Some standard content:
ICS07.100
National Standard of the People's Republic of China
GB15193.3—2003
Replaces GB15193.3—1994
Toxicity test
Acute toxicity test
Issued on September 24, 2003
Ministry of Health of the People's Republic of China
Standardization Administration of the People's Republic of China
Implemented on May 1, 2004
GB15193.3—2003
The full text of this standard is mandatory.
This standard replaces GB15193.3—1994 "Acute toxicity test". Compared with GB15193.3-1994, this standard has been revised as follows: the specific content of the test substances has been added to the scope: chemical, biological and physical factors involved in the production, processing, storage, transportation and sales of food that may cause harm to health; the test objects include food additives (including nutritional fortifiers), new food resources and their ingredients, new resource foods, irradiated foods, food containers and packaging materials, food tools, equipment, detergents, disinfectants, pesticide residues, veterinary drug residues, microorganisms used in the food industry, etc.; the content of "experimental animals" has been revised: "adult mice and rats" has been changed to "adult rats and/or mice"; in the "operation procedures", the original 4.2.4 content "if the toxicity of the test substance is estimated to be very low or the solubility is Very low, but as long as the dose reaches 10g/kg body weight and still does not cause the death of the animal, further administration can be stopped"; in several commonly used acute toxicity test design methods (original 4.3 method), a test method "maximum tolerated dose method" is added,
- In Appendix A, the 17th row of the confidence limit in Table A.1 "4.94~~1.67" is changed to *4.94~16.7"; - Appendix G "Acute toxicity median lethal dose (LDso) result report table" is deleted. Appendices A, B, C, D, E and F of this standard are normative appendices. From the date of implementation of this standard, GB15193.3-1994 will be abolished at the same time. This standard is proposed by the Ministry of Health of the People's Republic of China This standard was drafted by: Institute of Nutrition and Food Safety, Chinese Center for Disease Control and Prevention, Nanjing Medical College. The main drafters of this standard are: Dai Yin, Ma Fenglou, Guo Shiping. This standard was first issued in 1994 and this is the first revision. 18
1 Scope
Acute toxicity test
This standard specifies the basic technical requirements for acute toxicity tests. GB15193.3—2003
This standard is applicable to the evaluation of the acute toxic effects of chemical, biological and physical factors that may cause harm to health during the production, processing, storage, transportation and sales of food. The inspection objects include food additives (including nutritional fortifiers), new food resources and their ingredients, new resource foods, radiation Food, food containers and packaging materials, food tools, equipment, detergents, disinfectants, pesticide residues, veterinary drug residues, microorganisms used in the food industry, etc.
2 Terms and Definitions
The following terms and definitions apply to this standard. 2.1
medianlethaldose, LDso
The median lethal dose
is the single dose of the test substance that is expected to cause 50% mortality in animals after oral administration of the test substance. This dose is an estimated value obtained through statistics. Its unit is the number of milligrams, grams or milliliters of the test substance ingested per kilogram of body weight, that is, mg/kg body weight, g/kg body weight or mL/kg body weight.
Maximum tolerated dose method test of maximum tolerated dose After administering 20 animals with the maximum use concentration and maximum gavage volume, and observing them for 7 to 14 days in a row, if no animal dies, then the MTD is greater than ×Xg/kg body weight.
3 Principle
After oral administration of the test substance once or multiple times within 24 hours, observe the toxic reactions of the animals in a short period of time, including lethal and non-lethal index parameters. The lethal dose is usually expressed as the median lethal dose I.D5. 4 Experimental animals
Generally, adult mice or/and rats of both sexes are used. The weight of mice is 18g~22g, and the weight of rats is 180g~220g. If the toxicity of the test substance is already known, animals that are sensitive to it should also be selected for testing, such as ducks for aflatoxin and birds for cyanide. After the animals are purchased, they will adapt to the environment for 3 to 5 days. 5 Operation steps
5.1 Handling of test substances
The test substance should be dissolved or suspended in a suitable medium. Generally, water or edible vegetable oil is used as the solvent. Carboxymethyl cellulose, gelatin, starch, etc. can be considered to be prepared into a suspension; when it cannot be prepared into a suspension, it can be prepared into other forms (such as paste, etc.). Dimethyl sulfoxide can be used when necessary. However, organic chemical solvents with obvious toxicity cannot be used. If toxic solvents are used, a solvent control group should be set up for observation. 5.2 Administration of the test substance
