LD50计算方法

1实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•熟悉经口灌胃技术•了解急性毒性试验的设计LD50计算方法•改良寇氏法：计算简便，准确率高，常用•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<20%•最高剂量组死亡>80%•死亡不倒置二、原理•健康动物Î按体重随机分组Î一次或多次经口给药Î观察中毒特征或死亡Î剂量-反应关系Î求出LD50Î毒性分级三、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1.43.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)p·q12345i=∑p=计算•M=Xk-I(∑p-0.5)Sm=∑npqiM：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（M±1.96Sm）LD50=log-1MLD50计算方法之—Horn氏法原理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。0.386～25.93.160.122～8.181.0043120.858～36.95.620.271～11.71.7842121.96～50.910.00.621～16.13.1641120.837～11.93.160.265～3.781.0044021.49～21.25.620.471～6.721.7843022.65～37.810.00.837～11.93.1642020.779～12.83.160.246～4.061.0043211.20～18.01.07～9.301.40～15.42.05～22.63.38～29.62.78～7.743.03～15.34.12～24.31.40～7.141.63～6.151.75～10.22.20～14.42.37～7.502.49～12.73.11～18.14.43～22.6～4.22～13.35.14～19.40.379～5.680.338～2.960.442～4.870.649～7.151.07～9.360.880～2.450.959～4.841.30～7.670.443～2.260.514～1.940.554～3.210.695～4.550.750～2.370.788～4.010.984～5.711.40～7.14～1.33～4.221.63～6.15×10t×10t4.641.4742213.161.0044114.641.4743116.812.15421110.03.1641114.641.4744016.812.15430110.03.1642013.161.0043303.161.0044204.221.3343205.621.7842204.221.3344105.621.7843107.502.37421010.03.1641105.621.7844007.502.37430010.03.164200可信限LD50可信限LD504321剂量4＝31.6剂量4＝10.0剂量3＝10.0剂量3＝3.16剂量2＝3.16剂量2＝1.00剂量1＝1.00剂量1＝0.316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0.0316～3163.160.0100～1001.0032220.0472～2123.160.0149～66.91.0033120.149～66910.00.0472～2123.1632120.221～45.23.160.0700～14.31.0034020.385～26010.00.122～82.13.1633020.386～25.93.160.122～8.181.0033210.725～43.65.620.229～13.81.7832210.621～16.10.911～34.71.22～81.82.49～12.71.96～50.91.07～9.361.30～7.671.40～15.41.76～26.42.07～10.42.05～22.62.46～40.64.08～11.43.38～29.60.0472～212.00.385～260.00.122～82.10.700～1430.316～31.60.196～5.090.288～11.00.386～25.90.778～4.010.621～16.10.338～2.960.412～2.430.442～4.870.556～8.340.654～3.300.649～7.150.779～12.81.29～3.591.07～9.360.0149～66.90.122～82.10.0385～26.00.221～45.20.100～10.0×10t×10t3.161.0034115.621.78331110.03.1632115.621.78340110.03.1633013.161.0033303.161.0034204.641.4733206.812.1532224.641.4734106.812.15331010.03.1632106.812.15340010.03.1633003.161.00421310.03.1641133.161.00430310.03.1642033.161.004222可信限LD50可信限LD504321剂量4＝31.6剂量4＝10.0剂量3＝10.0剂量3＝3.16剂量2＝3.16剂量2＝1.00剂量1＝1.00剂量1＝0.316各剂量组动物死亡数(只)Horn氏表1-2每组4只动物，组距3.16倍0.385～2600.122～82.10.221～45.20.149～6690.0472～2120.100～10000.122～82.10.149～6690.700～1430.621～16.10.837～11.90.858～36.91.00～1001.49～21.21.22