IN VIVO AND IN VITRO TESTING

The underpinning of safety assessment has, for decades, been the in vivo testing programs that use laboratory animals. Starting in the early 1970s, in vitro testing was initiated and added to the battery of tests used to assess the safety of various substances. In vivo tests are carried out in several animal species for the development of drugs, food additives, pesticides, and industrial chemicals, and in humans primarily for drugs. In vivo studies range in duration from short-term dosing to lifetime exposure. They include studies to assess the potential for inducing birth defects, as well as multigenerational studies for assessing adverse reproductive outcomes. These studies are usually conducted under the Good Laboratory Practices (GLP) guidelines. These guidelines, promulgated by the U.S. Food and Drug Administration (FDA) and other regulatory agencies, lay out the boundaries within which toxicity studies that are to be used for regulatory purposes will be conducted. Most laboratories conduct toxicology studies within the spirit of the GLP guidelines even if the studies are not going to be used for regulatory purposes.

Short-term, acute studies are usually conducted in one or more rodent species with the purpose to determine the dose range for lethality of a particular substance, and to determine the shape of the dose-response curve. In addition to the LD50 (the dose lethal to half the animals), the results of the acute studies are used to set doses for longer-term, subchronic experiments. Acute studies can determine toxicity, time of onset of toxic signs, and recovery in the surviving animals. This information is extremely critical in an emergency situation where humans or domestic animals are exposed to high concentrations of a chemical. Occasionally, acute toxicity studies are used to establish antidotes to a given toxicant.

Subchronic studies are generally conducted in both sexes of two laboratory animal species, one of which is a nonrodent species. These studies are of longer duration, generally three to six months, and are conducted using multiple doses. The purpose of the subchronic study is to determine target-organ toxicity, to determine the effects of prolonged dosing, and to help establish margins of safety for food additives and drugs. At the end of a study all animals are autopsied, with complete gross and microscopic examination of tissues. Complete blood chemistries are evaluated and an overall clinical assessment is made on each animal.

The next level of toxicity study is the chronic bioassay. Again, the study is conducted in multiple species, in both sexes, and for a duration that approaches the lifespan of the animal. These are very large and complex studies that necessitate a great deal of day-to-day management. There are multiple intermediate clinical evaluations, including daily observations, weekly food and water consumption, and body weight determinations. At the termination of the study, or at times of interim sacrifices, all animals are autopsied.

Chronic toxicity studies provide a thorough examination of the dose effect of a given chemical on homeostasis, bodily function, induced diseases, and the effect on lifespan. Chronic toxicity studies provide the bulk of the preclinical information used for assessing safety and risk. Cancer potential can be determined in some of the aforementioned studies, but for many chemicals a definitive cancer bioassay is conducted. This bioassay is conducted in rodents and lasts for the major part of the natural lifespan of the animal. The study is generally carried out using at least two dose levels, and it is usually designed to mimic the route of human exposure.

The potential to induce adverse birth outcomes is tested in several species. Rodents, rabbits, and sometimes dogs are used depending on the end use of the candidate chemical. The studies are designed to determine if the chemical alters the reproductive cycle of the female or spermatogenesis in the male. The studies also examine the effects of the chemical during the first, second, and third trimester of pregnancy, and during parturition and lactation. Multigenerational studies are conducted to determine the overall effects of a given chemical on the parent generation, the offspring, and on the ability of the offspring to reproduce normally.

In the early 1970s, in vitro studies to determine the potential of a chemical or a mixture to induce point mutations in engineered strains of bacteria indicated that the mutagenic potency of a chemical was a reasonable predictor of its carcinogenic potential. The overall hypothesis was later shown to be less predictive than first thought, depending on the class of chemical tested, but the strength of the tests kept them in the battery of safety evaluation tests. Subsequently, in vitro tests have been developed to assess potential immunotoxicity, hormone action, eye irritation, and cellular and molecular events that are correlated with end-stage disease. The advantages of in vitro tests are that they are quick, relatively inexpensive, and specific mechanisms of action can be tested. The disadvantage of these tests is that the homeostatic mechanisms and pathways found in animals are not present. Hence, it is difficult, if not impossible, to determine injury repair in the same system in which toxicity is tested.

A tier testing approach has emerged that allows for an in-depth toxicity evaluation of a chemical, starting with in vitro and acute studies and ending with multigenerational studies and carcinogenesis bioassays. Specialty testing paradigms have also been developed to handle unique toxicology questions. An example of specialty testing is the battery of tests used to determine the potential for endocrine disruption (EDSTAC). The results of these studies are coupled with exposure assessments to the chemical, and the data are used to develop a safety evaluation, or a cancer risk assessment.

Michael Gallo

(see also: Ames Test; Food and Drug Administration; Maximum Tolerated Dose; Toxicology )