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quantal one (all or none), that the percentage responding in an experimental group is dose related, and that the cumulative effect follows a normal distribution. Data from a typical example (see Figure 11.3), their analysis, and implications are discussed in 11 on acute toxicity. As a result of much recent controversy, the LD50 test has been the subject of considerable regulatory attention and as a result changes in requirements have been promulgated. These changes are intended to obtain more information but, at the same time, use fewer animals.
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Criticism of the LD50 Test. The criticisms of the test include:
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Used uncritically, it is an expression of lethality only, not re ecting other acute effects. It requires large numbers of experimental animals to obtain statistically acceptable values. Moreover the results of LD50 tests are known to vary with species, strain, sex, age, and so on (Table 21.4); thus the values are seldom closely similar from one laboratory to another despite the numbers used. Because, for regulatory purposes, the most important information needed concerns chronic toxicity, little useful information is derived from the LD50 test. The small amount of information that is acquired could be obtained as well from an approximation requiring only a small number of animals. Extrapolation to humans is dif cult.
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Support of the LD50 Test. Continued use of the test has been advocated, however, on the grounds that it is of use in the following ways:
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Properly conducted, acute toxicity tests yield not only the LD50 but also information on other acute effects such as cause of death, time of death, symptomatology, nonlethal acute effects, organs affected, and reversibility of nonlethal effects. Information concerning mode of action and metabolic detoxication can be inferred from the slope of the mortality curve. The results can form the basis for the design of subsequent subchronic studies. The test is useful as a rst approximation of hazards to workers. The test is rapidly completed.
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For the previously listed reasons, there has been a concerted effort in recent years to modify the concept of acute toxicity testing as it is embodied in the regulations of many countries and to substitute more meaningful methods that use fewer experimental animals. The article by Zbinden and Flury-Roversi is an excellent summary of the
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Table 21.4 Factors Causing Variation in LD50 Values Species Strain Age Weight Gender Health Nutrition Gut contents Route of administration Housing Temperature Time of day Season Human error
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factors affecting LD50 determinations, the advantages and disadvantages of requiring such tests, and the nature and value of the information derived. It concludes that the acute toxicity test (single-dose toxicity) is still of considerable importance for the assessment of risk posed by new chemical substances, and for a better control of natural and synthetic agents in the human environment. It is not permissible, however, to regard a routine determination of the LD50 in various animal species as a valid substitute for an acute toxicity study.
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Current Status. Recently attention has been focused on developing alternatives to the classical LD50 test to reduce the number of animals used or re ne procedures to make exposures less stressful to animals. OECD adopted several alternative methods for determining acute oral toxicity: a limit test for materials with anticipated low toxicity, a xed dose procedure, an acute toxic class method, and an up-and-down procedure. The xed-dose procedure and the acute toxic class method estimate the LD50 within a dose range for use in classi cation and labeling. The up-and-down procedure generates point estimates and con dence intervals of the LD50 and therefore may be useful in a wider set of applications. The xed-dose procedure (Guideline OECD 420) aims to identify the appropriate hazard class for new chemicals; it does not provide a point estimate of the LD50. This method calls for testing animals sequentially at one of four doses: 5, 50, 300, or 2000 mg/kg body weight. The test begins with a sighting study in which animals are tested, one at a time, at doses selected from the set doses. Once clear signs of toxicity appear, additional animals (females, or the more sensitive sex) are dosed at that level for a total of ve animals. Subsequent groups of animals may receive doses at higher or lower levels, if necessary, depending on the outcome of the previous group. Decision criteria based on the number of animals surviving or showing evident toxicity provide for classi cation decisions. The acute toxic class method (guideline OECD 423) aims to identify the appropriate hazard and labeling classi cation and provides a range for lethality rather than a point estimate of the LD50. Groups of three animals (females, or the more sensitive sex) receive one of four or ve doses: 5, 50, 300, 2000 and if necessary 5000 mg/kg body weight. Depending on the survival or mortality of the rst group of animals, three or more animals may receive the same or a higher or lower dose. The number of animals that survive or die determines the classi cation decisions. The up-and-down procedure (Guideline OECD 425) employs sequential dosing, using only a single animal at each step, the dosage depending on whether the previously dosed animal lives or dies. The test provides a point estimate of lethality and con dence intervals, and can be used to evaluate lethality up to 5000 mg/kg. The main test incorporates elements of range nding and uses a exible stopping point. A sequential limit test uses up to ve animals. Default dose spacing is 3.2 times the previous dose. The starting dose should be slightly below the estimated LD50. If no information is available to estimate the LD50, the starting dose is 175 mg/kg. A computer program was developed by the US EPA to simplify both the experimental phase of the test and the calculation of the LD50 and con dence intervals. For all three guidelines, selection of a starting dose close to the actual LD50 should decrease the number of animals necessary, reduce study duration, and decrease the amount of test substance needed. Therefore it is desirable that all information on the test substance be made available to the testing laboratory for consideration prior to
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