Acute toxicity evaluation is one of the basic types of in vivo safety evaluation. Drug registration often requires acute toxicity evaluation. Acute toxicity test data has a special role in safety evaluation, and MTD or MFD can be determined in the initial toxicity description. The data obtained in these tests can provide a basis for the selection of doses for safety pharmacology tests and repeated dosing toxicity tests. Acute toxicity test can also be used for early drug candidate screening, or it can be used simultaneously with simple repeated dosing toxicity test. It should be noted that only the bioavailability and systemic exposure data based on exposure pathways and clinical indications can be provided to provide valuable information for acute toxicity studies.
Acute toxicity tests can take many forms, but the main purpose is the same, that is, the tested drugs can be tolerated at several multiples of the clinical dose. The advantage of the screening test is that several drug candidates can be compared in the same test at the same time. Tolerance indicators [such as changes in body weight and/or feed consumption and abnormal clinical symptoms] can provide a better reference for determining which drug candidate. At this time, the selection of animal species is generally not required to be all animal species, but at least one rodent is evaluated. For drugs that are taken every day for a predetermined period, it is reasonable to evaluate repeated administrations within a 5–7-day exposure period. This is helpful for preliminary evaluation of potential changes in clinical pathology endpoints, and is also useful for dose selection for other toxicity test items, but it cannot replace acute toxicity test for registration application submission.
For rodent experiments, 5 animals of each sex in each dose group is sufficient. The second animal species generally uses 2 to 3 animals. There should be a vehicle control group with at least 3 test product dose groups. The dose group can also be increased, especially when the toxicology profile of the acute toxicity test is poorly understood. After a single administration of the test product, the body weight, feed consumption and the clinical observation room were informed of key data on toxicological effects. The surviving animals were euthanized for the specified number of days after the administration (usually the 14th day after the administration). Gross anatomy is designed to evaluate the visible pathology of tissues and organs. However, in most cases, no further histopathological examinations will be done on these tissues. Toxicity evaluation is generally limited to observing obvious toxic reactions, such as changes in body weight, reduction in feed consumption and/or death, morbidity, and pharmacology of the test product. The acute toxicity test data can provide reference for the dosage design of the subsequent repeated dosing toxicity test.
Investigate the LD50 or maximal tolerance dose (MTD) of the tested drug in mice.
Median lethal dose (LD50): The dose that causes 50% of the test animals to die under certain test conditions. This value is the result of statistical processing.
Maximum tolerance dose (Maximal tolerance dose, MTD): The highest dose that does not cause death of the test animal.
Preliminary test of acute toxicity in mice to determine a reasonable dose design
The appropriate route of administration can be selected according to the characteristics of the tested drug, including gavage, intravenous injection, intraperitoneal injection, subcutaneous injection, etc.
Perform histopathological examination on some suspicious animals
The customer provides: the sample to be tested;
We submit: detailed experiment report;
Service period: Determined according to the experimental plan, please consult;
Service charge: Determined according to the experimental plan, please consult;
Service features: 1. Determine the dose and the dose ratio between groups through preliminary experiments; 2. Different routes of administration meet the needs of different tested drugs.
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Acute toxicity test (Single dose toxicity test) is to study the toxic reaction of animals within a certain period of time after an animal is given a test substance once or multiple times within 24 hours. Drugs intended for use in humans usually require animal acute toxicity tests. Acute toxicity test is in the early stage of drug toxicology research, and it is of great significance to clarify the toxic effects of drugs and understand their target organs of toxicity. The information obtained from the acute toxicity test has important reference value for the long-term toxicity test dose design and the selection of the initial dose of certain drugs in phase I clinical trials, and can provide some information related to the acute poisoning of human drug overdose.
The design of acute toxicity test should follow the principle of “specific analysis of specific problems” based on the knowledge of the test substance. A reasonable test method should be selected according to the structural characteristics, physical and chemical properties, indication characteristics and test purposes of the compound, an appropriate test program should be designed, and the test results should be comprehensively evaluated in combination with other pharmacological and toxicological research information.
