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The duration of such toxicity tests varies. In the USA, the FDA usually recommends a period of up to 2 years, while in Europe the recommended duration is usually much shorter. Chronic toxicity studies of biopharmaceuticals can also be complicated by their likely stimulation of an immune response in the recipient animals.
Reproductive toxicity and teratogenicity
All reproductive studies entail ongoing administration of the proposed drug at three different dosage levels (ranging from non-toxic to slightly toxic) to different groups of the chosen target species (usually rodents). Fertility studies aim to assess the nature of any effect of the substance on male or female reproductive function. The drug is administered to males for at least 60 days (one full spermatogenesis cycle). Females are dosed for at least 14 days before they are mated. Specific tests carried out include assessment of male spermatogenesis, female follicular development, as well as fertilization, implantation and early fetal development.
These reproductive toxicity studies complement teratogenicity studies, which aim to assess whether the drug promotes any developmental abnormalities in the fetus (a teratogen is any substance/agent which can induce fetal developmental abnormalities; examples include alcohol, radiation and some viruses). Daily doses of the drug are administered to pregnant females of at least two species (usually rats and rabbits). The animals are sacrificed close to term and a full autopsy on the mother and fetus ensues. Post-natal toxicity evaluation often forms an extension of such studies. This entails administration of the drug to females both during and after pregnancy, with assessment of mother and progeny not only during pregnancy, but also during the lactation period.
Mutagenicity, carcinogenicity and other tests
Mutagenicity tests aim to determine whether the proposed drug is capable of inducing DNA damage, either by inducing alterations in chromosomal structure or by promoting changes in nucleotide base sequence. While mutagenicity tests are prudent and necessary in the case of chemical-based drugs, they are less so for most biopharmaceutical substances. In many cases, biopharmaceutical mutagenicity testing is likely to focus more so on any novel excipients added to the final product, rather than the biopharmaceutical itself (‘excipient’ refers to any substances other than the active ingredient, present in the final drug formulation).
Mutagenicity tests are usually carried out in vitro and in vivo, often using both prokaryotic and eukaryotic organisms. A well-known example is the Ames test, which assesses the ability of a drug to induce mutation reversions in E. coli and Salmonella typhimurium.
Longer-term carcinogenicity tests are undertaken, particularly if (a) the product’s likely therapeutic indication will necessitate its administration over prolonged periods (a few weeks or more) or (b) if there is any reason to suspect that the active ingredient or other constituents could be carcinogenic. These tests normally entail ongoing administration of the product to rodents at various dosage levels for periods of up to (or above) 2 years.
Some additional animal investigations are also undertaken during pre-clinical trials. These include immunotoxicity and local toxicity tests. Again, for many biopharmaceuticals, immunotoxicity tests (i.e. the product’s ability to induce an allergic or hypersensitive response) are often inappropriate or impractical. The regulatory guidelines suggest that further studies should be carried out if a biotechnology drug is found capable of inducing an immune response. However, many of the most prominent biopharmaceuticals (e.g. cytokines) actually function to modulate immunological activities in the first place.
Many drugs are administered to localized areas within the body by, for example, subcutaneous (s.c.) or intramuscular (i.m.) injection. Local toxicity tests appraise whether there is any associated toxicity at/surrounding the site of injection. Predictably, these are generally carried out by s.c. or i.m. injection of product to test animals, followed by observation of the site of injection. The exact cause of any adverse response noted (i.e. active ingredient or excipient) is usually determined by their separate subsequent administration.
Pre-clinical pharmacological and toxicological assessment entails the use of thousands of animals. This is both costly and, in many cases, politically contentious. Attempts have been made to develop alternatives to using animals for toxicity tests, and these have mainly centred around animal cell culture systems. A whole range of animal and human cell types may be cultured, at least transiently, in vitro. Large-scale and fairly rapid screening can be undertaken by, for example, microculture of the target animal cells in microtitre plates, followed by addition of the drug and an indicator molecule.