LD50计算方法

1 实验实验 急性经口毒性试验急性经口毒性试验 一、目的 掌握LD50 计算方法和急性毒性分级标准 熟悉经口灌胃技术 了解急性毒性试验的设计原则 LD50计算方法 改良寇氏法：计算简便，准确率高，常用改良寇氏法：计算简便，准确率高，常用 霍恩法霍恩法 序贯法 Bliss法 改良寇氏法要求 每组动物数相等 各剂量组组距呈等比级数 死亡呈正态分布 最低剂量组死亡80% 死亡不倒置 二、原理 健康动物?按体重随机分组?一次或多次 经口给药?观察中毒特征或死亡?剂量-反 应关系?求出LD50 ?毒性分级 三、材料与试剂 动物：昆明种小白鼠 器材： 试剂：敌敌畏，苦味酸 2 四、步骤 1.动物选择 2.剂量设计：5-8个剂量（一般不设对照组） 最高、高、中、低、最低5个剂量, 中剂量组：100mg/kg，组距1.4 3.动物称重、编号、标记 4.受试物配制 5.灌胃：1ml/100g体重 6.观察：急性毒性试验原始记录 急性毒性试验原始记录 受试物名称： 受试物性状： 受试物来源： 动物种属品系： 动物来源及合格证号： 染毒途径： 室温： 日期： 剂量组别 （mg/kg） 动 物 编号 性 别 体重 （g） 染毒量 （ml） 染毒 时间 体征及出现时间 死 亡 时间 体重 记录 试验操作者： 实验记录者： 5. LD50计算：LD50及95%可信区间 敌敌畏小鼠经口染毒死亡情况 剂量 组别 mg/kg 对数 动物数 （n） 死亡数 (只) 死亡率 (p) 存活率 (q) pq 1 2 3 4 5 i= p= 计算公式 M = Xk - I (p - 0.5) Sm= n pq i M：logLD50i:相邻两剂量组对数值的差 n：每组动物数 Xk：最大剂量的对数值 P：死亡率q:存活率 p=各剂量组死亡率总和Sm：logLD50的标准误。 LD50的95%可信限=lg-1（M1.96Sm） LD50=log-1M LDLD50 50计算方法之 计算方法之HornHorn氏法氏法 原理原理 Horn氏法，又叫平均移动法、剂量递增法，是利 用剂量对数与死亡率(反应率)的转换数(即几率单位) 呈直线关系而设计的方法。 设 计 原 则设 计 原 则 4个剂量组； 每组动物数相等； 每组4只或5只； 设计剂量时可根据化学物致死剂量范围的宽窄应 用两个剂量系列，其组距分别为2.15倍和3.16倍。 根据每组动物数、组距和每组动物死亡数，即可 从Horn氏表中查得LD50及其95可信限。 3 优 缺 点优 缺 点 1、1、使用动物数少； 2、可直接从Horn氏表查出LD50及其95可信 限，不需计算，甚为简便。 3、但LD50的95可信区间范围较大，方法精确 度尚不够。 0.38625.93.160.1228.181.004312 0.85836.95.620.27111.71.784212 1.9650.910.00.62116.13.164112 0.83711.93.160.2653.781.004402 1.4921.25.620.4716.721.784302 2.6537.810.00.83711.93.164202 0.77912.83.160.2464.061.004321 1.2018.0 1.079.30 1.4015.4 2.0522.6 3.3829.6 2.787.74 3.0315.3 4.1224.3 1.407.14 1.636.15 1.7510.2 2.2014.4 2.377.50 2.4912.7 3.1118.1 4.4322.6 4.2213.3 5.1419.4 0.3795.68 0.3382.96 0.4424.87 0.6497.15 1.079.36 0.8802.45 0.9594.84 1.307.67 0.4432.26 0.5141.94 0.5543.21 0.6954.55 0.7502.37 0.7884.01 0.9845.71 1.407.14 1.334.22 1.636.15 10t10t 4.641.474221 3.161.004411 4.641.474311 6.812.154211 10.03.164111 4.641.474401 6.812.154301 10.03.164201 3.161.004330 3.161.004420 4.221.334320 5.621.784220 4.221.334410 5.621.784310 7.502.374210 10.03.164110 5.621.784400 7.502.374300 10.03.164200 可 信 限LD50可 信 限LD504321 剂量431.6剂量410.0 剂量310.0剂量33.16 剂量23.16剂量21.00 剂量11.00剂量10.316 各剂量组动物死亡数 (只) Horn氏表1-1 每组4只动物，组距3.16倍Horn氏表1-1 每组4只动物，组距3.16倍 0.03163163.160.01001001.003222 0.04722123.160.014966.91.003312 0.14966910.00.04722123.163212 0.22145.23.160.070014.31.003402 0.38526010.00.12282.13.163302 0.38625.93.160.1228.181.003321 0.72543.65.620.22913.81.783221 0.62116.1 0.91134.7 1.2281.8 2.4912.7 1.9650.9 1.079.36 1.307.67 1.4015.4 1.7626.4 2.0710.4 2.0522.6 2.4640.6 4.0811.4 3.3829.6 0.0472212.0 0.385260.0 0.12282.1 0.700143 