5.2.1 Route: oral.
5.2.2 Fasting before the test: The animals should be fasted overnight (generally fasting for about 16 hours, and drinking water is not restricted). 5.2.3 Capacity: The gavage capacity of each dose group is the same (mL/kg body weight), the commonly used capacity for mice is 0.4mL/20g body weight; the commonly used capacity for rats is 2.0mL/200g body weight.
GB15193.3—2003
5.2.4 Method: Generally, the test substance is administered once. It can also be administered multiple times within a day (each time with an interval of 4h to 6h, no more than 3 times within 24h, and the maximum dose is reached as much as possible, and it is combined as a single dose). 5.3 Several commonly used acute toxicity test design methods 5.3.1 Horn method
5.3.1.1 Preliminary test
The following methods can be selected according to the properties of the test substance and known data: Generally, 10100 and 1000 mg/kg body weight are used, and 2 to 3 animals are used for preliminary test. According to the death within 24 hours, the possible range of LDso is estimated, and the dose group for the formal test is determined. It is also possible to simply use a dose, such as 215 mg/kg body weight, and use 5 animals for preliminary test. Observe the poisoning symptoms of the animals within 2 hours. If the symptoms are severe and it is estimated that most animals may die, a dose series lower than 215 mg/kg body weight can be used. On the contrary, if the symptoms are mild, a dose series higher than this dose can be used. If there is relevant literature, no preliminary test is required. 5.3.1.2 Number of animals
Usually 5 animals are used in each group.
5.3.1.3 Commonly used dose series
t=0,±1,±2,±3
Because the dose interval is smaller than 3.16
X10°, t=0,±1,土2,士3, the result is more accurate. In general experiments, 5 groups can be designed according to the above dose series, that is, one group is added below the lowest dose group or above the highest dose group compared with the original method, so that it is easy to get the result when looking up the table.
5.3.1.4 Formal test
The animals are kept and observed in the experimental animal room for 3 to 5 days to adapt to the environment. After proving that they are healthy animals, they are randomly divided into groups. After the test substance is administered, it is generally observed for 7 or 14 days. If there are still deaths on the 4th day after administration, it is necessary to observe for 14 days, and if necessary, it is extended to 28 days. Record the number of deaths, find the LD50 by looking up the table, and record the time of death and poisoning symptoms. 5.3.1.5 Advantages and Disadvantages of this Method
The advantage is that it is simple and easy to use, and it saves animals; the disadvantage is that the confidence limit range of the obtained LDso is large and not accurate enough. However, after years of practical application and verification, the results obtained by the same test substance and the Korbor method are very similar. Therefore, the results of its determination should be considered reliable and valid. 5.3.2 Korbor Method
5.3.2.1 Preliminary Test
Unless otherwise required, the dose that causes all animals to die or more than 90% of the animals to die and the dose that causes no animals to die or less than 10% of the animals to die should generally be obtained in the preliminary test, which will be used as the highest and lowest doses for the formal test, respectively. 5.3.2.2 Number of Animals
Unless otherwise required, 5 to 10 dose groups are generally set up, with 6 to 10 animals in each group. 5.3.2.3 Dose
Convert the highest and lowest doses obtained from the preliminary test into common logarithms, and then divide the logarithmic difference between the highest and lowest doses into several dose groups with equal (or unequal) logarithmic intervals according to the required number of groups. 5.3.2.4 Calculation and statistics of test results
5.3.2.4.1 List the test data and its calculation table, including the dose of each group (mg/kg body weight, g/kg body weight), the logarithm of the dose (X), the number of animals (n), the number of animal deaths (r), the percentage of animal deaths (P, expressed as a decimal), and other calculation data items required in the statistical formula. 5.3.2.4.2LDso calculation formula
