Acute systemic toxicity-prospects for tiered testing strategiesHistorical perspectiveA strategy for acute oral toxicity testing in the ‘‘Post LD50 Test Era’’A future strategy for acute toxicity testing-the prospects for further reduction and refinement and/or replacementReferencesAcute systemic toxicity—prospects for tiered testing strategiesP.A. Botham*Syngenta Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire SK10 4TJ, UKAccepted 15 July 2003AbstractAfter many years of controversy and debate, the LD50test was finally deleted by the end of 2002. Three alternative animal tests,the Fixed Dose Procedure, the Acute Toxic Class Method and the Up and Down Procedure have been developed which give rise tosignificant improvements in animal welfare. They have recently undergone revision to improve their scientific performance but moreimportantly to increase their regulatory acceptance. They can now be used within a strategy for acute toxicity testing for all types oftest substances and for all regulatory and in-house purposes. In vitro cytotoxicity tests could be used as adjuncts to these alternativeanimal tests within the next year or so to improve dose level selection and thus give further modest improvements in the numbers ofanimals used. However, the total replacement of animal tests requires a considerable amount of further test development, followedby validation, and is at least 10 years away.# 2003 Elsevier Ltd. All rights reserved.Keywords: LD50 test; Alternative methods; In vitro cytotoxicity assays; Validation1. Historical perspectiveThe LD50 test was first introduced in 1927 by Trevanfor testing substances intended for human use such asdigitalis and insulin. However, by the 1970s, the test,which aims to find the single lethal dose of a substancewhich kills half the animals in a test group, had becomegenerally accepted as a basis of comparing and classify-ing the toxicities of chemicals, and gradually became arequired test for various regulatory bodies concernedwith new drugs, food additives, cosmetic ingredients,household products, industrial chemicals and pesticides.The test required up to 100 animals, sometimes for eachof two species (normally the rat but also the mousewhen a second species was needed) for each substancetested.In 1981, the Organisation for Economic Cooperationand Development (OECD) incorporated the LD50testinto its new Test Guidelines. However, by this time, itwas generally agreed that the statistical precision of theLD50value, together with its confidence intervals andthe slope of the dose–mortality curve, which this classi-cal LD50test could provide, were not needed for normalhazard and risk assessment purposes. Hence, the 1981guideline for acute oral toxicity (OECD 401) requiredthe use of only five animals per sex per dose group, withthree dose groups per test which were chosen, fromsighting studies or from historical data, to span theLD50value. An upper dose level limit of 5000 mg/kgwas also introduced and, for essentially non-toxic sub-stances, the concept of a Limit Test was included whichrequired, for those substances with LD50values greaterthan 5000 mg/kg, only this upper dose level to be tested.Similar guidelines were also published for acute dermal(OECD 402) and inhalation (OECD 403) toxicity.In 1987, OECD 401 was revised, predominantly foranimal welfare reasons. The test could now be con-ducted using animals of only one sex, with confirmationthat there were no sex differences in the acute toxicity ofa test material by testing at just one dose level in thesecond sex. This reduced the number of animalsrequired from 30 to 20. The limit dose was also reducedto 2000 mg/kg.In 1984, Iain Purchase facilitated the setting up of aworking party of the British Toxicology Society. Thisgroup proposed a new method for acute oral toxicitytesting which avoided using the death of animals as anendpoint, and relied instead on the observation of clearsigns of toxicity developed at one of a series of fixeddose levels. This method, which became know as theFixed Dose Procedure (FDP) was assessed in both a0887-2333/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.doi:10.1016/S0887-2333(03)00143-7Toxicology in Vitro 18 (2004) 227–230www.elsevier.com/locate/toxinvit* Tel.: +44-1625-515491; fax: +44-1625-586396.E-mail address: [email protected] (P.A. Botham).national and international validation study, the latterbeing published in 1990 (van den Heuvel et al., 1990).These studies showed that the FDP was able to provideresults that enable substances to be ranked according tothe EU system of classification in a way which isbroadly compatible with how they would have beenallocated by LD50values derived from classical acuteoral toxicity tests. The FDP also provided the necessaryinformation on the nature, time to onset, duration andoutcome of signs of toxicity that is required for riskassessment purposes. In so doing, the FDP uses feweranimals than OECD 401, causes less compound-relatedmortality and subjects those animals which are used toless pain and distress.The FDP was adopted as an OECD Guideline(OECD 420) in 1992 but as an alternative for OECD401, not a replacement. In 1996, a second alternativemethod, the Acute Toxic Class Method (ATC) wasadopted (OECD 423) and this was followed in 1998 bythe Up and Down Procedure (UDP; OECD 425). TheATC also uses the concept of fixed dose levels butretains mortality as a principal endpoint; the UDP, asits name suggests, aims to estimate the LD50value bytesting individual animals sequentially, with the dose foreach animal being adjusted up or down depending uponthe outcome for the previous animal.The use in practice of these three alternative methodshas not been as widespread as originally envisaged bythe animal welfare community and also by many tox-icologists. In Europe, the FDP and ATC have been usedextensively for the notification of new industrial chemi-cal substances under the Dangerous Substances Direc-tive, as the methods were incorporated into the EUAnnex V Test Guidelines (European Commission,1997). However, in the USA, although they were inclu-ded in the 1996 revision of the EPA’s Office of Preven-tion, Pesticides and Toxic Substances (OPPTS)Guidelines, their use has been restricted by the require-ments of key legislation governing the classification andrisk assessment needs for substances such as consumerproducts and pesticides. This legislation still