0.31631.6 0.1965.09 0.28811.0 0.38625.9 0.7784.01 0.62116.1 0.3382.96 0.412 2.43 0.4424.87 0.5568.34 0.6543.30 0.6497.15 0.77912.8 1.293.59 1.079.36 0.014966.9 0.12282.1 0.038526.0 0.22145.2 0.10010.0 10t10t 3.161.003411 5.621.783311 10.03.163211 5.621.783401 10.03.163301 3.161.003330 3.161.003420 4.641.473320 6.812.153222 4.641.473410 6.812.153310 10.03.163210 6.812.153400 10.03.163300 3.161.004213 10.03.164113 3.161.004303 10.03.164203 3.161.004222 可 信 限LD50可 信 限LD504321 剂量431.6剂量410.0 剂量310.0剂量33.16 剂量23.16剂量21.00 剂量11.00剂量10.316 各剂量组动物死亡数 (只) Horn氏表氏表1-2 每组每组4只动物，组距只动物，组距3.16倍倍 0.385260 0.12282.1 0.22145.2 0.149669 0.0472212 0.1001000 0.12282.1 0.149669 0.700143 0.62116.1 0.83711.9 0.85836.9 1.00100 1.4921.2 1.2281.8 2.6537.8 0.12282.1 0.038526.0 0.070014.3 0.0472212 0.014966.9 0.0316316 0.038526.0 0.0472212 0.22145.2 0.196 5.09 0.2653.78 0.27111.7 0.31631.6 0.4716.72 0.38625.9 0.83711.9 10t10t 10.03.161410 3.161.001330 3.161.001420 10.03.161320 3.161.002321 10.03.162221 3.161.002411 10.03.162311 10.03.162401 3.161.002330 3.161.002420 5.621.782320 10.03.162220 5.621.782410 10.03.162310 10.03.162400 可 信 限LD50可 信 限LD504组3组2组1组 剂量431.6剂量410.0 剂量310.0剂量33.16 剂量23.16剂量21.00 剂量11.00剂量10.316 各剂量组动物死亡数 (只) Horn氏表氏表1-3 每组每组4只动物，组距只动物，组距3.16倍倍 剂量221.5 剂量410.0 剂量34.64 10t 1.1418.8 1.9523.9 3.3829.6 1.9111.3 2.8116.5 4.1224.3 1.8211.8 2.4314.8 2.259.57 2.6913.3 3.8417.2 4.8520.6 4.268.43 4.5113.3 5.5418.1 2.707.99 2.987.23 3.1310.1 3.6412.7 3.838.25 3.9611.7 4.5914.8 5.8117.2 5.6212.1 6.4215.6 可 信 限 4.64 6.81 10.0 4.64 6.81 10.0 4.64 5.99 4.64 5.99 7.74 10.0 5.99 7.74 10.0 4.64 4.64 5.62 6.81 5.62 6.81 8.25 10.0 6.81 8.25 10.0 LD50 剂量22.15 0.5318.752.150.2464.061.004312 0.90311.13.160.4195.141.474212 1.5713.74.640.7286.382.154112 0.8885.232.150.4122.431.004402 1.307.673.160.6053.561.474302 1.9111.34.640.8885.232.154202 0.8465.482.150.3932.551.004321 1.136.86 1.044.44 1.256.19 1.618.00 2.259.57 1.983.91 2.106.16 2.578.38 1.253.71 1.383.36 1.454.69 1.695.92 1.783.83 1.845.44 2.136.89 2.707.99 2.615.62 2.987.23 0.5243.18 0.4852.06 0.5802.87 0.7503.71 1.044.44 0.9181.82 0.9732.86 1.193.89 0.5811.72 0.6421.56 0.6742.18 0.7842.75 0.8251.78 0.8532.53 0.9893.20 1.253.71 1.212.61 1.383.36 10t10t 2.781.294221 2.151.004411 2.781.294311 3.591.674211 4.642.154111 2.781.294401 3.591.674301 4.642.154201 2.151.004330 2.151.004420 2.611.214320 3.161.474220 2.611.214410 3.161.474310 3.831.784210 4.642.154110 3.161.474400 3.831.784300 4.642.154200 可 信 限LD50可 信 限LD504321 剂量410.0剂量44.64 