The acute toxicity test should comply with the basic principles of general animal testing, namely randomization, control and repetition.
Maximum non-toxic reaction dose: No observed adverse effect level (NOAEL), which refers to the test substance in contact with the body in a certain way within a certain period of time, and no damage is found with sensitive modern detection methods and observation indicators The highest dose of effect.
Minimal toxic reaction dose: The smallest dose that an animal has a toxic reaction.
Maximum tolerance dose (Maximal tolerance dose, MTD): The highest dose that does not cause death of the test animal.
Minimal lethal dose (Minimal lethal dose, MLD): The dose that causes death in individual test animals.
Median lethal dose (LD50): The dose that causes 50% of the test animals to die under certain test conditions. This value is the result of statistical processing.
The test substance for the acute toxicity test should be a sample with a stable preparation process and conforming to the quality standards for clinical trials, and the name, source, batch number, content (or specification), storage conditions and preparation method of the test substance should be indicated, and The self-inspection report of the research and development unit is attached. The auxiliary materials, solvents, etc. used should be marked with batch numbers, specifications and manufacturers, and meet the test requirements.
Species: Animals of different species have their own characteristics, and their response to the same drug will be different. There will be differences in the results of acute toxicity tests between rodents and non-rodents in terms of quality and quantity. From the perspective of fully exposing the toxicity of the test substance, sufficient safety information should be obtained from rodents and non-rodents. Therefore, the acute toxicity test should use at least two mammals. Generally, one rodent and one non-rodent should be selected for acute toxicity test. If the acute toxicity test is not performed with non-rodent animals, its rationality should be clarified.
Gender: usually two sexes of animals are used for testing, with half males and half males. If single-sex animals are used for testing, its rationality should be clarified.
Age: Normally, healthy adult animals are used for testing. If the test substance is intended to be used in children or may be used in children, it is recommended to use juvenile animals for testing if necessary.
Number of animals: The number of animals used should be determined according to the species of the animals and the purpose of the research. The number of animals should meet the needs of the test method and the analysis and evaluation of the results. The minimum number of animals should be used on the premise of obtaining as much information as possible.
Body weight: The initial body weight of the animal should not exceed or fall below 20% of the average body weight.
The sex and age of the experimental animal can be determined according to the purpose of the experiment and the characteristics of the test substance. Animals should meet the level requirements of relevant national regulations, have a clear source, strain, and genetic background, and have a laboratory animal quality certificate.
Depending on the route of administration, the absorption rate, absorption rate and exposure of the test substance will be different, so it is necessary to use different routes of administration for acute toxicity tests. In addition, by comparing the results obtained from different routes of administration, some preliminary bioavailability information can be obtained. Generally, the route of administration should include at least the intended clinical route and a route (such as intravenous injection) that allows the original drug to enter the circulation more completely. If the proposed clinical route is intravenous administration, only this one route is sufficient. Animals should generally be fasted for a period of time (usually overnight) before oral administration, and can’t help but water. Because the stomach content will affect the administration volume of the test substance, and the length of the rodent’s fasting time will affect the activity of drug metabolizing enzymes and the intestinal absorption of the test substance, thereby affecting the exposure of toxicity.
The focus of the acute toxicity test is to observe the toxicity of animals. Appropriate methods can be selected to conduct acute toxicity studies. For non-rodents, a dose that exhibits obvious toxicity is sufficient, and the dose does not need to reach the lethal level.
In general, the dose should be never seen to be toxic to severely toxic (life-threatening) dose, and a blank and/or vehicle (excipient) control group should be set up at the same time.
The maximum administration volume under different animals and administration routes can be determined with reference to relevant literature and actual conditions.
