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GB 15193.3-2014 English PDF (GB15193.3-2014)

GB 15193.3-2014 English PDF (GB15193.3-2014)

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GB 15193.3-2014: National Food Safety Standard -- Acute toxicity test

This standard specifies the basic test methods and technical requirements, for acute oral toxicity test. This standard applies to the evaluation of the acute oral toxicity of the test substance.
GB/T 15193.3-2014
NATIONAL STANDARD OF THE
PEOPLE REPUBLIC OF CHINA
National food safety standard - Acute toxicity test
ISSUED ON: DECEMBER 24, 2014
IMPLEMENTED ON: MAY 01, 2015
Issued by: Health and Family Planning Commission of PRC
Table of Contents
Foreword ... 3
1 Scope ... 4
2 Terms and definitions... 4
3 Test purpose and principle ... 4
4 Test methods ... 5
5 Data processing and result evaluation ... 14
6 Report ... 14
7 Interpretation of the test ... 15
Appendix A Calculation of LD50 value in Horn's method (calculation table for the determination of LD50 by dose escalation method) ... 16
Appendix B Dose gradient table for up-down procedure (UDP) with different slopes ... 21
Appendix C Calculation of LD50 point estimates and confidence limits for formal test ... 23
Appendix D Response rate-probability unit table ... 25
Appendix E Probability units equivalent to 0% and 100% response rates ... 26 Appendix F Observation items in experimental animal poisoning performance ... 27 Appendix G Dose grading of acute toxicity (LD50) ... 28
National food safety standard - Acute toxicity test
1 Scope
This standard specifies the basic test methods and technical requirements, for acute oral toxicity test.
This standard applies to the evaluation of the acute oral toxicity of the test substance. 2 Terms and definitions
2.1 Acute oral toxicity
The toxic effects on animals, in a short period of time, after one or more oral administration of the test substance to experimental animals within 24 h. 2.2 The half lethal dose (LD50)
The dose of the test substance, that can cause animal mortality to be 50%, after oral administration of the test substance once or multiple times within 24 h. It is the calculated value, which is obtained through statistics. Its unit is the number of milligrams or grams of the test substance, which is ingested per kilogram of body weight, that is, mg/kg body weight or g/kg body weight.
3 Test purpose and principle
The acute oral toxicity test is the most basic test, for detecting and evaluating the toxic effects of the test substance, that is, after the test substance is administered orally once or multiple times within 24 hours, the toxic reactions produced by animals, including poisoning sign and death, are observed in a short period of time. It is usually expressed as LD50.
This test can provide information on health hazards, which are caused by oral exposure to the test substance in the short term. It serves as the basis for acute toxicity grading. It provides the basis for dose selection and observation indicators for further toxicity tests. It preliminarily estimates the target organs of toxic effects AND the possible toxic action mechanism.
4 Test methods
4.1 Test substance
4.1.1 Preparation of test substances
It shall dissolve or suspend the test substance in a suitable solvent. The preferred solvent is water. The test substance, which is insoluble in water, can use vegetable oil (such as olive oil, corn oil, etc.). The test substance, which is insoluble in water or oil, can also use carboxymethyl cellulose, starch, etc., to prepare a suspension or paste. The test substance shall be freshly prepared, unless there is data showing that its solution or suspension is stable during storage.
4.1.2 Administration of test substance
4.1.2.1 Approach
Oral gavage.
4.1.2.2 Fasting before the test
Animals need to fast before the test. Generally, rats need to fast overnight (generally about 16 h); mice need to fast for 4 h ~ 6 h; BUT they can drink water freely. After administration of the test substance, rats need to continue fasting for 3 h ~ 4 h; mice need to continue fasting for 1 h ~ 2 h. If the test substance is administered in batches multiple times, the animals can be given a certain amount of feed, according to the length of the exposure interval.
4.1.2.3 Gavage volume
The gavage volume of each test substance group shall be the same, which is 10 mL/kg body weight for rats AND 20 mL/kg body weight for mice. If the solvent is water, the maximum gavage volume of rats can reach 20 mL/kg body weight and that of mice can reach 40 mL/kg body weight.
