GB 15193.16-2014National Food Safety Standard -- Toxicokinetic test (English PDF)

2.1 Test substance

Items for testing, refers specifically to meet the test requirements toxicokinetics the test.

2.2 Toxicokinetics

Test substance in the body absorption, distribution, biotransformation and excretion process time-varying dynamic characteristics.

2.3 Absorption

From the site of the test substance is usually administered biofilm transport outer or inner surface of the body's blood circulation to the process.

2.4 Distribution

After the test substance by absorption into the blood and body fluids in the body circulation and distribution process.

2.5 Metabolism

Test substance in the body through enzymatic or non-enzymatic reactions, the process of structural change occurs.

2.6 Excretion

The test substance and (or) its metabolites are cleared from the body process.

2.7 Bioavailability

The extent of the test substance is absorbed by the body.

2.8 Rate process

By the rate of change of the amount of poison dynamics of the test substance in unit time, usually with the amount of change per unit time through the process of representation Rate process. Rate process toxicokinetics, including zero order, first order and nonlinear three types.

2.9 Concentration - time curve

After administration of the test substance with time as the horizontal to the blood concentration of the test substance is made ordinate arithmetic graph reflects the test substance in the body Disposal within the state, the rate of change over time and content of the test, the area under the curve reflects into the systemic circulation of the subject matter content.

2.10 Apparent volume of distribution

When tested in vivo was partitioned Buda homeostasis hypothesis test substance concentration in the body fluid uniformly and plasma concentration of a test substance Thus, this body was dissolved test substance required amount of fluid volume is apparent volume of distribution. It was tested in vivo test volume and plasma concentration the ratio of representation.

2.11 Total body clearance

Test substance by metabolism and (or) excretion from the body to clear the way of speed; within per unit time in vivo clearance of the test substance is apparent from points Cloth volume fraction.

2.12 Elimination half-life

Vivo blood test substance concentration required for half fall time, which is a parameter indicating the subject matter of the elimination rate.

2.13 Peak concentration

After administering the test substance in the blood to reach the maximum concentration.

2.14 Peak time

After the test is administered to achieve maximum blood concentration time.

5.1 Basic information of the test substance

The test substance should provide the name, CAS number, batch number, source, purity, character, physical and chemical properties, storage conditions and preparation method, etc.

data.

5.2 Experimental Animals

5.2.1 Animal species, the Department of choice

Experimental animals should be selected in line with the relevant provisions of GB GB 14922.2 and 14922.1 of. Possible selection and other toxicology testing phase the same species, the Department, and to the typical toxicity of the test substance the animals. Rats generally preferred, usually six weeks weeks to 12 weeks, but if certificate it proved its metabolic pathways to human proximity, should select the appropriate animal. General should use young, healthy adult animals. Selection of rodent when objects, start of the test animal weight difference should not exceed ± 20% of average weight. Select other animals shall state the reasons.

5.2.2 Number and sex

Experimental animals sex without special provisions, such as toxicology studies have shown that there are significant gender differences in toxicity, should set up different gender group. In general, female animals should be used in nulliparous and non-pregnant too young; each test group of animals should not be less than 5, in a non-rodent Test substance, the discretion to reduce the number of animals.

5.2.3 Animal Feeding

Before the test, animals in the experimental animal room should be at least 3d ~ 5d acclimatization and quarantine observation. Animal rearing conditions, drinking Water and feed should be in line with national standards and regulations (animal breeding conditions should be consistent with GB 14925, drinking water should be consistent with GB 5749, feeding Material shall comply with the relevant provisions of GB 14924).

5.3 Dose

Trials need to use at least two dose levels, so that each dose level should metabolite test or the test substance is sufficient to drain it was measured out. Dose setting should take full account of existing toxicological data provided the information. If the lack of toxicological data, the High dose levels should be less than the LD50, acute toxicity or below the dose range lower value. Low dose levels should be a high dose levels section.

If the test set only one dose level, the dose should theoretically make the test substance or metabolite sufficient test substance in excrement the measure does not produce significant toxicity, and should provide a reasonable justification is not provided reasons for two dose levels.