After administration, it is generally observed continuously for at least 14 days, and the interval and frequency of observation should be appropriate so that the time of occurrence of toxic reaction and its recovery time, animal death time, etc. can be observed. Observed indicators include general indicators (such as animal appearance, behavior, response to stimuli, secretions, excrement, etc.), animal death (time of death, pre-death reaction, etc.), and changes in animal weight (before administration, end of test) The animals are weighed once before the animals are sacrificed, and the animals can be weighed several times during the observation period) and so on. Record all deaths, symptoms, and the time of onset, severity, and duration of symptoms.
All experimental animals should be grossly dissected, including the animals that were put to death due to dying during the experiment, the animals that died, and the animals that were still alive at the end of the experiment. Any changes in volume, color, texture, etc. of any tissue or organ shall be recorded and histopathological examination shall be performed.
According to the time, severity and duration of the various reactions observed, analyze the incidence and severity of various reactions at different doses. Summarize and analyze the observation results to determine the dose-response and time-response relationship of each reaction.
Determine the tissues, organs or systems that may be involved in various reactions.
Based on the gross anatomy and the results of histopathological examinations, the possible toxic target organs can be preliminarily judged. Histopathological examination should be accompanied by a report signed by the person in charge of the pathological examination and stamped by the drug registration applicant and pathological photos of the diseased tissue.
According to the various toxic reactions and incidence of different dose groups, animal deaths, etc., determine the animal’s non-toxic reaction dose and severe toxic reaction dose of the test substance. For this reason, it is recommended to adopt appropriate test methods to determine the maximum non-toxic reaction dose or the minimum toxic reaction dose, the maximum tolerated dose or the approximate lethal dose or the minimum lethal dose, etc., in order to preliminarily judge the safety range of the test substance.
For drugs that need to be determined for LD50, reasonable statistical methods should be used to calculate them.
Explain the calculation method and statistical method used, and provide the basis for the rationality of the selected method.
According to the time of occurrence of various reactions at different doses, incidence, dose-response relationship, historical background data of different species of animals and laboratories, the results of pathological examinations and the characteristics of similar drugs, determine the response and the drug The relevance of the role. Summarize the safety range of the test substance, the severity of toxicity, and the recoverability; according to the possible parts of the toxicity, the results of gross anatomy and histopathological examination are combined to initially judge the target organs of toxicity.
The results of the acute toxicity test can be used as a reference for the dose selection of follow-up toxicology studies, and can also indicate some indicators that need to be observed in follow-up toxicity studies. In addition, the bioavailability of the test substance can be preliminarily judged according to the animal’s response to different routes of administration, which provides a reference for the development of dosage forms.
Because the chemical structure of the test substance, the content of active ingredients are different, and the strength of the toxic reaction is also different, the researcher should choose the following method or the test method recognized at home and abroad according to the characteristics of the test substance.
This method is mainly used for non-rodent animal experiments. The test method is as follows:
Generally, 6 healthy Beagle dogs or monkeys are used. Dogs are generally 4-6 months old, and monkeys are generally 2-3 years old. Reasons should be given when choosing other animals.
Based on the results of small animal toxicity tests, the chemical structure of the test substance and other relevant information, the dose range that may cause toxicity and death is estimated. According to the 50% increasing method, a dose sequence table containing several doses was designed.
According to estimates, find the possible lethal dose range from the dose sequence table. Within this range, give one animal every interval of dose, measure the lowest lethal dose and the highest non-lethal dose, and then use the dose between the two to give An animal. If the animal does not die at this dose, the range between the dose and the lowest lethal dose is the approximate lethal dose range; if the animal dies at this dose, the range between the dose and the highest non-lethal dose is the approximate lethal dose range.
This method can be used for some low-toxic test substances. Under the premise of a reasonable maximum dose concentration and dose volume, a single dose or multiple doses within 24 hours at the maximum allowable dose (the dose generally does not exceed 5 g/kg body weight), and observe the reaction of the animal. Generally, 10-20 animals are used for continuous observation for 14 days.
This method was first proposed by the British Toxicology Society in 1984. It does not use death as the end point of observation, but uses obvious signs of toxicity as the end point for evaluation.