剂量34.64剂量32.15 剂量22.15剂量21.00 剂量11.00剂量10.464 各剂量组动物死亡数 (只) Horn氏表氏表2-1 每组每组4只动物，组距只动物，组距2.15倍倍 可 信 限LD50可 信 限LD50可 信 限LD504321 剂量221.5 剂量410.0 剂量34.64 10t 0.215100 0.28276.5 0.607165 0.78927.3 1.1487.7 1.1418.8 1.7426.7 1.5713.7 2.0222.9 2.4640.6 3.9611.7 3.3829.6 2.259.57 2.578.38 2.6913.3 3.1419.1 3.5010.3 3.8417.2 3.9325.5 5.5010.9 4.8520.6 0.28276.5 1.1487.7 0.52940.7 1.7058.9 1.0021.5 4.64 4.64 10.0 4.64 10.0 4.64 6.81 4.64 6.81 10.0 6.81 10.0 4.64 4.64 5.99 7.74 5.99 7.74 10.0 7.74 10.0 4.64 10.0 4.64 10.0 4.64 剂量22.15 0.10046.42.150.046421.51.003222 0.13135.52.150.060716.51.003312 0.28276.54.640.13135.52.153212 0.36612.72.150.1705.891.003402 0.52940.74.640.24618.92.153302 0.5318.752.150.2464.061.003321 0.80712.43.160.3755.751.473221 0.7286.38 0.94010.6 1.1418.8 1.845.44 1.5713.7 1.044.44 1.193.89 1.256.19 1.468.86 1.624.77 1.618.00 1.8211.8 2.565.05 2.259.57 0.13135.5 0.52940.7 0.24618.9 0.78927.3 0.46410.0 0.3382.96 0.4364.94 0.5318.75 0.8532.53 0.7286.38 0.4852.06 0.5541.81 0.5802.87 0.6764.11 0.7532.21 0.7503.71 0.8465.48 1.192.35 1.044.44 0.060716.5 0.24618.9 0.1148.77 0.36612.7 0.2154.64 10t10t 2.151.003411 3.161.473311 4.642.153211 3.161.473401 4.642.153301 2.151.003330 2.151.003420 2.781.293320 3.591.673220 2.781.293410 3.591.673310 4.642.153210 3.591.673400 4.642.153300 2.151.004213 4.642.154113 2.151.004303 4.642.154203 2.151.004222 剂量410.0剂量44.64 剂量34.64剂量32.15 剂量22.15剂量21.00 剂量11.00剂量10.464 各剂量组动物死亡数 (只) Horn氏表氏表2-2 每组每组4只动物，组距只动物，组距2.15倍倍 4 可 信 限LD50可 信 限LD50可 信 限LD504321 剂量221.5 剂量410.0 剂量34.64 10t 1.1487.7 0.52940.7 0.78927.3 0.607165 0.28276.5 0.464215 0.52940.7 0.607165 1.7058.9 1.5713.7 1.9111.3 1.9523.9 2.1546.4 2.8116.5 2.4640.6 4.1224.3 10.0 4.64 4.64 10.0 4.64 10.0 4.64 10.0 10.0 4.64 4.64 6.81 10.0 6.81 10.0 10.0 剂量22.15 0.52940.7 0.24618.9 0.36612.7 0.28276.5 0.13135.5 0.215100 0.24618.9 0.28276.5 0.78927.3 0.7286.38 0.8885.23 0.90311.1 1.0021.5 1.307.67 1.1418.8 1.9111.3 0.24618.9 0.1148.77 0.1705.89 0.13135.5 0.060716.5 0.10046.4 0.1148.77 0.13135.5 0.36612.7 0.3382.96 0.4122.43 0.4195.14 0.46410.0 0.6053.56 0.5318.75 0.8885.23 10t10t 4.642.151410 2.151.001330 2.151.001420 4.642.151320 2.151.002321 4.642.152221 2.151.002411 4.642.152311 4.642.152401 2.151.002330 2.151.002420 3.161.472320 4.642.152220 3.161.472410 4.642.152310 4.642.152400 剂量410.0剂量44.64 剂量34.64剂量32.15 剂量22.15剂量21.00 剂量11.00剂量10.464 各剂量组动物死亡数 (只) Horn氏表2-3 每组4只动物，组距2.15倍Horn氏表2-3 每组4只动物，组距2.15倍 2.2913.85.620.7234.371.785402 3.1821.48.251.016.772.615302 