- Email: [email protected]
To be, or not to be .. the ld 50
rurlcun. 1~N~6. Vul. 4. pp . öl-öJ. Pcrtwmon Press Ltd., Pdnled in (irrot Britain
EDITORIAL TO BE, OR NOT TO BE . . . . . . . . THE
LDSp
PROBLEM Of deCldlIIg which of the several methods one should employ for determining the lethality of a venom is not as difficult as it might appear from a study of the literature. Examining some 200 papers on venoms and antivenins published during the past decade, one finds no less than 24 different techniques being described and used for determining lethal doses. These techniques include the use of the "Las", "AU.n", "mld", "Lnl", "LDgo", "LD y p", "LD loo", "LDlpoxg", "LDspxg", "ALD", "CLD", defined and undefined "mOUSe units", and several others of more humble origins. In examining the articles a little more closely one finds that the authors have usually developed their choice of standards on the basis of several factors ; the amount of toxin or antitoxin available ; the number of test animals available ; the form of the toxin ; or as in a few cases, a desire to continue with a technique previously employed by the author. There is also an occasional report in which some rather obscure standard is used, when one of the more acceptable methods might have been employed . It would be presumptuous to speculate on the reasons behind the uses of these obscure standards, since there are often extenuating circumstances unknown to the reader, but editors in the course of their correspondence with authors gather some general impressions worthy of comment, although some are admittedly venturesome. It is apparent that some investigators are not at all familiar with the requirements necessary for a biological assay, nor have they familiarized themselves sufficiently with the literature to know whether or not a particular assay is suitable for their study. The worker who submits figures on an Las so based on five animals must consider a percentage mortality obtainable by chance of between 0 and 100 per cent ; with ten animals the chance mortality is still between 5 and 95 per cent ; with approximately twenty animals it is between 20 and 80 per cent; with thirty animals, between 23 and 77 per cent ; with fifty animals, 28 and 72 per cent and with one hundred animals, 35 and 65 per cent . It can be seen that the chance mortality diminishes as the square root of the number of animals used . if one draws a characteristic from the above figures it is obvious that when between thirty and one hundred animals are used, that part of the characteristic between these two points is almost a straight line. Below 30 animals the curve for the dose error increases sharply. During studies with Crotalus viridis helleri venom it was found that even under those conditions where experimental variants were almost non-existent, differences in the Lasso with varying numbers of animals were impressive. The intravenous Lasso as determined in ten mice was 1 ~50 mg per kg body weight, while the dose for thirty animals was 1 ~34 and for one hundred animals, 129. The intraperitoneal LDso showed much greater differences, the figures being 1 ~96 for ten mice, 1 ~66 for thirty mice and 1 ~60 for one hundred mice. Occasionally, an author submits a characteristic plotted for the values between the LD aa and the LD,s based on a study in ten animals, five at the Las so and five at the LD,s . On 81
THE
82
Editorial
the basis of a similar study in a larger group of animals the probability that the Ln, b will kill more than the Ln~ is 0922 ; the probability that the same number of animals will die with each dose is 0058; the probability that more animals will die with the lower dose is 0020, and the probability that either all will live or all will die with each dose is 00005. From these and other evidences it is apparent that the most practical number of animals to use in the determination of a lethal dose of a venom is thirty . In general, the relatively small increases in the degree of accuracy obtained with larger numbers of animals is not of sufficient importance to warrant their use. "Some of my best friends" use the minimal lethal dose (mld). This is defined as the smallest dose of a substance necessary to kill. With any dose smaller than the mld the animal is expected to live. The per cent error associated with the use of this expression is far greater than most investigators appreciate . It might be acceptable to use the mld where the quantity of toxin is so small that only four or five animals could be used in a determination, but even under these circumstances it is far wiser to state what dosages killed and what dosages did not. Such data usually provide adequate information for those who need some knowledge of the lethality of a toxin for physiopharmacological or biochemical studies. At the same time they encourage more accurate work on the toxin without eliciting subsequent retractions or revisions. The difficulty with permitting an mld to stand in the literature is that it implies to the uninitiated that it has been established that there is a definite amount of the toxin that always kills, and that below this dose the toxin never kills. Any worker familiar