4.1.2.4 Method
The test substance is generally administered at one time. It can also be administered multiple times in one day (each interval is 4 h ~ 6 h, no more than 3 times within 24 h, the maximum dose is reached as far as possible, which is calculated as a single dose). 4.1.2.5 Observation period
General observation period is 14 d, which may be extended to 28 d if necessary. It shall be observed for at least 7 d, in special emergency situations.
4.2 Experimental animals
4.2.1 Animal selection
The selection of experimental animals shall comply with national standards and relevant regulations (GB 14923, GB 14922.1, GB 14922.2). Choose healthy adult rats (180 g ~ 220 g) and/or mice (18 g ~ 22 g) of two genders, OR choose other experimental animals. Females shall be unmated and unpregnant. The difference in body weight, between animals of the same gender, shall not exceed ??20% of the average body weight. 4.2.2 Animal preparation
Before the test, the experimental animals shall undergo at least 3 d ~ 5 d environmental adaptation and quarantine observation, in the test environment.
4.2.3 Animal feeding
The feeding conditions, drinking water, feed of experimental animals shall comply with national standards and relevant regulations (GB 14925, GB 5749, GB 14924.1, GB 14924.2, GB 14924.3). Animals in each test group are raised in separate cages by gender. The number of animals per cage shall not affect the free movement of animals AND it can observe the physical signs of animals. For the special biological characteristics and toxic reactions (such as irritability, cannibalism, etc.), which are often caused by some test substances, it can be raised in a single cage. During the test, the experimental animals are fed with basal feed AND allowed for free access to water.
4.3 Design methods for several commonly used acute toxicity tests
4.3.1 Horn's method
4.3.1.1 Pre-test
According to the nature of the test substance and the known data, the following methods are used: Generally, it uses the dose of 100 mg/kg body weight, 1000 mg/kg body weight, 10000 mg/kg body weight, each of which uses 2 ~ 3 animals for pretest. According to the death situation within 24 h, estimate the possible range of LD50. Determine the dose group of the formal test. A single dose, such as 215 mg/kg body weight, can also be simply used directly in a pretest, with 5 animals. Observe the poisoning performance of animals, within 2 hours. If the signs of poisoning are severe, it is estimated that most animals may die, THEN the dose series below 215 mg/kg body weight can be used to perform the formal test. Otherwise, the dose series above this dose can be used if the signs of poisoning are mild. Pretests are not required, if there are relevant documents.
4.3.1.2 Formal test
4.3.1.2.1 Number of animals
Generally, there are 10 animals in each group, half male and half female. 4.3.3.1 Scope of application
This method is mainly applicable to the test substance, which has high purity, high toxicity, low intake, AND the animal will die, within 1 d ~ 2 d after administration of the test substance. For the test substance, which causes the animal to die at 5 d and above, after administration, it is not applicable. The test can be carried out, according to the dose sequence selected by the tester OR under the guidance of a special software package; the test results can be counted.
4.3.3.2 Up-down procedure limit test
Use the dose of 2000 mg/kg body weight, to administer the test substance to 1 animal. If the animal dies within 48 h, a formal test shall be carried out. If the animals survive within 48 hours, take another 4 animals, to make them administered of the test substance at the same dose. If 3 out of 5 animals die, a formal test shall be carried out; if 3 or more animals survive, the test will be terminated. THEN, the test substance has a LD50 > 2000 mg/kg body weight.
If the dose of 5000 mg/kg body weight is required, administer the test substance to 1 animal; if the animal dies within 48 h, a formal test shall be carried out. If the animal survives within 48 hours, take another 2 animals, to administer the test substance, at the same dose. If all animals survive within the 14-day observation period, the test ends, the test substance has a LD50 > 5000 mg/kg body weight. However, if 1 or 2 of the 2 animals dies, after the 14-day observation period, administer the test substance to another 2 animals; if more than 3 of the 5 animals survive, within the 14-day observation period, the test ends; the test substance has a LD50 > 5000 mg/kg. If 3 or more of the 5 animals die within the 14-day observation period, a formal test shall be carried out.
