Toxicology in Vitro 18 2004 227 230 www elsevier com locate toxinvit Acute systemic toxicity prospects for tiered testing strategies P A Botham Syngenta Central Toxicology Laboratory Alderley Park Maccles eld Cheshire SK10 4TJ UK Accepted 15 July 2003 Abstract After many years of controversy and debate the LD50 test was nally 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 to signi cant improvements in animal welfare They have recently undergone revision to improve their scienti c performance but more importantly to increase their regulatory acceptance They can now be used within a strategy for acute toxicity testing for all types of test substances and for all regulatory and in house purposes In vitro cytotoxicity tests could be used as adjuncts to these alternative animal tests within the next year or so to improve dose level selection and thus give further modest improvements in the numbers of animals used However the total replacement of animal tests requires a considerable amount of further test development followed by validation and is at least 10 years away 2003 Elsevier Ltd All rights reserved Keywords LD50 test Alternative methods In vitro cytotoxicity assays Validation 1 Historical perspective The LD50 test was rst introduced in 1927 by Trevan for testing substances intended for human use such as digitalis and insulin However by the 1970s the test which aims to nd the single lethal dose of a substance which kills half the animals in a test group had become generally accepted as a basis of comparing and classifying the toxicities of chemicals and gradually became a required test for various regulatory bodies concerned with new drugs food additives cosmetic ingredients household products industrial chemicals and pesticides The test required up to 100 animals sometimes for each of two species normally the rat but also the mouse when a second species was needed for each substance tested In 1981 the Organisation for Economic Cooperation and Development OECD incorporated the LD50 test into its new Test Guidelines However by this time it was generally agreed that the statistical precision of the LD50 value together with its con dence intervals and the slope of the dose mortality curve which this classical LD50 test could provide were not needed for normal hazard and risk assessment purposes Hence the 1981 Tel 44 1625 515491 fax 44 1625 586396 E mail address phil botham syngenta com P A Botham 0887 2333 see front matter 2003 Elsevier Ltd All rights reserved doi 10 1016 S0887 2333 03 00143 7 guideline for acute oral toxicity OECD 401 required the use of only ve animals per sex per dose group with three dose groups per test which were chosen from sighting studies or from historical data to span the LD50 value An upper dose level limit of 5000 mg kg was also introduced and for essentially non toxic substances the concept of a Limit Test was included which required for those substances with LD50 values greater than 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 for animal welfare reasons The test could now be conducted using animals of only one sex with con rmation that there were no sex di erences in the acute toxicity of a test material by testing at just one dose level in the second sex This reduced the number of animals required from 30 to 20 The limit dose was also reduced to 2000 mg kg In 1984 Iain Purchase facilitated the setting up of a working party of the British Toxicology Society This group proposed a new method for acute oral toxicity testing which avoided using the death of animals as an endpoint and relied instead on the observation of clear signs of toxicity developed at one of a series of xed dose levels This method which became know as the Fixed Dose Procedure FDP was assessed in both a 228 P A Botham Toxicology in Vitro 18 2004 227 230 national and international validation study the latter being published in 1990 van den Heuvel et al 1990 These studies showed that the FDP was able to provide results that enable substances to be ranked according to the EU system of classi cation in a way which is broadly compatible with how they would have been allocated by LD50 values derived from classical acute oral toxicity tests The FDP also provided the necessary information on the nature time to onset duration and outcome of signs of toxicity that is required for risk assessment purposes In so doing the FDP uses fewer animals than OECD 401 causes less compound related mortality and subjects those animals which are used to less pain and distress The FDP was adopted as an OECD Guideline OECD 420 in 1992 but as an alternative for OECD 401 not a replacement In 1996 a second alternative method the Acute Toxic Class Method ATC was adopted OECD 423 and this was followed in 1998 by the Up and Down Procedure UDP OECD 425 The ATC also uses the concept of xed dose levels but retains mortality as a principal endpoint the UDP as its name suggests aims to estimate the LD50 value by testing individual animals sequentially with the dose for each animal being adjusted up or down depending upon the outcome for the previous animal The use in practice of these three alternative methods has not been as widespread as originally envisaged by the animal welfare community and also by many toxicologists In Europe the FDP and ATC have been used extensively for the noti cation of new industrial chemical substances under the Dangerous Substances Directive as the methods were incorporated into the EU Annex V Test Guidelines European Commission 1997 However in the USA although they were included in the 1996 revision of the EPA s O ce of Prevention Pesticides and Toxic Substances OPPTS Guidelines their use has been restricted by the requirements of key legislation governing the classi cation and risk assessment needs for substances such as consumer products and pesticides This legislation still requires a point estimate of the LD50 together with slope and con dence intervals which the FDP and ATC are not able to provide In addition this same legislation sometimes requires the use of a limit dose of 5000 mg kg and the alternative method guidelines had adopted the lower limit dose of 2000 mg kg In Japan also even in the latest revision