Ethics of Research involving animals
Principal types of animal-based toxicity tests - continuation I
9.16 These studies have three main objectives (i) to identify toxicity that develops only after a certain length of continuous exposure to the chemical, (ii) to identify the organs most affected and (iii) to determine the doses at which each effect occurs.
9.17 Repeated-dose studies are conducted for various periods of time. The 28-day (sub-acute) study is most common, but studies of 90 days to one year are also regularly carried out. Rats and mice are generally used but for certain classes of chemicals, such as agrochemicals and pharmaceuticals, the tests may also be conducted in non-rodent animals such as the beagle dogs, pigs, marmosets or macaques (see paragraphs 9.26 and 9.30). The test data allow an assessment of the highest dose without significant effects (the ‘no observed adverse effect level’, or NOAEL). This is used in risk assessment and risk management, by limiting the acceptable exposure of humans to a fraction of the NOAEL. For example, in the case of agrochemicals and food additives, these studies are used to assign a reference dose to which safety factors are applied to give an acceptable daily intake (ADI) that is typically a hundredfold less than the observed NOAEL. This can be defined as the dose level to which humans may be exposed, through residues on foodstuffs and in drinking water, with the practical certainty that no adverse health effects will ensue.
9.18 Repeated-dose studies are also used to give an insight into any species differences in toxicity that could be relevant to the assessment of risk in human health. Depending on the use and physico-chemical properties of a chemical, different routes of administration, such as oral, by inhalation or dermal contact, may be used to give a more appropriate risk assessment. For pharmaceuticals, results of these studies support investigations requiring the first administration of the test substance to humans.
Carcinogenicity
9.19 For the assessment of carcinogenicity, rats and mice are dosed for up to two years (the typical lifespan for these species) and the incidence and type of the tumours that develop is evaluated (see paragraph 9.33). This knowledge is used to assess the risk of cancer induction by the chemical in exposed humans. In practice, the assessment of repeated-dose studies and carcinogenicity is often combined into a single study in rodents thus reducing the use of experimental animals.
Genotoxicity
9.20 Short-term studies investigating interactions with genetic material (DNA and chromosomes) are widely used to screen chemicals for the potential to cause cancer or heritable mutations. Most of these studies involve the use of in vitro assays for mutation in bacteria or isolated mammalian cells that have been shown to predict the potential for a substance to be carcinogenic or mutagenic through interaction with DNA. In the pharmaceutical industry tests are performed as high-throughput screens (see paragraphs 8.9 and 8.21), both early in drug discovery and also to support drug registration. Animal studies, usually in the mouse, are used only when one or more of these in vitro tests has given a positive result, and with the purpose of demonstrating that the chemical can or cannot reach a sensitive tissue and cause genetic changes in the intact animal. In practice, very few chemicals that have been confirmed to be mutagenic in vitro are tested any further in animals. However, in the case of pharmaceuticals, regulatory requirements demand that an in vivo test be completed before the start of Phase II clinical studies in humans.
9.21 In vivo tests include the rodent bone marrow micronucleus test, which is an early predictor of carcinogenic activity. A single dose of compound is administered to rats or mice which are killed either 24 or 48 hours later for examination of chromosomal changes in bone marrow cells. It is expected that the highest dose level used will show evidence of adverse effects if the substance is genotoxic, and the MTD is normally used to set this dose level.
Effects on reproduction and development
9.22 Studies within this category are intended to determine the effects of compounds on various aspects of the reproductive capacity of the adult, and on the development of the offspring. The most comprehensive test method for reproduction (the two-generation reproduction study) involves repeated oral doses to young rats through the period of sexual maturation into young adulthood when the animals are mated to treated females. The females are dosed throughout pregnancy and until the offspring are weaned. The pups are dosed until adulthood and mated and the second-generation young evaluated. These tests provide information on fertility, mating behaviour, parental behaviour and development of the neonate to adulthood. The results are used in hazard classification and in risk assessment. More-limited information on fertility and reproductive performance can also be obtained from a one-generation study or a screening test, which combines reproductive investigations with a 28-day repeated-dose toxicity test.
9.23 Studies on developmental toxicity provide specific information on the potential hazards to the unborn that may arise from exposure of the mother to a particular substance during pregnancy. Typically, groups of pregnant rats or rabbits are treated orally for up to the whole period of gestation, and the uterine contents are then examined and evaluated just prior to parturition. An evaluation is made of maternal toxicity relative to that in non-pregnant females, embryo or fetal death, altered growth and structural changes in the fetus. Rabbits are used in addition to rats and mice because rodents do not generally respond, or respond variably, to the effects of potent human teratogens such as thalidomide (see Box 8.4). The results of both tests are used in classification by hazard and in risk assessment.