1.2011.23.650.3783.531.155331 1.2910.33.650.4083.271.155421 1.6914.14.870.5344.441.545321 2.3418.06.490.7405.702.055221 1.707.853.650.5372.481.155511 2.0011.9 2.5516.5 3.4821.6 3.406.98 3.5112.0 4.2617.6 1.717.35 2.019.92 2.026.24 2.169.25 2.5312.5 3.1915.7 2.827.08 2.9310.8 3.4214.7 4.3018.5 4.4711.2 5.0715.7 0.6323.75 0.8065.23 1.106.82 1.072.21 1.113.80 1.355.56 0.5422.32 0.6363.14 0.6381.97 0.6822.93 0.8013.95 1.014.97 0.8912.24 0.9273.41 1.084.64 1.365.84 1.413.55 1.604.95 10t10t 4.871.545411 6.492.055311 8.662.745211 4.871.545501 6.492.055401 8.662.745301 3.551.125430 4.471.415330 3.551.125520 4.471.415420 5.621.785320 7.082.245220 4.471.415510 5.621.785410 7.082.245310 8.912.825210 5.621.785500 7.082.245400 8.912.825300 可 信 限LD50可 信 限LD504321 剂量431.6剂量410.0 剂量310.0剂量33.16 剂量23.16剂量21.00 剂量11.00剂量10.316 各剂量组动物死亡数 (只) Horn氏表氏表3-1 每组每组5只动物，组距只动物，组距3.16倍倍 0.87116.83.830.2765.331.214331 0.96615.23.830.3054.801.214421 1.3423.65.620.4237.481.784321 1.8836.38.250.59411.52.614221 1.4010.53.830.4423.321.214511 1.6718.95.620.5285.981.784411 2.0832.78.250.65810.42.614311 3.279.68 3.0122.6 1.479.10 1.7613.5 1.807.42 1.9112.4 2.2518.7 2.8326.5 2.63 9.01 2.67 15.3 3.06 24.5 4.53 9.31 4.03 18.6 0.81917.9 1.3723.0 1.0014.6 1.5320.7 2.4328.1 1.758.39 1.033.06 0.9537.15 0.4632.88 0.5554.27 0.5682.35 0.6043.92 0.7115.93 0.8968.37 0.8332.85 0.8435.00 0.9687.75 1.432.94 1.275.88 0.2595.67 0.4347.28 0.3184.62 0.4846.53 0.7688.87 0.5542.65 10t10t 5.621.784501 8.252.614401 3.651.154430 4.871.544330 3.651.154520 4.871.544420 6.492.054320 8.662.744220 4.871.544510 6.492.054410 8.662.744310 6.492.054500 8.662.744400 3.831.215322 5.621.785222 3.831.215412 5.621.785312 8.252.615212 3.831.215502 可 信 限LD50可 信 限LD504321 剂量431.6剂量410.0 剂量310.0剂量33.16 剂量23.16剂量21.00 剂量11.00剂量10.316 各剂量组动物死亡数 (只) Horn氏表氏表3-2 每组每组5只动物，组距只动物，组距3.16倍倍 0.46738.14.220.14812.11.333331 0.54532.6 0.77173.0 0.95818.6 1.0553.4 2.5122.4 1.1313.0 1.3723.0 1.489.94 1.5320.7 1.7638.6 2.2913.8 2.1631.5 3.7718.1 0.43341.0 0.83067.8 0.59230.0 0.97058.0 1.4112.6 1.7033.0 0.17210.3 0.24423.1 0.3035.87 0.33316.9 0.7937.10 0.3564.12 0.4347.28 0.4673.14 0.4846.53 0.55812.2 0.7234.37 0.6849.95 1.195.71 0.13713.0 0.26221.4 0.1879.49 0.30718.3 0.4463.99 0.53910.4 10t10t 4.221.333421 7.502.373321 4.221.333511 7.502.373411 7.502.373501 3.831.213430 5.621.783330 3.831.213520 5.621.783420 8.252.613320 5.621.783510 8.252.613410 8.252.613500 4.221.334322 7.502.374222 4.221.334412 7.500.37 ?4312 4.221.334502 7.502.374402 可 信 限LD50可 