～81.82.65～37.80.122～82.10.0385～26.00.0700～14.30.0472～2120.0149～66.90.0316～3160.0385～26.00.0472～2120.221～45.20.196～5.090.265～3.780.271～11.70.316～31.60.471～6.720.386～25.90.837～11.9×10t×10t10.03.1614103.161.0013303.161.00142010.03.1613203.161.00232110.03.1622213.161.00241110.03.16231110.03.1624013.161.0023303.161.0024205.621.78232010.03.1622205.621.78241010.03.16231010.03.162400可信限LD50可信限LD504组3组2组1组剂量4＝31.6剂量4＝10.0剂量3＝10.0剂量3＝3.16剂量2＝3.16剂量2＝1.00剂量1＝1.00剂量1＝0.316各剂量组动物死亡数(只)Horn氏表1-3每组4只动物，组距3.16倍剂量2＝21.5剂量4＝10.0剂量3＝4.64×10t1.14～18.81.95～23.93.38～29.61.91～11.32.81～16.54.12～24.31.82～11.82.43～14.82.25～9.572.69～13.33.84～17.24.85～20.64.26～8.434.51～13.35.54～18.12.70～7.992.98～7.233.13～10.13.64～12.73.83～8.253.96～11.74.59～14.85.81～17.2～5.62～12.16.42～15.6可信限4.646.8110.04.646.8110.04.645.994.645.997.7410.05.997.7410.04.644.645.626.815.626.818.2510.06.818.2510.0LD50剂量2＝2.150.531～8.752.150.246～4.061.0043120.903～11.13.160.419～5.141.4742121.57～13.74.640.728～6.382.1541120.888～5.232.150.412～2.431.0044021.30～7.673.160.605～3.561.4743021.91～11.34.640.888～5.232.1542020.846～5.482.150.393～2.551.0043211.13～6.861.04～4.441.25～6.191.61～8.002.25～9.571.98～3.912.10～6.162.57～8.381.25～3.711.38～3.361.45～4.691.69～5.921.78～3.831.84～5.442.13～6.892.70～7.99～2.61～5.622.98～7.230.524～3.180.485～2.060.580～2.870.750～3.711.04～4.440.918～1.820.973～2.861.19～3.890.581～1.720.642～1.560.674～2.180.784～2.750.825～1.780.853～2.530.989～3.201.25～3.71～1.21～2.611.38～3.36×10t×10t2.781.2942212.151.0044112.781.2943113.591.6742114.642.1541112.781.2944013.591.6743014.642.1542012.151.0043302.151.0044202.611.2143203.161.4742202.611.2144103.161.4743103.831.7842104.642.1541103.161.4744003.831.7843004.642.154200可信限LD50可信限LD504321剂量4＝10.0剂量4＝4.64剂量3＝4.64剂量3＝2.15剂量2＝2.15剂量2＝1.00剂量1＝1.00剂量1＝0.464各剂量组动物死亡数(只)Horn氏表2-1每组4只动物，组距2.15倍可信限LD50可信限LD50可信限LD504321剂量2＝21.5剂量4＝10.0剂量3＝4.64×10t0.215～1000.282～76.50.607～1650.789～27.31.14～87.71.14～18.81.74～26.71.57～13.72.02～22.92.46～40.63.96～11.73.38～29.62.25～9.572.57～8.382.69～13.33.14～19.13.50～10.33.84～17.23.93～25.55.50～10.94.85～20.60.282～76.51.14～87.70.529～40.71.70～58.91.00～21.54.644.6410.04.6410.04.646.814.646.8110.06.8110.04.644.645.997.745.997.7410.07.7410.04.6410.04.6410.04.64剂量2＝2.150.100～46.42.150.0464～21.51.0032220.131～35.52.150.0607～16.51.0033120.282～76.54.640.131～35.52.1532120.366～12.72.150.170～5.891.0034020.529～40.74.640.246～18.92.1533020.531～8.752.150.246～4.061.0033210.807～12.43.160.375～5.751.4732210.728～6.380.940～10.61.14～18.81.84～5.441.57～13.71.04～4.441.19～3.891.25～6.191.46～8.861.62～4.771.61～8.001.82～11.82.56～5.052.25～9.570.131～35.50.529～