According to the test conditions and test results, one of the following three formulas can be selected to calculate logLDso, and then find its natural number, that is, I.Dso (mg/kg body weight, g/kg body weight). 20
5.3.2.4.2.1 Any result obtained according to this test design can be calculated using formula (1). logLDso = (X: +XμI).(P+1 - P)2
Where:
X, and Xi+1 and P:+1 and P.--are the logarithms of the doses of two adjacent groups and the percentage of animal deaths respectively. 5.3.2.4.2.2 According to this test design and when the logarithms of the doses between the groups are equidistant, formula (2) can be used. logLD5o XK
Where:
-The logarithm of the highest dose, the others are the same as formula (1). (P,+P1)
GB15193.3—2003
5.3.2.4.2.3 If the test conditions are the same as those in 5.3.2.4.2.2 and the death percentages of animals in the highest and lowest dose groups are 100 (all dead) and 0 (all alive), respectively, the convenient formula (3) can be used. logLDso = XK d(ZP- 0.5)
Where:
The sum of the death percentages of animals in each group, and the rest are the same as formula (2). 5.3.2.4.3 Standard error and 95% confidence limits
5.3.2.4.3.1 Standard error of logLD5o (S): SiogLDso
5.3.2.4.3.2 95% confidence limits (X):
/ZP(1- P,)
X = log-1(logLDso ± 1. 96 . SlogLDs. i（3）
（4）
·（5)
This method is easy to understand, simple to calculate, has a small confidence limit, and reliable results. It is particularly applicable when the acute toxicity of the test substance is not well known before the test.
5.3.3 Probability unit
5.3.3.1 Preliminary test
Logarithmic graphical method
Use 2 to 3 animals in each group to find the dose that causes total mortality and total survival. 5.3.3.2 Number of animals
Generally, each group should have no less than 10 animals, and the number of animals in each group does not necessarily have to be equal. 5.3.3.3 Dosage and grouping
-Generally in the preliminary test Six or more dose groups with geometric ratios are proposed between the two dose groups obtained. This method does not require that the dose groups be in geometric ratio, but the geometric ratio can make the distances between the points equal, which is conducive to drawing. 5.3.3.4 Drawing calculation
5.3.3.4.1 Graph each group according to the dose and mortality rate on logarithmic probability paper. Except for the mortality rates of 0% and 100%, the dose can also be converted into logarithm, and the percentage can be found in the probability unit table to obtain its corresponding probability unit and plotted on ordinary arithmetic grid paper. Theoretically, 0% and 100% mortality rates do not exist. For calculation needs: 0% is changed to 0.25×100%,
100% Change to (N-α. 25)×100%
N is the number of animals in the group, which is equivalent to the probability unit for drawing 0% and 100%. 5.3.3.4.2 Draw a straight line, visually inspect with a transparent ruler, and take into account the probability. 5.3.3.5 Calculate the standard error
SE = 2SN2N\
Where:
N—the sum of the number of animals in each group between probability units 3.5 and 6.5 (the reaction percentage is between 6.7 and 93.7); · (6)
GB15193.3—2003
SE—standard error;
2S—the difference between -LDg and LD16, that is, 2S=LDa—LDi6 (or EDg4 —EDi6). The dose equivalent to ID&4 and LD1 can be found from the straight line. Ordinary graph paper can also be used to draw a graph, and the dose can be converted into a logarithm by looking up a table, and the mortality rate can be converted into a probability unit. The horizontal axis of the graph paper is the logarithm of the dose, and the vertical axis is the probability unit. According to the logarithm of the dose and the probability unit, a line is drawn to connect the points. A horizontal line is drawn from the probability unit 5 to intersect the straight line. A vertical line is drawn from the intersection point to the horizontal axis. The intersection point on the horizontal axis is the logarithm of the dose. The anti-log lethal dose (LDso) value is calculated. 5.3.4 Maximum Tolerated Dose Method
5.3.4.1 Suitable conditions: For test substances with extremely low toxicity or no toxicity shown in the relevant data, no death occurs when the animals are given the maximum concentration and maximum gavage volume.