The test selects four fixed doses of 5, 50, 500 and 2000mg/kg for the test, and the dose of 5000mg/kg can be increased under special circumstances. Rats are the first choice for experimental animals, fasting for 6-12 hours before administration, and then fasting for 3-4 hours after administration of the test substance. It is carried out by means of one-time administration. If there is no data to prove that male animals are more sensitive to the test substance, the female animals should be used for pre-testing first. According to the relevant information of the test substance, choose one of the above four doses as the initial dose; if there is no relevant information for reference, 500mg/kg can be used as the initial dose for pre-test, if there is no toxic reaction, use 2000mg/kg Carry out a pre-test, if there is no death at this dose, the pre-test can be ended. If there is a serious toxicity reaction at the initial dose, that is, reduce the dose by one level for pre-test. If the animal survives, choose an intermediate dose test between the two fixed doses. Each dose is given to one animal, and the pre-test generally does not exceed 5 animals. There should be at least 24 hours between each dose test. The observation period after the administration of the test substance is at least 7 days. If the toxic reaction of the animal still exists on the 7th day, the observation should be continued for another 7 days.
A formal test will be conducted on the basis of the above-mentioned pre-test. At least 10 animals are used for each dose, half of the male and female. According to the results of the pre-test, choose one of the above four doses that may cause obvious toxicity but does not cause death for the test. If the pre-test results show that 5.0 mg/kg causes death, then the test will be lowered by a dose level.
After giving the test substance, it should be observed for at least 2 weeks, and it can be appropriately extended according to the specific characteristics of the toxic reaction. Each animal should be carefully observed and recorded in detail the time of the appearance and disappearance of various toxic reactions. The test substance should be observed and recorded at least twice on the same day, and once a day thereafter. The contents of the observation record include skin, mucous membrane, coat color, eyes, breathing, circulation, autonomous activities, and central nervous system behavior. The record of the time of animal death must be accurate. The weight of the animal should be weighed before and after the administration of the test substance for 1 week, when the animal dies and the end of the experiment. All animals, including dead or executed animals, should undergo autopsy, and organs with abnormal autopsy should be histopathologically examined. The results obtained by the fixed-dose test method are evaluated with reference to the following standards.
This method was first proposed by Dixon and Mood, and it was improved by Bruce in 1985. It is currently one of the methods recommended by OECD and EPA. Its biggest feature is to save experimental animals. At the same time, it can not only observe the toxicity performance, but also estimate the LD50 and its credibility limit, which is suitable for drugs that can cause rapid death of animals. This method is divided into limit test and main test. The limit test is mainly used when there is data suggesting that the toxicity of the test substance may be less. Relevant toxicity data can be obtained from compounds or similar mixtures or products related to the test substance. When there is little or no relevant toxicity data, or when the test substance is expected to be toxic, the main test should be carried out.
Limit test: It is a sequence test with a maximum of 5 animals. The test dose is 2000 mg/kg, and 5000 mg/kg can also be used under special circumstances.
Limit test at a dose level of 2000 mg/kg: The test substance is administered to 1 animal. If the animal dies, the main test will be carried out; if the animal is alive, the test substance will be given to another 4 animals in sequence, and the total number of animals will be 5. If one animal died at the end of the experiment and other animals survived, the administration to other animals should be stopped, and all animals should be observed to see if they also died during the similar observation period. Animals that died later should be counted the same as other dead animals, and the results should be evaluated as follows: When 3 or more animals die, the LD50 is less than 2000mg/kg; when 3 or more animals survive, the LD50 is greater than 2000 mg/kg; if 3 animals die, the main test will be carried out.
