Some standard content:
ICS.07.100
National Standard of the People's Republic of China
GB15193.16--2003
Replaces GB15193.16-1994
Metabolic study
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
GB15193.16—2003
The full text of this standard is mandatory.
This standard replaces GB15193.16—1994 Metabolic study. Compared with GB15193.16-1994, this standard has the following major revisions: "Experimental animals" and "Dosage and grouping" are listed as chapter titles; - In "Scope", the following are added: biological substances, veterinary drugs, food containers, food tools, equipment, detergents, disinfectants, new food resources and their ingredients, etc., as well as the metabolic transformation pathways and fate of known chemical substances or derivatives with basically the same structure as known chemical substances produced and/or contaminated in these processes; "drug" in the original text is changed to "test substance". 6-1994 is abolished at the same time.
From the date of implementation of this standard, GB15193.16 is proposed and managed by the Ministry of Health of the People's Republic of China. The drafting unit of this standard: Institute of Nutrition and Food Hygiene, Chinese Academy of Preventive Medicine. The main drafters of this standard: Zhu Jiaqi, Chen Junshi. This standard was first issued in 1994, and this is the first revision. 104
1 Scope
Metabolic test
This standard specifies the basic technical requirements for metabolic tests. GB 15193.16-2003
This standard is applicable to the evaluation of my country's innovative chemical and/or biological substances (food additives, food containers and packaging materials, food tools, equipment, detergents, disinfectants, pesticide residues, veterinary drug residues, new food resources and their ingredients), as well as the metabolic transformation pathways and fate of known chemical substances or derivatives with basically the same structure as known chemical substances produced and/or contaminated in these processes. 2 Principle
The test substance can undergo a series of complex biochemical changes in the body. After being absorbed by the gastrointestinal tract, the test substance is transported to various tissues and organs throughout the body through the blood, and then undergoes biotransformation and is excreted from the body through various pathways. Therefore, the prototype of the test substance is gradually metabolized and degraded, while its metabolites are continuously generated. The content of the prototype of the test substance or its metabolites in the blood, tissues or excreta at different times after intragastric administration is determined to understand the toxicokinetic characteristics of the test substance in animals, including the characteristics of absorption, distribution, elimination, tissue accumulation and possible target organs, etc., and various toxicokinetic parameters are calculated based on mathematical models. At the same time, separation and purification methods are used to determine the chemical structure of the main metabolites, test their toxicity and infer the specific metabolic pathway of the test substance in the body. Through the observation of this experiment, a correct evaluation can be made on the process of the test substance in the body, providing a scientific basis for clarifying the nature and extent of the toxic effects of the test substance. 3 Instruments and reagents
3.1 According to laboratory conditions, high-performance thin-layer chromatography, high-performance liquid chromatography, gas chromatography, gas chromatography-mass spectrometry and ultraviolet light, fluorescence detection and other instruments and equipment are equipped.
3.2 Radioactive measurement instruments: liquid scintillation counter and scintillation fluid. 3.3 Commonly used laboratory instruments and reagents.
4 Experimental animals
In principle, animal strains with the same metabolic pathway as humans should be used as much as possible. Generally, adult mice of two sexes with a weight of 22g to 28g or rats of 170g to 200g are selected. The difference in animal weight at the beginning of the experiment should not exceed ±20% of the average weight. 5 Dosage and grouping
Use a dose lower than the maximum no-observed-adverse-effect dose, and high and low doses can be used when necessary. The test substance can be administered once or multiple times. If a labeled compound is used, in addition to determining the chemical dose, the radioactive dose is generally 10uCi/mouse to 20μCi/mouse (0.4MBq/mouse to 0.8MBq/mouse) for mice and 100μCi/kg to 250μCi/kg (4MBq/mouse to 9MBq/mouse) for rats. 6 Operation steps
6.1 Preparation of test substance
Generally, distilled water is used as the solvent. If the test substance is insoluble in water, edible oil, medical starch, and carboxymethyl cellulose can be used to prepare an emulsion or suspension. The test substance should be freshly prepared before oral administration, unless there is information indicating that it is stable when stored as a solution (or suspension, emulsion, etc.). 6.2 Route of administration of the test substance
Mainly oral administration. Animals should be fasted for 16h to 18h before oral administration and have free access to water. When conducting toxicokinetic analysis, it is best to use oral administration and intravenous injection at the same time.