信 限LD504321 剂量431.6剂量410.0 剂量310.0剂量33.16 剂量23.16剂量21.00 剂量11.00剂量10.316 各剂量组动物死亡数 (只) Horn氏表氏表3-3 每组每组5只动物，组距只动物，组距3.16倍倍 可 信 限LD50可 信 限LD50可 信 限LD504321 剂量221.5 剂量410.0 剂量34.64 10t 3.7412.4 4.6616.6 2.4310.8 2.5510.2 3.0512.5 3.8014.8 3.078.51 3.4211.2 4.0214.0 4.9416.7 4.877.87 4.9811.3 5.6614.6 3.088.14 3.439.95 3.447.30 3.609.50 4.0011.6 4.6713.5 4.307.94 4.4110.5 4.8912.9 5.7015.0 5.8410.8 6.3613.5 6.81 8.80. 5.11 5.11 6.19 7.50 5.11 6.19 7.50 9.09 6.19 7.50 9.09 5.01 5.84 5.01 5.84 6.81 7.94 5.84 6.81 7.94 9.26 6.81 7.94 9.26 剂量22.15 1.745.763.160.8062.671.475402 2.167.714.081.003.581.905302 1.134.992.370.5322.321.105331 1.194.742.370.5502.201.105421 1.425.822.870.6582.701.335321 1.766.873.480.8183.191.625221 1.423.952.370.6611.831.105511 1.595.20 1.876.49 2.297.75 2.263.65 2.315.24 2.636.76 1.433.78 1.594.62 1.603.39 1.674.41 1.865.38 2.176.28 2.003.69 2.054.88 2.275.99 2.656.98 2.715.01 2.956.26 0.7372.41 0.8663.01 1.063.60 1.051.70 1.072.43 1.223.14 0.6651.75 0.7402.14 0.7411.57 0.7752.05 0.8622.50 1.012.91 0.9261.71 0.9512.27 1.052.78 1.233.24 1.262.33 1.372.91 10t10t 2.871.335411 3.481.625311 4.221.965211 2.871.335501 3.481.625401 4.221.965301 2.331.085430 2.711.265330 2.331.085520 2.711.265420 3.161.475320 3.691.715220 2.711.265510 3.161.475410 3.691.715310 4.302.005210 3.161.475500 3.691.715400 4.302.005300 剂量410.0剂量44.64 剂量34.64剂量32.15 剂量22.15剂量21.00 剂量11.00剂量10.464 各剂量组动物死亡数 (只) Horn氏表氏表4-1 每组每组5只动物，组距只动物，组距2.15倍倍 可 信 限LD50可 信 限LD50可 信 限LD504321 剂量221.5 剂量410.0 剂量34.64 10t 1.9714.2 13.2 17.7 23.6 10.3 15.3 22.0 9.78 17.2 9.39 12.2 8.19 11.5 15.2 4.3119.1 4.119.33 4.1413.6 4.5418.2 5.909.53 5.4615.1 1.8914.8 2.6617.4 2.1612.9 2.8616.2 3.8919.9 3.138.89 5.28 5.28 6.81 8.80 5.28 6.81 8.80 6.81 8.80 5.11 6.19 5.11 6.19 7.50 9.09 6.19 7.50 9.09 7.50 9.09 5.28 6.81 5.28 6.81 8.80 5.28 剂量22.15 0.9126.572.450.4233.051.144331 0.9776.132.450.4542.851.144421 1.218.243.160.5643.821.474321 1.5211.04.080.7065.091.904221 1.254.792.450.5812.221.144511 1.417.103.160.6543.301.474411 1.6310.24.080.7574.751.904311 2.204.54 2.097.99 1.294.36 1.465.67 1.483.80 1.545.36 1.727.06 2.008.88 1.914.33 1.926.30 2.118.44 2.744.42 2.537.02 0.8756.85 1.248.10 1.005.98 1.337.53 1.819.23 1.454.13 1.022.11 0.9693.71 0.5992.02 0.6762.63 0.6861.77 0.7152.40 0.7973.28 0.9304.12 0.8852.01 0.8932.92 0.9783.92 1.272.05 1.183.26 0.4063.18 0.5733.76 0.4662.77 0.6163.50 0.8394.29 0.6741.92 10t10t 3.161.474501 4.081.904401 2.371.104430 2.871.334330 2.371.104520 2.871.334420 3.481.