5.3.4.2 Animals: At least 10 females and males. 5.3.4.3 Dose: The maximum concentration and gavage volume of the test substance (one dose group). 5.3.4.4 Method: After the animals are purchased, they are observed for 3 to 5 days, and the test substance with the maximum concentration and maximum oral gavage volume is given (given once or more times a day, and not more than 3 times a day). The animals are observed for 7 to 14 days continuously. If the animals do not die, it is considered that the maximum tolerated oral acute toxicity dose of the test substance to a certain animal is greater than a certain value (g/kg body weight). The maximum oral gavage volume is 0.4mL/20g body weight for mice and 4.0ml/200g body weight for rats.
5.3.5 Acute combined toxicity test
5.3.5.1 Principle
When two or more test substances exist at the same time, three different combined modes of action may occur: antagonism, addition or synergy. These three different actions can be determined according to certain formula calculations and judgment criteria. 5.3.5.2 Steps
5.3.5.2.1 Determine the LD50 of each test substance separately. The method is the same as above. 5.3.5.2.2 Prepare a mixture of test substances of equal toxicity according to the ratio of the LD5 values of each test substance. 5.3.5.2.3 Determine the LD50 of the mixture. When using other LDs determination methods, the sum of half of the LDs values of each test substance can be used as the middle group, and then several groups can be calculated upward and downward in geometric progression, which is the same as the design of the LD5 determination of a single test substance. If it is estimated to be an additive effect, two groups can be calculated upward and downward; if it is likely to be a synergistic effect, several more groups can be set downward; if it is likely to be an antagonistic effect, several more groups can be set upward.
5.3.5.3 Calculation
5.3.5.3.1 The test substances in the mixture are mixed in an equitoxic ratio, so the LD50 obtained is multiplied by the ratio of each test substance to obtain the dose of each test substance.
5.3.5.3.2 Use formula (7) to calculate the ratio of the expected LD50 values of the mixture, and determine the mode of action according to the ratio. 1
Expected LD value of the mixture = LD value of test substance A + LD value of test substance B + ... + LD value of test substance N. (7)
Where:
The mass ratio of each test substance in the mixture. a+6++n=1. a, b......n
5.3.5.3.3 The ratio for determining the combined action mode of the test substances adopts the provisions of Smith.HF, that is, less than 0.4 is antagonistic, 0.4-2.70 is additive, and greater than 2.7 is synergistic. 5.4 Observation of poisoning reaction