with lethality determinations has discovered that this is not the true condition of affairs. As data on the mld of venoms are being replaced by data on the Lnbu, it is becoming increasingly apparent how great the degree of error the former determination presented. The mld for Crotalus viriclis helleri venom, for instance, is given as 1 ~58, while three different investigators over a period of five years have found the Lnsu to be 150-186. In studies over a seven-year period, with approximately eight hundred mice in groups of thirty to fifty each, we have found the LD 6o of the same venom to be 168. The ALD in ten mice for one Crotalus venom was found to be the Lneo when determined in thirty mice and the Lnbo when determined by another investigator in fifty mice. Accurate determinations of the Lnl and LD sy can be made on the basis of studies on the Lnao and these should be preferred when data on extreme end-points are needed . The Lnbo of a venom is a far more reliable statistical end-point to work with in determining the lethality or effects of a drug than any other point. Further, use of 2 times the Ln 9s or 2 times the nLn, or some other such multiple of death, must certainly be viewed with caution for what is the criterion for predicting the dose necessary to kill the same animal twice? The problems involved in calculating doses on the basis of body weight and surface areas, and the influences of age, sex, time of injection, number of animals in a cage, etc., must always be taken into consideration when determining lethal dosages. Fortunately, there is sufficient literature on these subjects to guide the investigator into techniques for obtaining reliable and reproducible results. It is not the task of an Editor to direct methodology, nor to seek to standardize techniques, but it is his duty, in so far as possible, to assure his readers that the utmost care has been exercised in the reviewing and editing of those manuscripts which appear in his Journal. With this thought firmly in mind, the Editorial Boards of Toxicon respectfully urge that all contributors give careful thought to the methods they employ for determining the lethality of a venom or toxin. The Editorial Boards will continue to accept lethality
~ditorial
83
data based on a mld, ALD, or cL~, or on small groups of animals, but sufficient explanation will need to be given in each manuscript to indicate why the method at hand has been selected . It is hoped that faced with the problem of presenting such data, the investigator will refresh his memory by reading the classic paper by TREVAN [1] . REFERENCE [l] TREVAN, J . W ., Prac . R. Soc., B, 101, 483, 1927 . F.E .R.
EDITORIAL TO BE, OR NOT TO BE . . . . . . . . THE
LDSp
PROBLEM Of deCldlIIg which of the several methods one should employ for determining the lethality of a venom is not as difficult as it might appear from a study of the literature. Examining some 200 papers on venoms and antivenins published during the past decade, one finds no less than 24 different techniques being described and used for determining lethal doses. These techniques include the use of the "Las", "AU.n", "mld", "Lnl", "LDgo", "LD y p", "LD loo", "LDlpoxg", "LDspxg", "ALD", "CLD", defined and undefined "mOUSe units", and several others of more humble origins. In examining the articles a little more closely one finds that the authors have usually developed their choice of standards on the basis of several factors ; the amount of toxin or antitoxin available ; the number of test animals available ; the form of the toxin ; or as in a few cases, a desire to continue with a technique previously employed by the author. There is also an occasional report in which some rather obscure standard is used, when one of the more acceptable methods might have been employed . It would be presumptuous to speculate on the reasons behind the uses of these obscure standards, since there are often extenuating circumstances unknown to the reader, but editors in the course of their correspondence with authors gather some general impressions worthy of comment, although some are admittedly venturesome. It is apparent that some investigators are not at all familiar with the requirements necessary for a biological assay, nor have they familiarized themselves sufficiently with the literature to know whether or not a particular assay is suitable for their study. The worker who submits figures on an Las so based on five animals must consider a percentage mortality obtainable by chance of between 0 and 100 per cent ; with ten animals the chance mortality is still between 5 and 95 per cent ; with approximately twenty animals it is between 20 and 80 per cent; with thirty animals, between 23 and 77 per cent ; with fifty animals, 28 and 72 per cent and with one hundred animals, 35 and 65 per cent . It can be seen that the chance mortality diminishes as the square root of the number of animals used . if one draws a characteristic from the above figures it is obvious that when between thirty and one hundred animals are used, that part of the characteristic between these two points is almost a straight line. Below 30 animals the curve for the dose error increases sharply. During studies with Crotalus viridis helleri venom it was found that even under those conditions where experimental variants were almost non-existent, differences in the Lasso with varying numbers of animals were impressive. The intravenous Lasso as determined in ten mice was 1 ~50 mg per kg body weight, while the dose for thirty animals was 1 ~34 and for one hundred animals, 129. The intraperitoneal LDso showed much greater differences, the figures being 1 ~96 for ten mice, 1 ~66 for thirty mice and 1 ~60 for one hundred mice. Occasionally, an author submits a characteristic plotted for the values between the LD aa and the LD,s based on a study in ten animals, five at the Las so and five at the LD,s . On 81