GB15193.16—2003
6.3 Test items
Before conducting a metabolic test, a microchemical analysis method for determining the content of the test substance in biological samples or an isotope tracer method for labeling the test substance needs to be established.
6.3.1 Determination of the content or radioactivity level of the test substance in plasma Blood is collected at 6 to 10 different time phases after oral gavage of the animal, and the number of animals in each time phase should not be less than 3. The results are plotted on semi-logarithmic paper with the content of the test substance or the radioactivity intensity per milliliter of plasma as the ordinate and time as the abscissa, and a drug-time curve is drawn. If the content of the test substance is determined by a chemical analysis method, the curve is fitted using a compiled pharmacokinetic computer program, and the toxicokinetic equation and various metabolic kinetic parameters are obtained according to the compartment model. If the total radioactivity level in plasma is determined by an isotope tracer method, caution should be exercised when performing metabolic kinetic analysis. 6.3.2 Gastrointestinal absorption
The animals were killed at different times after oral gavage, and the gastrointestinal tract and its contents (including feces) were taken out to make a slurry, and the content of the test substance or the level of radioactivity was determined. The gastrointestinal recovery of the animals killed immediately after oral gavage was taken as 100%, and the disappearance of the test substance or radioactivity from the gastrointestinal tract in each group of animals at different time phases was observed. The percentage of the recovery at different time phases was used as the ordinate and the time was used as the abscissa. The disappearance rate of the test substance or radioactivity in the gastrointestinal tract was obtained by plotting on semi-logarithmic paper. In order to determine whether the disappearance rate of the test substance in the gastrointestinal tract can reflect the absorption in vivo, an in vitro gastrointestinal tract incubation test is required, that is, the test substance is injected into the in vitro gastrointestinal tract, the two ends are tied, and the test substance is incubated in Kreb's solution at 37℃ for 1 hour with shaking, and the recovery rate of the test substance is determined to observe whether the test substance is damaged in the gastrointestinal tract, thereby estimating the absorption rate of the test substance in the gastrointestinal tract.
6.3.3 Distribution in major organs and tissuesbzxz.net
After oral gavage, take 2 to 3 animals that were killed at different times, and measure the content or radioactivity of the test substance in the liver, kidney, brain and other organs and tissues to find out the tissue and time with the highest content of the test substance. 6.3.4 Excretion
6.3.4.1 Urine and feces excretion: After oral gavage of the test substance, place the animal in a plexiglass metabolic cage, and collect urine and feces at the prescribed time within 3 to 7 days. If urine and feces are found to be mixed, discard the specimen and collect it separately. When analyzing the structure of metabolites, the urine collection container should be placed in an ice bath and protected from light.
6.3.4.2 Bile excretion: Under mild ether anesthesia, perform bile duct intubation on the animal, wait for the animal to wake up, gavage the test substance, and collect bile at different times (not less than 24 hours).
Calculate the cumulative excretion (percentage of the oral dose) from the test substance content or radioactivity intensity in urine, feces, and bile samples collected at different times.