4.3.3.3 Formal test
4.3.3.3.1 Number of animals
For a single gender, the number of experimental animals is generally 6 ~ 9. 4.3.3.3.2 Dosage
When selecting the starting dose and dose gradient coefficient, if there is no data on the estimated value of the LD50 of the test substance, the default starting dose is 175 mg/kg body weight. If there is no data on the slope of the dose-response curve of the test substance, the default dose gradient coefficient is 3.2 (which is the gradient coefficient, when the slope is 2); the dose series set are 1.75 mg/kg body weight, 5.5 mg/kg body weight, 17.5 mg/kg body weight, 55 mg/kg body weight, 175 mg/kg body weight, 555 mg /kg body weight, 2000 mg/kg body weight, OR 1.75 mg/kg body weight, 5.5 mg/kg body weight, 17.5 mg/kg body weight, 55 mg/kg body weight, 175 mg/kg body weight, 555 mg/kg body weight, 1750 mg/kg body weight, 5000 mg/kg weight. For the test substance, which has a gentle or steep slope of the dose-response curve, the dose gradient coefficient can be increased or decreased; the initial dose can be adjusted appropriately. Appendix B lists dose gradients, which have slopes from 1 ~ 8. 4.3.3.3.3 Methods
At the beginning of the test, weigh the body weight of the fasted animals; calculate the gavage volume. Perform oral gavage for one animal at a time, which has an interval of 48 hours for each animal. The dose of the second animal depends on the toxicity results of the first animal; if the animal is moribund or dead, the dose is lowered by one level; if the animal survives, the dose is raised by one level.
4.3.3.3.4 Provisions for termination of test
Whether to continue to administer the test substance, depends on the survival status of all animals, within a fixed time interval. The test can be terminated, when any of the following conditions are reached, for the first time:
a) 3 consecutive animals survived in the test at higher doses;
b) 5 opposite results appear after 6 consecutive animals are administered the test substance;
c) After the first occurrence of the opposite result, continue to administer the test substance to at least 4 animals; calculate the likelihood value of each dose, from the first occurrence of the opposite result; the given likelihood ratio exceeds the critical value.
The LD50 of the test substance can be calculated, according to the animal?€?s survival state, at the end of the test. Appendix C describes the calculation method of LD50 estimates and confidence limits for formal tests, as well as the handling method of special cases.
If the animals died in the later stage of the test after the test substance is administered, whilst the animals with higher doses are still alive, the continued administration of the test substance shall be temporarily stopped, to observe whether other animals also experience delayed death. Continue the exposure when all animals, that have been administered the test substance, have a clear outcome. If the latter animals also show delayed death, it means that all exposure dose levels have exceeded LD50; two dose levels that are more appropriate and lower than the lowest dose that causes death shall be selected, to restart the test; meanwhile it shall extend the observation period. Animals with delayed death are counted as death.
4.3.4 Korbor method
4.3.4.1 Pre-test
2S - The difference between LD84 and LD16, that is, 2S = LD84 - LD16 (or ED84 - ED16);
N' - The sum of the number of animals in each group, within the probability unit of 3.5 ~ 6.5 (response percentage is 6.7% ~ 93.7%).
Note: The dose, which is equivalent to LD84 and LD16, can be found from the straight line. Ordinary graph paper can also be used to make a graph; look up the Table to convert the dose into logarithmic value; convert the percentage rate into probability unit. Take the abscissa of the quadrille paper as the dose logarithm AND the ordinate as the probability unit; connect the points of the dose logarithm and probability unit into a line; make a horizontal line from the probability unit 5, to intersect the straight line; draw a vertical line from the intersection point to the abscissa. The intersection point on the abscissa is the dose logarithm value. Find the antilog LD50 value.
4.3.6 Acute combined toxicity test
4.3.6.1 Principle
When two or more test substances exist at the same time, three different combined action modes of antagonism, addition or synergy may occur. These three different actions can be determined, according to certain formulas and judgement criteria. 4.3.6.2 Procedure
4.3.6.2.1 Determine the LD50 of a single test substance respectively; the method is the same as 4.3.1, 4.3.2, 4.3.3, 4.3.4, 4.3.5.
4.3.6.2.2 Prepare mixed test substances, which have equal toxicity, according to the proportion of the LD50 values of each test substance.
4.3.6.2.3 For the determination of the LD50 of the mixture, when other LD50 determination methods are used, the sum of half of the LD50 value of each test substance can be used as the middle group; then several groups can be calculated upward and downward, according to the proportional series, which are same as the design of the LD50 determination of a single test substance. If it is estimated to be an additive action, two groups can be calculated upward and downward; if it may be a synergistic action, several more groups can be set downward; if it may be an antagonistic action, several more groups can be set upwards.