40.70.246～18.90.789～27.30.464～10.00.338～2.960.436～4.940.531～8.750.853～2.530.728～6.380.485～2.060.554～1.810.580～2.870.676～4.110.753～2.210.750～3.710.846～5.481.19～2.351.04～4.440.0607～16.50.246～18.90.114～8.770.366～12.70.215～4.64×10t×10t2.151.0034113.161.4733114.642.1532113.161.4734014.642.1533012.151.0033302.151.0034202.781.2933203.591.6732202.781.2934103.591.6733104.642.1532103.591.6734004.642.1533002.151.0042134.642.1541132.151.0043034.642.1542032.151.004222剂量4＝10.0剂量4＝4.64剂量3＝4.64剂量3＝2.15剂量2＝2.15剂量2＝1.00剂量1＝1.00剂量1＝0.464各剂量组动物死亡数(只)Horn氏表2-2每组4只动物，组距2.15倍4可信限LD50可信限LD50可信限LD504321剂量2＝21.5剂量4＝10.0剂量3＝4.64×10t1.14～87.70.529～40.70.789～27.30.607～1650.282～76.50.464～2150.529～40.70.607～1651.70～58.91.57～13.71.91～11.31.95～23.92.15～46.42.81～16.52.46～40.64.12～24.310.04.644.6410.04.6410.04.6410.010.04.644.646.8110.06.8110.010.0剂量2＝2.150.529～40.70.246～18.90.366～12.70.282～76.50.131～35.50.215～1000.246～18.90.282～76.50.789～27.30.728～6.380.888～5.230.903～11.11.00～21.51.30～7.671.14～18.81.91～11.30.246～18.90.114～8.770.170～5.890.131～35.50.0607～16.50.100～46.40.114～8.770.131～35.50.366～12.70.338～2.960.412～2.430.419～5.140.464～10.00.605～3.560.531～8.750.888～5.23×10t×10t4.642.1514102.151.0013302.151.0014204.642.1513202.151.0023214.642.1522212.151.0024114.642.1523114.642.1524012.151.0023302.151.0024203.161.4723204.642.1522203.161.4724104.642.1523104.642.152400剂量4＝10.0剂量4＝4.64剂量3＝4.64剂量3＝2.15剂量2＝2.15剂量2＝1.00剂量1＝1.00剂量1＝0.464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2.29～13.85.620.723～4.371.7854023.18～21.48.251.01～6.772.6153021.20～11.23.650.378～3.531.1553311.29～10.33.650.408～3.271.1554211.69～14.14.870.534～4.441.5453212.34～18.06.490.740～5.702.0552211.70～7.853.650.537～2.481.1555112.00～11.92.55～16.53.48～21.63.40～6.983.51～12.04.26～17.61.71～7.352.01～9.922.02～6.242.16～9.252.53～12.53.19～15.72.82～7.082.93～10.83.42～14.74.30～18.5～4.47～11.25.07～15.70.632～3.750.806～5.231.10～6.821.07～2.211.11～3.801.35～5.560.542～2.320.636～3.140.638～1.970.682～2.930.801～3.951.01～4.970.891～2.240.927～3.411.08～4.641.36～5.84～1.41～3.551.60～4.95×10t×10t4.871.5454116.492.0553118.662.7452114.871.5455016.492.0554018.662.7453013.551.1254304.471.4153303.551.1255204.471.4154205.621.7853207.082.2452204.471.4155105.621.7854107.082.2453108.912.8252105.621.7855007.082.2454008.912.825300可信限LD50可信限LD504321剂量4＝31.6剂量4＝10.0剂量3＝10.0剂量3＝3.16剂量2＝3.16剂量2＝1.00剂量1＝1.00剂量1＝0.316各剂量组动物死亡数(只)Horn氏表3-1每组5只动物，组距3.16倍0.871～16.83.830.276～5.331.2143310.966～15.23.830.305～4.801.2144211.34～23.65.620.423～7.481.7843211.88～36.38.250.594～11.52.6142211.40～10.53.830.442～3.321.2145111.67～18.95.620.528～5.981.7844112.