Ethics of Research involving animals
Principal types of animal-based toxicity tests
Acute toxicity
9.9 Acute toxicity refers to the adverse effects that occur on first exposure to a single dose of a substance. Separate tests are needed to detect the effects of contact with the skin and eye (corrosion, irritancy and sensitisation; topical or local toxicity) and the effects on internal organs of a substance that is swallowed, inhaled, absorbed through the skin or injected (systemic toxicity; see paragraph 9.28).
9.10 In the case of local toxicity, skin irritancy is normally assessed by applying the test substance to shaved areas of the backs of rabbits and observing the development of redness, swelling, erosion and ulceration over a period of 72 hours (see paragraph 9.36). Eye-irritancy tests involve administering the test substance directly into the eye of the rabbit and observing corneal opacity, swelling, reddening and other signs of irritation.
9.11 In the case of skin-sensitisation testing, multiple doses of the test substance are applied to the skin of guinea pigs to see if a later dose will cause a strong immune reaction, indicating sensitisation to the chemical (see paragraph 9.36). Tests using guinea pigs are increasingly being replaced by a test involving mice called the local lymph node assay. The test material is applied to the ears of the mice. After an interval the mice are euthanised and the early stages of sensitisation are detected by measuring the level of induced DNA synthesis in the lymph nodes. This test provides more useful information, uses fewer animals than the guinea pig test, and causes substantially less pain and distress to the animals involved.
9.12 The main purpose of skin and eye testing is to allow classification and labelling of corrosive, irritant and sensitising chemicals. The current systems of classification now follow a progressive, step-wise strategy that allows chemicals to be classified as corrosive or irritant to the skin by using physico-chemical properties, such as pH value. Tests that use isolated human or animal tissue cells or ex vivo tissues or organs to identify chemicals with the potential to cause severe irritation or corrosion are known as non-animal pre-screens (see paragraph 11.9). For sensitisation, analysis of chemical structure (structure–activity relationships) can identify many potential sensitisers. Therefore, in many cases it is now possible to classify chemicals without the need for animal tests. The value of these approaches is illustrated by a decrease in rabbit eye tests in the UK from approximately 4,000 in 1995 to 1,100 in 2003.6
9.13 Acute systemic toxicity is assessed by the administration of a single dose of compound, typically to rats and mice, orally, dermally or by inhalation. For pharmaceuticals, the main aims of these studies are to determine the nature (including delayed toxicity) and duration of any acute toxic response. They also determine the maximum non-lethal dose and provide preliminary information relevant to single exposure or over-dosage in humans (see paragraph 9.39).7
9.14 For industrial chemicals and agrochemicals, testing covers acute toxicity by oral, dermal and inhalation routes of exposure. The information obtained is used primarily to ascribe a chemical to bands of acute toxic effect, which restricts how the materials may be used, and thus the extent of human exposure by the routes of exposure which have been evaluated. In the past, in the UK and elsewhere, acute systemic toxicity was investigated by the use of lethal-dose tests, in which the oral dose causing the death of 50 percent of the treated animals (the LD50 value) was determined.8 Such tests used at least 30 animals per test chemical and required death of the animals as an endpoint, regardless of the suffering caused. In 2001 the OECD agreed that the LD50 test for acute oral toxicity should be abolished and deleted from the OECD manual of internationally accepted test guidelines by the end of 2002 (see paragraphs 9.4 and 12.8).9 Several alternative methods have been developed which use fewer animals and in some cases replace death as the endpoint with signs of significant toxicity instead. Information on similar chemicals is used to guide the selection of initial dose levels and the tests are designed to avoid or minimise lethality or severe toxicity. These methods have replaced the LD50 test for acute oral toxicity, but several acute tests such as those involving inhalation, dermal and eye exposure have yet to be modified. They are still used internationally for tests on birds and, for some purposes, also on mammals.10 Lethal-dose tests are also still used to assess the safety of biological products, such as vaccines (Box 8.5), and certain foods, such as shellfish, for the presence of toxins (see paragraph 9.37).
9.15 The approach to assessing the acute toxicity of pharmaceuticals differs from that described above, in that maximum tolerated dose (MTD) studies are carried out to aid the later process of dose selection. These tests often replace acute studies, especially in the case of larger species such as the dog and primates which are used to complement and verify earlier findings in rodents. They involve steadily increasing the dose given to an animal (single or a number of consecutive doses), until adverse effects indicate that an MTD has been reached. This is normally determined by careful observation of the animals, but there is no universally accepted definition of the MTD and effects such as vomiting and convulsions may occur and are sometimes used as signs of the MTD (see paragraphs 9.34–9.45).
Footnotes6 See Home Office (2004) Statistics of Scientific Procedures on Living Animals Great Britain 2003 (Norwich: HMSO).
7 The studies provide information that may support selection of dose levels for repeated-dose toxicity studies, in vivo genotoxicity tests (see paragraphs 9.20-9.21) and, subsequently, first human exposure studies.
8 The OECD gives the definition as the dose that can be expected to cause death in 50 percent of animals when administered by the oral route.
9 OECD (2001) OECD Test Guideline 401 will be deleted: A Major Step in Animal Welfare: OECD Reaches Agreement on the Abolishment of the LD50 Acute Toxicity Test, available at:
http://www.oecd.org/document/52/0,2340,en_2649_34377_2752116_1_1_1_1,00.html. Accessed on: 27 Apr 2005; The UK ceased
the practice of the LD50 test in 1999 (APC (1999) Press release: LD 50 Test - Changes To Licensing Procedures, available at:
http://www.apc.gov.uk/press_releases/991021.htm. Accessed on: 27 Apr 2005). In the US, Environmental Protection Agency guidelines describe the LD50 test as standard for pesticides and toxic substances although state that it may be unnecessary in certain circumstances (Environment Protection Agency (1998) Health Effects Test Guidelines OPPTS 870.1100
Acute Oral Toxicity, available at: http://www.epa.gov/docs/OPPTS_Harmonized/870_Health_Effects_Test_Guidelines/Series/870-
1100.pdf. Accessed on: 27 Apr 2005.
10 OECD (2002) OECD guidelines for the testing of chemicals: Proposal for a new guideline 223 – Avian acute oral toxicity test,
available at: http://www.oecd.org/dataoecd/16/41/1836204.pdf. Accessed on: 27 Apr 2005.