After administering the test substance, the poisoning manifestations and deaths of the experimental animals should be observed and recorded. The observation record should be as accurate, specific and complete as possible, including the degree and time of occurrence. The dead animals can be roughly dissected. 6 Evaluation of results
The toxicity classification of the test substance is determined based on the LDso value. The toxicity symptoms initially indicate the characteristics of the toxic effect. 22
Appendix A
(Normative Appendix)
Calculation of LDso value by Horn's method (Table for calculation of LDso by dose escalation method) GB 15193.3—2003
A.1 Table A.1 is used for 5 animals per group, and the common dose escalation ratio is V10, that is, 10×V10=21.5, 21.5×V0=46.4, and so on. The dose series is arranged as follows: 10.07
Group 2 Group 3 Group 4
Dose 1=0.464
Dose 2=1.00
Dose 3=2.15
Dose 4=4.6.4
Confidence limits
1.37~2.91
1.26-2.33
1.23~3.24
1.05~2.78
t=0,±1,±2,±3.··
Dose 1=1.00／
Dose 2=2.15
Dose 3=4.64
Dose 4=10.0
0. 951 -~ 2. 27
0.9261.71
1.01~2.91
0. 862 ~2. 50
0. 7752.05 | |tt | 01 | | tt | 65~6.98
2.27~5.99
2, 05~4. 88
2.00~3.69
2. 17 ~6. 28
1. 86~~5. 38
1. 69~~4. 41
1.60～3.99
1.59~~4.62
1.43~3.78
2. 63~~6. 76
2. 26~3. 65
2. 29~7. 75 | | tt |
Dose 4=21.5)
Confidence limit
5. 84 ~10. 8
5.70~15.0
4.89~12.9
4.41~10.5
4. 30~7. 94
4.67~13.5
4.00~13.5
3.60~9.50
3.44~~7.30
3.43~9.95
3.08~8.14
5. 66 ~14. 6
4.87~7.87
4.94~16.7
4. 02~16. 7
3.42~11.2
3.07~~8.51
3. 80 ~~14. 8
3. 05 ~~12. 5
GB 15193.3-—2003
Dose 1=0.464
Dose 2=1.00
Table A.1 (continued)
Dose 1=1.00
Dose 3-2.15
Dose 4=4.64
Confidence limits
0.550~2.20
0.523~2.32
1.00~3.58
0.806~2.67
0.674~1.92
0.839~4.29
0.6163.50
0.466~2.77
0 .573~3.76
0.406~3.18
1.27~2.05
0.978~3.92
0.893~2.92
0. 885 ~~2. 01
0.930~4.12
0.797~3.28
0.715~2.49
0.68 6~1.77
0.6762.63
0.599~2.02
0.969~3.71
0. 757 ~~4, 75
0.654~3.30
0. 581~2.22
D. 706 ~5. 09
0.564~~3.82
0.454~2.85
0.423~3.05
0.662~4,78||t t||Dose 2=2.15
Dose 3=4.64
Dose 4=10.0
Confidence limit
1.13~4.99
2. 16 ~7. 71
1.74~5.76
1.33~7.53
1. 00~5. 98
1.24~8.10
0.875~6.85
2.53~7.02
2.74~4.42
1.92~6.30
1. 9) ~4.33
2. 00 ~8. 88 | |tt | 41-7.10
1.25~-4.79
1. 52 ~11. 0
1.21 ~8. 24
0.977~6.13
0.912~6.57
1. 43~10. 3 | | tt | 6
3. 74~12, 4
3.13~8.89
3.89~19.9
2,16~12.9
2. 66~17. 4
1.89~14.8
5. 46~15.1
5.90~9.53
4. 11 ~9. 33
4.31~19.1
3. 70~15. 2
3.32-11.5
2, 78~9. 39
4.50-17.2
4. 74 ~9. 78 | |tt | 2
dose1=0.464
dose2=1.00
dose3-2. 15
dose4= 4. 64
Table A.1 (continued)
Dose 1=1.00
Dose 2=2.15
Dose 3=4,64
Dose 4=10.0／
Confidence limit
0. 583~2. 52 | |tt | 806~2. 67
0. 678~5. 30
0. 616 -~3. 50
0. 602~2, 15
0. 573~3. 76
0.503~~2.57
0. 856-~3. 69
0.481-6,58
0, 451~3. 25 | | tt | 88316, 6
0, 573～11.9
2. 42~~6. 89
1.67 ~~ 9. 97
1. 74 ~5. 76
1.46~11.4
1. 33~~7. 53
1,30~4. 62
1. 24~~8. 10