08～32.78.250.658～10.42.6143113.27～9.683.01～22.61.47～9.101.76～13.51.80～7.421.91～12.42.25～18.72.83～26.52.63～9.012.67～15.33.06～24.54.53～9.314.03～18.60.819～17.91.37～23.01.00～14.61.53～20.72.43～28.11.75～8.391.03～3.060.953～7.150.463～2.880.555～4.270.568～2.350.604～3.920.711～5.930.896～8.370.833～2.850.843～5.000.968～7.751.43～2.941.27～5.880.259～5.670.434～7.280.318～4.620.484～6.530.768～8.870.554～2.65×10t×10t5.621.7845018.252.6144013.651.1544304.871.5443303.651.1545204.871.5444206.492.0543208.662.7442204.871.5445106.492.0544108.662.7443106.492.0545008.662.7444003.831.2153225.621.7852223.831.2154125.621.7853128.252.6152123.831.215502可信限LD50可信限LD504321剂量4＝31.6剂量4＝10.0剂量3＝10.0剂量3＝3.16剂量2＝3.16剂量2＝1.00剂量1＝1.00剂量1＝0.316各剂量组动物死亡数(只)Horn氏表3-2每组5只动物，组距3.16倍～～～～～～～～～～～～0.467～38.14.220.148～12.11.3333310.545～32.60.771～73.00.958～18.61.05～53.42.51～22.41.13～13.01.37～23.01.48～9.941.53～20.71.76～38.62.29～13.82.16～31.53.77～18.10.433～41.00.830～67.80.592～30.00.970～58.01.41～12.61.70～33.00.172～10.30.244～23.10.303～5.870.333～16.90.793～7.100.356～4.120.434～7.280.467～3.140.484～6.530.558～12.20.723～4.370.684～9.951.19～5.710.137～13.00.262～21.40.187～9.490.307～18.30.446～3.990.539～10.4×10t×10t4.221.3334217.502.3733214.221.3335117.502.3734117.502.3735013.831.2134305.621.7833303.831.2135205.621.7834208.252.6133205.621.7835108.252.6134108.252.6135004.221.3343227.502.3742224.221.3344127.500.37?43124.221.3345027.502.374402可信限LD50可信限LD504321剂量4＝31.6剂量4＝10.0剂量3＝10.0剂量3＝3.16剂量2＝3.16剂量2＝1.00剂量1＝1.00剂量1＝0.316各剂量组动物死亡数(只)Horn氏表3-3每组5只动物，组距3.16倍可信限LD50可信限LD50可信限LD504321剂量2＝21.5剂量4＝10.0剂量3＝4.64×10t3.74～12.44.66～16.62.43～10.82.55～10.23.05～12.53.80～14.83.07～8.513.42～11.24.02～14.04.94～16.74.87～7.874.98～11.35.66～14.63.08～8.143.43～9.953.44～7.303.60～9.504.00～11.64.67～13.54.30～7.944.41～10.54.89～12.95.70～15.0～5.84～10.86.36～13.56.818.80.5.115.116.197.505.116.197.509.096.197.509.095.015.845.015.846.817.945.846.817.949.266.817.949.26剂量2＝2.151.74～5.763.160.806～2.671.4754022.16～7.714.081.00～3.581.9053021.13～4.992.370.532～2.321.1053311.19～4.742.370.550～2.201.1054211.42～5.822.870.658～2.701.3353211.76～6.873.480.818～3.191.6252211.42～3.952.370.661～1.831.1055111.59～5.201.87～6.492.29～7.752.26～3.652.31～5.242.63～6.761.43～3.781.59～4.621.60～3.391.67～4.411.86～5.382.17～6.282.00～3.692.05～4.882.27～5.992.65～6.98～2.71～5.012.95～6.260.737～2.410.866～3.011.06～3.601.05～1.701.07～2.431.22～3.140.665～1.750.740～2.140.741～1.570.775～2.050.862～2.501.01～2.910.926～1.710.951～2.271.05～2.781.23～3.24～1.26～2.331.37～2.91×10t×10t2.871.3354113.481.6253114.221.9652112.871.3355013.481.6254014.221.9653012.331.0854302.711.2653302.331.