Limit test of 5000mg/kg dose level: In special circumstances, a dose of 5000mg/kg can be considered. The test substance was given to 1 animal. If the animal dies, the main test is performed; if the animal survives, the test substance is administered to two other animals. If the two animals are alive, the LD50 is greater than 5000mg/kg, stop the test (that is, no more administration to other animals, observe for 14 days). If one of the two animals died or both died, the test substance was given to the other two animals, one at a time. If one animal died at the end of the experiment and other animals survived, the administration to other animals should be stopped, and all animals should be observed to see if they also died during the similar observation period. Animals that died later should be counted the same as other dead animals, and the results were evaluated as follows. When 3 or more animals die, the LD50 is less than 5000 mg/kg; when 3 or more animals survive, the LD50 is greater than 5000 mg/kg.
Main test: It consists of a set dosing program, in which one animal is administered at a time with an interval of at least 48 hours. The interval between dosing depends on the time when the toxicity occurs, the duration and the severity of the toxicity. The next dose should be postponed until the animal can survive the previous dose. The time interval can be adjusted appropriately, but when a single time interval is used, the experiment will be easier.
The dose administered to the first animal was lower than the closest estimate of LD50. If the animal survives, the second animal is given a higher dose; if the first animal dies or appears dying, the second animal is given a lower dose. The dose series factor should be selected as the antilog of 1/(the estimated slope of the dose-response curve) (the series factor corresponding to slope 2 is 3.2), and should remain unchanged throughout the experiment. When there is no relevant information on the slope of the test substance, 3.2 is used as the dose series factor. When using the default progression factor, the dose should be selected from the following sequence: 1.75, 5.5, 17.5, 55, 175, 550, 2000 mg/kg (or 1.75, 5.5, 17.5, 55, 175, 550, 1750, 5000 mg if there are special requirements /kg). If there is no fatality estimate for the test substance, it should start at 175 mg/kg. If the animal’s tolerance to the test substance is expected to vary significantly (ie, the estimated slope is less than 2.0), then before starting the experiment, it should be considered to increase the dose series factor beyond the logarithmic dose calculation of the default value of 0.5 (ie, the series The factor is 3.2). Similarly, for a test substance with a known steep slope, a progression factor smaller than the default value should be selected.
Each animal should be carefully observed for 48 hours before deciding whether and how to administer the next animal. When one of the criteria for stopping the test is met, the administration is stopped, and the estimated LD50 value and the confidence interval are calculated according to the state of all animals at the time of termination. Calculate the LD50 value with the maximum probability method (the US EPA has developed the corresponding calculation software, AOT425StatPgm).
When one of the following stop test criteria is met, stop the test:
(A) 3 consecutive animals survive;
(B) In any consecutive 6 experimental animals, 5 consecutively undergo a survival/death transition;
(C) At least 4 animals entered the experiment after the first animal was converted, and the estimated LD50 range exceeded the critical value (2.5 times). (After the 4th animal of the first conversion, each administration is calculated).
For various combinations of LD50 and slope, 4 to 6 animals can meet the stopping test criterion (C) after the death/survival transition of the animal occurs. However, in some cases, the slope of the compound’s dose-response curve is small, and additional animals may be needed (up to 15 in total).
This method can be used for acute toxicity tests in non-rodent animals. The classic experimental design requires 8 animals, divided into a control group and an administration group, with 4 animals in each group, 2 males and 2 males.
The dosage design can be 1, 3, 10, 30, 100, 300, 1000, 3000 mg/kg, or 10, 20, 40, 80, 160, 320, 640, 1280 mg/kg, and usually the next high is given every other day. The dose, the dose is gradually increased, until the death of the animal or the upper limit of the dose is reached.
When no animal died, the MLD (minimum lethal dose) and LD50 were greater than the highest dose or restricted dose. When all animals died at a certain dose, MLD and LD50 should be between the last two doses. When part of the animals died at a certain dose, part of the deaths occurred in the next high dose. At this time, the MLD is between the dose of the first death and the previous low dose, and the LD50 should be between the dose of the first death of the animal and the next high dose. All animals died between the doses. If no animal deaths occur, the animals are often given the highest dose for 5-7 days to determine the choice of high dose in subsequent repeated dosing trials.
It is a classic acute toxicity test method. The LD50 of the test substance can be obtained by statistical processing of the test results.
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