5.4 Test procedure and outcome measures

5.4.1 Prepare the test substance

Purity of the test substance should not be less than 98%. Test can be "unlabeled" or "flag" test substances. If the use of radioisotopes Labeled test substance, the radiochemical purity of not less than 95% and should be marked on the radioisotope test molecule or a skeleton Important functional groups on.

5.4.2 Was tested routes of administration

When selecting a vehicle or other media, the test substance should be fully dissolved or uniformly suspended therein, the selected vehicle or medium for the test substance poison power Science does not have any effect. Commonly used oral route, and in some cases may also be employed to swallow capsules, administered by the incorporated feed Test substance.

Using intravenous administration of the test substance, should select the appropriate injection site and injection volume administration of the test substance, the selected vehicle or medium should not affect the integrity of the blood or blood flow.

5.4.3 Establishment and validation of biological sample analysis methods

5.4.3.1 Since biological sample generally from whole blood, serum, plasma, urine, organ or tissue, with less sampling, low concentrations of the test substance, Interfering substances and more individual differences and other characteristics, it is necessary according to the structure of the test substances, biological agents and the expected range of concentrations, the establishment of the Spirit Sensitive, specific, accurate, and reliable quantitative biological sample analysis method, and the method for confirmation.

5.4.3.2 biological sample analysis methods are.

a) chromatography. Gas chromatography (GC), high performance liquid chromatography (HPLC), chromatography - mass spectrometry (LC-MS, LC-MS-MS, GC-MS, GC-MS-MS), biological sample analysis is generally preferred chromatography;

b) Immunological methods. radioimmunoassay, enzyme immunoassay, fluorescence immunoassay method;

c) microbiological method;

d) isotope tracer method.

Confirmatory methods in general should investigate the following aspects.

a) Specificity. must prove analyte is expected analyte, endogenous substances and other metabolites can not interfere with the determination of the sample. Correct Chromatography to analyze at least six different individuals in a biological sample chromatogram blank, blank samples plus biological control substance chromatogram After administration of the test substance and a biological sample chromatogram.

b) a standard curve and quantitative range. According to the correlation with the analyte concentration in response, with the regression analysis (such as weighted least square Multiplication) to obtain a standard curve. Low concentration range of the standard curve for the quantitative range in the concentration range of quantitative measurement results should be achieved Test precision and accuracy requirements.

c) precision and accuracy. asked to choose three different concentrations of quality control samples at the same time to inspect the precision and accuracy of the method.

Choose low concentrations near the lower limit of quantification in less than three times the lower limit of quantification of the concentration; close to the upper limit of the standard curve of the high concentration;

Choose a middle concentration.

d) Quantitative limit. the limit of quantification is the lowest concentration point on the calibration curve, the determination to meet the requirements of at least three to five elimination half-life Test substance concentration 1/10 to 1/20 when the test substance concentration in the sample, or peak concentration, its accuracy should be in a real concentration 80% to 120% range, and inter-assay relative standard deviation should be less than 20% within a batch.

e) Sample stability. depending on the circumstances of the biological sample containing the test substance at room temperature, frozen or freeze-thaw conditions and different storage Time stability study to determine the biological sample storage conditions and time. Note also that the stability of the stock solution and the like Stability of the post-disposal solution analysis.

f) extraction recovery. study should be high, medium and low concentrations of extraction recoveries, the results should be accurate and reproducible.

5.4.4 OUTCOME MEASURES

5.4.4.1 Test blood concentration - time curve

5.4.4.1.1 Number of test animals

Select 9 to 11 different time points blood after administration of the test animals, animal per time point of not less than five. the best Sampled repeatedly from the same individual animal. As indicated by the data of multiple animals together constitute a test substance in blood concentration - time curve up accordingly the number of animals, in order to reflect the impact of individual differences in the test results.