0855202.711.2654203.161.4753203.691.7152202.711.2655103.161.4754103.691.7153104.302.0052103.161.4755003.691.7154004.302.005300剂量4＝10.0剂量4＝4.64剂量3＝4.64剂量3＝2.15剂量2＝2.15剂量2＝1.00剂量1＝1.00剂量1＝0.464各剂量组动物死亡数(只)Horn氏表4-1每组5只动物，组距2.15倍可信限LD50可信限LD50可信限LD504321剂量2＝21.5剂量4＝10.0剂量3＝4.64×10t1.97～14.2～13.2～17.7～23.6～10.3～15.3～22.0～9.78～17.2～9.39～12.2～8.19～11.5～15.24.31～19.14.11～9.334.14～13.64.54～18.25.90～9.535.46～15.11.89～14.82.66～17.42.16～12.92.86～16.23.89～19.93.13～8.895.285.286.818.805.286.818.806.818.805.116.195.116.197.509.096.197.509.097.509.095.286.815.286.818.805.28剂量2＝2.150.912～6.572.450.423～3.051.1443310.977～6.132.450.454～2.851.1444211.21～8.243.160.564～3.821.4743211.52～11.04.080.706～5.091.9042211.25～4.792.450.581～2.221.1445111.41～7.103.160.654～3.301.4744111.63～10.24.080.757～4.751.9043112.20～4.542.09～7.991.29～4.361.46～5.671.48～3.801.54～5.361.72～7.062.00～8.881.91～4.331.92～6.302.11～8.442.74～4.422.53～7.020.875～6.851.24～8.101.00～5.981.33～7.531.81～9.231.45～4.131.02～2.110.969～3.710.599～2.020.676～2.630.686～1.770.715～2.400.797～3.280.930～4.120.885～2.010.893～2.920.978～3.921.27～2.051.18～3.260.406～3.180.573～3.760.466～2.770.616～3.500.839～4.290.674～1.92×10t×10t3.161.4745014.081.9044012.371.1044302.871.3343302.371.1045202.871.3344203.481.6243204.221.9642202.871.3345103.481.6244104.221.9643103.481.6245004.221.9644002.451.1453223.161.4752222.451.1454123.161.4753124.081.9052122.451.145502剂量4＝10.0剂量4＝4.64剂量3＝4.64剂量3＝2.15剂量2＝2.15剂量2＝1.00剂量1＝1.00剂量1＝0.464各剂量组动物死亡数(只)Horn氏表4-2每组5只动物，组距2.15倍5可信限LD50可信限LD50可信限LD504321剂量2＝21.5剂量4＝10.0剂量3＝4.64×10t～～～～～～1.30～24.41.44～22.01.81～37.62.09～15.12.23～30.53.98～17.12.33～11.92.66～17.42.79～9.962.86～16.23.15～24.63.74～12.43.60～21.55.22～14.81.23～25.61.90～35.81.52～20.82.11～32.32.71～11.73.07～22.25.625.628.255.628.258.255.286.815.286.818.806.818.808.805.628.255.628.255.628.25剂量2＝2.15～～～～～～～～～～～～0.602～11.32.610.279～5.261.2133310.668～10.20.840～17.50.972～7.011.04～14.21.85～7.961.08～5.541.24～8.101.30～4.621.33～7.531.46～11.41.74～5.761.67～9.972.42～6.890.573～11.90.883～16.60.705～9.660.980～15.01.26～5.421.43～10.30.310～4.740.390～8.110.451～3.250.481～6.580.856～3.690.503～2.570.573～3.760.602～2.150.616～3.500.673～5.300.806～2.670.777～4.631.12～3.200.266～5.520.410～7.720.327～4.480.455～6.950.583～2.520.662～4.78×10t×10t2.611.2134213.831.7833212.611.2135113.831.7834113.831.7835012.451.1434303.161.4733302.451.1435203.161.4734204.081.9033203.161.4735104.081.9034104.081.9035002.611.2143223.831.7842222.611.2144123.831.7843122.611.2145023.831.784402剂量4＝10.0剂量4＝4.64剂量3＝4.64剂量3＝2.15剂量2＝2.15剂量2＝1.00剂量1＝1.00剂量1＝0.464各剂量组动物死亡数(只)Horn氏表4-3每组5只动物，组距2.15倍