6.3.5 Biotransformation
According to the chemical structure of the test substance and literature data, estimate the possible metabolites. After the test substance is given to the animal, collect urine, bile and other samples, or use liver microsomes, enzyme activity system and the test substance to shake and culture at 37°C under in vitro metabolic conditions to extract and purify the metabolites for structural identification. Analytical methods include thin layer chromatography, gas chromatography, liquid chromatography, mass spectrometry, infrared spectroscopy, etc. There must be a predicted metabolite pure product as a standard. If a labeled compound is used, the sample is separated by thin layer chromatography and the radioactivity is measured by a radioactive thin layer scanner or by scraping silica gel in sections. The amount of the test substance and possible metabolites are determined and measured by the R value, and further analysis is performed. From the separation and identification of metabolites, inferences can be made about the possible metabolic pathways of the test substance in the body. 6.4 Precautions in isotope experiments
Isotope method is one of the indispensable means in toxicant metabolism experiments and is often listed as the preferred experimental method. It has the advantages of high sensitivity, simple sample preparation, not easily interfered by impurities in biological materials, and can trace and observe the fate of the test substance after entering the body. Combined with chemical analysis methods such as thin layer chromatography and liquid chromatography, the parent substance and metabolites can be separated to preliminarily determine the possible existence form of the metabolites. Radioautography can be used to locate and observe the distribution of the test substance and metabolites in the whole animal or certain tissues. 6.4.1 Requirements for labeled compounds
6.4.1.1 Labeling nuclides: Determined by the purpose of the experiment, the molecular structure of the test substance, half-life, funding and other factors. Commonly used are \H, 14C, 3S, etc. 6.4.1.2 Labeling position: It should be labeled on the biologically active group in the structure of the test substance, that is, positioning labeling. If the biologically active group is unclear, uniform labeling or full labeling can be used. The labeling position should be stable in the chemical structure. According to different research purposes, it can be single-labeled, 106
double-labeled or multi-labeled.
GB 15193.16—2003
6.4.1.3 Radiochemical purity: The labeling substance should ensure a high radiochemical purity (at least 90%), and it should be purified by thin layer chromatography when necessary.
6.4.1.4 Radioactivity ratio: Determined by the toxicity of the test substance. For highly toxic test substances, labeling substances with high radioactivity ratio are required. Dilute the unlabeled test substance to prepare the chemical dose required by the experiment. 6.4.2 Measurement of radioactive samples
Most of the labeled radionuclides commonly used in metabolic tests are soft beta rays, and the liquid scintillation counter is mainly used to measure food. 6.4.2.1 Sample preparation: acid digestion method. Two portions of 0.1g tissue, 0.1mL plasma, and 0.1mL fecal mixture (feces are added to water to make a homogenate, or feces are dried under an infrared lamp and ground into powder, weighing 0.1g) are added with 0.2mL perchloric acid, 0.4mL hydrogen peroxide, and a drop of n-octanol, and digested in a water bath at 80℃ for 45min, and distilled water is added to make the volume 1mL, 0.1mL is taken out, and 3mL~~5mL scintillation liquid is added to measure the radioactivity (bile and urine can be directly sampled and measured after centrifugation). The formula of scintillation fluid is 0.4%～0.6% 2,5-diphenyloxazole (PPO), 0.01%～0.03% 1,4-bis-[5-phenyloxazolyl-2]benzene (POPOP), xylene (or toluene) and ethylene glycol polyoxyethylene isooctylphenol ether (TritonX-100) (ratio 2:1). Solid flash crystal can be used if conditions permit. 6.4.2.2 Sample measurement method: homogeneous measurement method. After quenching correction, the sample is expressed as the number of radioactive decays per minute (DpM) per gram of tissue or per milliliter of plasma.
6.4.2.3 Radioactivity measurement error: The relative standard error of radioactivity measurement should not exceed 5%～10%. The relative standard error of a measurement result is expressed as:
SE(%) =±
Where:
r—calculation rate, times/min;
t measurement time, min.
6.4.2.4 Comply with the radioactive health protection operation procedures. 6.5 Requirements for the analytical method of the test substance in biological samples Establish a sensitive, specific and reproducible determination method. 7 Result determination
7.1 Estimate the metabolic rate and accumulation of the test substance in the body based on the absorption rate, tissue distribution and excretion. 7.2 Based on the structure and properties of the main metabolites, infer the possible metabolic pathways of the test substance in the body and the generation of toxic metabolites.
Tip: This standard content only shows part of the intercepted content of the complete standard. If you need the complete standard, please go to the top to download the complete standard document for free.