THE
82
Editorial
the basis of a similar study in a larger group of animals the probability that the Ln, b will kill more than the Ln~ is 0922 ; the probability that the same number of animals will die with each dose is 0058; the probability that more animals will die with the lower dose is 0020, and the probability that either all will live or all will die with each dose is 00005. From these and other evidences it is apparent that the most practical number of animals to use in the determination of a lethal dose of a venom is thirty . In general, the relatively small increases in the degree of accuracy obtained with larger numbers of animals is not of sufficient importance to warrant their use. "Some of my best friends" use the minimal lethal dose (mld). This is defined as the smallest dose of a substance necessary to kill. With any dose smaller than the mld the animal is expected to live. The per cent error associated with the use of this expression is far greater than most investigators appreciate . It might be acceptable to use the mld where the quantity of toxin is so small that only four or five animals could be used in a determination, but even under these circumstances it is far wiser to state what dosages killed and what dosages did not. Such data usually provide adequate information for those who need some knowledge of the lethality of a toxin for physiopharmacological or biochemical studies. At the same time they encourage more accurate work on the toxin without eliciting subsequent retractions or revisions. The difficulty with permitting an mld to stand in the literature is that it implies to the uninitiated that it has been established that there is a definite amount of the toxin that always kills, and that below this dose the toxin never kills. Any worker familiar with lethality determinations has discovered that this is not the true condition of affairs. As data on the mld of venoms are being replaced by data on the Lnbu, it is becoming increasingly apparent how great the degree of error the former determination presented. The mld for Crotalus viriclis helleri venom, for instance, is given as 1 ~58, while three different investigators over a period of five years have found the Lnsu to be 150-186. In studies over a seven-year period, with approximately eight hundred mice in groups of thirty to fifty each, we have found the LD 6o of the same venom to be 168. The ALD in ten mice for one Crotalus venom was found to be the Lneo when determined in thirty mice and the Lnbo when determined by another investigator in fifty mice. Accurate determinations of the Lnl and LD sy can be made on the basis of studies on the Lnao and these should be preferred when data on extreme end-points are needed . The Lnbo of a venom is a far more reliable statistical end-point to work with in determining the lethality or effects of a drug than any other point. Further, use of 2 times the Ln 9s or 2 times the nLn, or some other such multiple of death, must certainly be viewed with caution for what is the criterion for predicting the dose necessary to kill the same animal twice? The problems involved in calculating doses on the basis of body weight and surface areas, and the influences of age, sex, time of injection, number of animals in a cage, etc., must always be taken into consideration when determining lethal dosages. Fortunately, there is sufficient literature on these subjects to guide the investigator into techniques for obtaining reliable and reproducible results. It is not the task of an Editor to direct methodology, nor to seek to standardize techniques, but it is his duty, in so far as possible, to assure his readers that the utmost care has been exercised in the reviewing and editing of those manuscripts which appear in his Journal. With this thought firmly in mind, the Editorial Boards of Toxicon respectfully urge that all contributors give careful thought to the methods they employ for determining the lethality of a venom or toxin. The Editorial Boards will continue to accept lethality
~ditorial
83
data based on a mld, ALD, or cL~, or on small groups of animals, but sufficient explanation will need to be given in each manuscript to indicate why the method at hand has been selected . It is hoped that faced with the problem of presenting such data, the investigator will refresh his memory by reading the classic paper by TREVAN [1] . REFERENCE [l] TREVAN, J . W ., Prac . R. Soc., B, 101, 483, 1927 . F.E .R.