1.08~5.54
1. 85~7. 96
1.04-~14.2
0, 972~~7, 01
0. 840 ~~17. 5
0. 668 ~ 10.2 | | tt | | 0.602 ~ 11.3 | | tt | 71~~11. 7
2. 11~32. 3
1.52~20.8
1. 90~~35. 8
1.23~25.6
5.22~14.8
3. 60~~21. 5
3. 74~12. 4
3. 15 ~24. 6
2. 86~16. 2
2. 79~~9. 96
2.66~17.4
2. 3311. 9
3. 98~17. 1
2.23~30.5
2. 09 ~15. 1
1. 81 ~~37. 6
1. 44 ~~22. 0
1.30~～24.4
A.2 Table A.2 is used for 5 animals in each group, and the common dose escalation ratio is V10, that is, 10×V10=31.6, 31.6×V10=100, and so on. This dose series can be arranged as follows: 1.00
Group 3 Group 2
t—0,±1,±2,±3
Dose 1=0.316)
Dose 2=1.00
Dose 3-3.16
Dose 4=10.0
Confidence limit
1. 60~~4. 95
1.41~3.55
Dose 1=1. 00
Dose 2=3.16
Dose 310.0㎡
Dose 4=31.6,
Confidence limit
5. 07 ~~15. 7
4. 47~~11.2
GB 15193.3—2003
Table A.2 (continued)
Dose 1=0.316
Dose 2=1.00
Dose 3=3.16
Dose 4=10.0
Confidence limits
1.36~5.84
1.08~~4.64
0. 927~3. 41
0.8912.24
0.801~~3.95
0.6822.93
0.6381.97
0.636~3,14
0.542~2.32|| tt||1.35~5.56
1.07~2.21
1.10~6.82
0.806~5.23
0.632~~3.75
0.537~2.48
0 .740~5.70
0.5344.44
0.408~3.27
0.3783.53
1.01~6.77
0.723~4.37
0.554~ ~2.65
0.768~8.87
0.484~6.53
0.318~4.62
0.434~7.28
0.259~5.67
1.27~~5, 88
dose1=1. 00／
Dose 2=3.16
Dose 3=10.0
Dose 4=31.6
Confidence limit
4.30~~18.5
3.42-~14.7
2.93~10.8
2.82~7. 08 | |tt | 0
3.40~6.98
3.48~21.6
2.55~16.5
2. 00~11. 9
1. 70 ~~7. 85
2.34~18.0
1.69~14.1
1.29~~10. 3
3. 18~21. 4
2. 29~~13. 8
1. 75 ~8. 39
2.43~28.1
1.53~20.7
1. 00 ~~14. 6
1.37~23. 0
0. 819~17. 9
4.03~18. 6
Group 2Group 3
Group 3Group 2
Table A.2 (continued)
Dose 1=0.316
Dose 2-1. 00
Dose 3=3.16
Dose 4=10.0
Confidence limits
1. 43~~2. 94
0.968~7.75
0.843~~5, 00
0. 833~~2.85
0.8968.37
0. 711~~5. 93 | | tt | 4 | | tt | 539~10. 4
0. 446~3.99
0.307~~18.3
0.187~9.49
0. 262~~21. 4
0.137~13.0
1. 195. 71
0.684~9.95
0.723~~4.37
0. 558~~12. 2
0.484~6,53
0.467~3.14
GB 15193.3—2003
Dose 1=1.00
Dose 23.16
Dose 310.0
Dose 4=31.6
Confidence limit
3.06~24.5
2. 67~15. 8
2.63~9.01
2. 25 ~~18. 7
1.91~12. 4
1.80~7. 42
1, 76~13. 5
1.47~9.10
3. 01 ~22. 6
3. 27~9. 68
2. 08～32. 7
1.67~18.9
1. 40~10. 5
1.88~36.3
1.34~23.6
0. 966 ~~15. 2
0.871~~16. 8
1.70~33.0
1. 41~12. 6
0.970~~58.0
0.592~30.0
0.830~67.8
0.433~~41.0
3. 77 ~~18. 1
2.16~31.5
2. 29~13. 8
1. 76 ~ 38. 6
1.53~~20.7
GB 15193.3—2003
Response rate
Table A.2 (continued)
Dose 1=0.316)
Dose 2=1.00
Dose 33.16
Dose 410.0
Confidence limit
0.4347.28
0.356~4.12
0. 793 ~～7. 10
0. 333~~16. 9www.bzxz.net
0.303~5.87
0. 244 ~~23. 1
0. 172~～10. 3
0.14812.1
Appendix B
(Normative Appendix)
Response rate-probability unit table
Response rate-probability unit table
Dose 1=1.00／
Dose 2=3.16
Dose 3=10.0
Dose 4=31.6
Confidence limit
1.37~23.0
1.13~13.0
2.51~22. 4
1.05~53.4
0. 958~~18. 6
0.771~~73. 0
0.545~~32.6
0.467~38.1
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