4.3.6.3 Calculation
4.3.6.3.1 Each test substance in the mixture is mixed in an equal toxic ratio, so the calculated LD50 is multiplied by the ratio of each test substance, to obtain the dose of each test substance.
4.3.6.3.2 Calculate the ratio of the expected LD50 value of the mixture, by the formula (9); determine the mode of action, according to the ratio.
1 / Expected LD50 value of mixture = ?? / LD50 value of test substance A + b / LD50 value of test substance B + ?€? + n / LD50 value of test substance N ?€??€??€??€??€?.?€??€??€??€? (9) Where:
??, b?€?n - The mass ratio of each test substance of A, B?€?N in the mixture, where ?? + b +?€?+ n = 1.
4.3.6.3.3 The ratio of the combined action mode of the test substances is determined, according to Keplinger's provisions, that is, less than 0.57 is antagonistic action, 0.57 ~ 1.75 is additive action, greater than 1.75 is synergistic action.
4.4 Observation indicators
4.4.1 Clinical observation
Observations include skin, hair, eyes, mucous membranes, respiratory system, genitourinary system, digestive system, nervous system, etc. Special attention shall be paid to the presence or absence of tremor, convulsions, salivation, diarrhea, sluggishness, lethargy, coma (see Appendix F). The body weight of the animals is weighed and recorded, at the beginning and end of the test; the body weight of the animals is weighed at least once a week, during the observation period. Comprehensively observe and record the time, degree, duration of changes in animals; evaluate possible toxic target organs. Animals shall be sacrificed, if found to be moribund or showing severe pain and persistent distress. Time-of-death records shall be as accurate as possible.
4.4.2 Pathological examination
All animals, including those died during the test, humanely sacrificed, sacrificed at the end of the test, shall undergo gross anatomical examination. Record the gross pathological changes of each animal. It shall perform histopathological observation, where there are gross anatomical pathological changes.
5 Data processing and result evaluation
Describe the characteristics of the toxic effects, which are initially suggested by the poisoning performance. Determine the acute toxicity grading of the test substance, according to the LD50 value (see Appendix G).
6 Report
6.1 Test name, test organization name, contact information, report number. b) If all animals at the higher dose die AND all animals at the lower dose survive, then the LD50 would be between the doses for the total death and the total survival. An accurate LD50 cannot be provided, at this time. If there is sigma, the maximum possible value of LD50 can still be estimated;
c) If death and survival occur at a certain dose, all animals above this dose die AND all animals below this dose survive, THEN, the LD50 is equal to the dose. If the toxicity test is performed with the congener of the above-mentioned test substance, a smaller dose gradient factor shall be used.
C.3 Calculation of confidence limits (CI)
C.3.1 The AOT425StatPgm software package can complete the calculation of the confidence limit; it will finally evaluate the reliability and validity of the formal test results. A wide range of LD50 confidence limits indicates that there are more uncertainties in estimating LD50; the reliability and validity of the estimated LD50 are lower. When the confidence limit range is narrow, the obtained LD50 has less uncertainties; the reliability and validity are relatively high. The significance is that when the formal test is repeated, the estimated LD50 obtained is closer to the estimated value of the original determination, meanwhile both are closer to the true LD50. C.3.2 According to the test results of the formal test, two methods can be used, to estimate the true LD50 confidence limit.
a) In the test results of 3 different doses for poisoning, at least 1 animal die and 1 animal survive, in the middle dose. The maximum likelihood method can be used, to calculate the true LD50 and 95% confidence limit. However, confidence limits are generally inaccurate, due to the desire to minimize the number of animals used. Provisions for randomized termination of tests improve this somewhat, BUT there will still be some differences from the true confidence limits.
b) If all animals, at a certain dose and below this dose, survive, whilst all animals above this dose die, the interval is the total survival dose and the total death dose. It is only an approximate range, which can not determine the confidence limit. However, when the dose-response curve is steep, the true LD50 confidence limit is very close to this interval.
C.3.3 In some cases, such as the response slope is relatively flat, the confidence limit may be reported to infinity, as low as infinitely small and as high as infinitely large, OR between the two, which occurs when the response is relatively flat.
C.3.4 If the calculation process needs a special program to complete, it can be completed using the free software special program, which is provided by EPA and OECD.

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