OF TOXICOLOGY

290 CHEMICAL CARCINOGENESIS
Increasing the number of animals tested might increase the sensitivity of the test, but as the
number of animals is increased, the cost of the experiment rises and could render the test
cost-prohibitive.
• The exposure and observation periods should last a lifetime, if possible, so that the latency
of the response does not become an issue.
• At least two doses should be tested. One should be the maximally tolerated dose (MTD), the
second dose should be some fraction (usually 50% or 25%) of the MTD. The MTD is defined
as the highest dose that can be reasonably administered for the lifetime of the animal without
producing serious, life-threatening toxicity to the animal that might compromise completion
of the study. In the past the MTD has been defined as a dose that causes no more than a 10
percent decrease in body weight gain and does not lead to lethality over time.
• A detailed pathologic examination of all tissues should be held at termination of the
experiment (and sometimes at 6-month intervals).
In addition to these recommended guidelines, this test is normally performed following good laboratory
practice (GLP) procedures. These and other procedures ensure proper animal care during the
extended period of the test, that no cross-contamination with other chemicals being tested will occur,
and the possibility of having infectious agents or disease affect the outcome of the test is limited.
Using these basic guidelines, any positive result obtained in at least one sex of one species is
generally considered sufficient evidence to classify the chemical, for regulatory and public health
purposes, as a carcinogen. Four different types of tissue response might be observed in a chronic test
and considered positive evidence of carcinogenicity:
1. An increase in the incidence of a tumor type that occurs in control animals but at a significantly
lower rate
2. The development of tumors at a significantly earlier period than is observed in the control
animals
3. The presence of tumor types that are not seen in control animals
4. An increased multiplicity of tumors (although generally speaking, differences in total tumor
load between exposed and unexposed animals is not considered reliable evidence)
Positive results in a test with a more limited power to detect carcinogenicity (e.g., tests of shorter
duration or fewer animals), but where the overall test procedures employed are considered adequate,
may also become accepted as sufficient evidence of carcinogenicity, particularly where other relevant
evidence (e.g., mechanistic data, structural alerts, structure–activity relationships) are also available.
In contrast, because it is well recognized that important species differences exist in regard to response,
negative results (an observed lack of a tumorigenic response), might not be considered definitive
evidence that a chemical is not a carcinogen in other species that were not tested.
The Issue of Generating False-Negative or False-Positive Results
Both false-negative and false-positive results are a potential problem in carcinogen bioassays. Ideally,
the number of animals required to provide adequate negative evidence should be great enough that
even a false-negative test (a test failing to detect existent carcinogenicity) will not allow an excessive
risk to go unnoticed. The likelihood that such a risk will not be detected during the evaluation of
bioassay data is dependent on two factors (excluding species differences in response): the number of
animals tested and the extent to which the test dose exceeds the usual level of human exposure, therefore
increasing either parameter tends to lessen the chance of obtaining a false negative response (with
respect to humans).
The probability that a test will generate a false-negative result is also affected by the background
tumor rate in the control animals. As the background incidence of tumorigenesis increases, so does the