5.4.4.1.2 Sampling point

Before administration of the test substance needed for blood collection as a blank sample. To obtain a complete administration of the test substance in the blood after the test substance concentration - time Curve, sampling time points should be designed to take into account the absorption of the test substance phase, the distribution phase (near the peak concentration) and the elimination phase. The entire sampling time shall be at least Until three to five elimination half-life, or until the blood test substance concentration peak concentration of 1/10-1/20.

5.4.4.1.3 Toxicokinetics parameters

According to blood tests measured the animals tested in each test concentration - time data obtained by the test substance is mainly toxicokinetics parameters.

Intravenous administration of the test substance should be provided elimination half-life, the apparent volume of distribution, the area under the curve parameters, total body clearance rate; extravascular Administration of the test substance, in addition to providing the above parameters, there should provide peak concentration and peak time and other parameters, the test was taken in order to reflect the law.

5.4.4.1.4 a single administration of the test substance After single administration of different doses of the test substance (or radioisotope labels), plasma or whole blood of the test substance at different times Concentration (or the total radioactivity intensity), providing each test animal blood test substance concentration - time data and curves and mean, standard deviation its curve; poison each major subject animal pharmacokinetic parameters and mean, standard deviation.

5.4.4.1.5 Repeated administration of the test substance

Repeated administration of the test substance, should be combined with a single test, select a general administration of the test dose was repeated at least three times to provide stability State trough concentrations of the test substance, after reaching a steady state were the last administration of the test substance test. Plasma or whole blood concentration of the test substance at different times or the total radioactivity intensity, compared with the single administration of the test was to determine the toxicokinetics of repeated administration of the test substance when.

5.4.4.2 Absorption

Test substance absorption rate depends on the extent of the test substance and route of administration. It is generally believed that the test was administered intravenously precursor chemicals Transient absorption rate calculated as 100%, the test substance shall be administered orally to determine the peak concentration, time to peak and area under the curve. Chemical analysis of the parent Concentration versus time curve can be determined orally administered absorption coefficient of the test substance.

The ratio of the area under the curve and the area under the curve of intravenous bioavailability of orally administered the test substance.

5.4.4.3 Distribution

After selecting the appropriate doses of the test substance in experimental animals, measured in the blood, heart, liver toxicity based on physical and chemical properties and characteristics of the test substance, the concentration of the spleen, lung, kidney, gastrointestinal tract, gonads, brain, body fat, skeletal muscle and other tissues in order to understand the distribution of the test substance in the main organs of the body.

Particular attention to high concentrations of the test substance, long storage time of tissues and organs, as well as distribution in the target organ toxicity. Reference blood test concentration -Time trend curve, select at least three time points represent the relative absorption, distribution and elimination phase of the phase distribution of the test substance. If an organization Higher concentrations of the test substance, should be added to the observation point, and further research in the organization of the test was eliminated. Each time point, there should be at least 5 animal data.

To organize the distribution of the test, it should pay attention to a representative sampling and consistency.

Tissue distribution of isotope markers test, the test substance should be provided labeled radiochemical purity, labeling efficiency (specific activity), marking position, to They test substance dose and other parameters; detailed radiometric method employed to provide; biological tests using radioactive tracer too detailed Cheng, and the correction equation when the biological sample measurement of radioactive decay were like. Under the experimental conditions permit, to the extent possible Overall radiation after administration of the test substance at different phases of self-developed image.

5.4.4.4 Metabolism

We should adopt appropriate technical analysis of biological samples to determine metabolic pathways and extent of the test substance. It should clarify metabolite structure. body Field test also helps to get tested metabolic pathways of information.

5.4.4.5 Excretion

After excretion test, the administration of the test substance selected the appropriate dose, according to a certain time interval segment collect urine, feces, exhaled breath measured Given the concentration of the test substance, the test substance is calculated by this route of excretion and rate of excretion. It should also be collected if necessary bile excretion detected by this route Rate and excretion.

In the administration of the test dose was at least 90% has been eliminated, or in the above sample has been collected from the test substance is not detected, or the detection time Up to 7d, to stop the collection of excreta. If exhaled test substance and (or) metabolite concentrations <=1%, to stop the animals exhaled Collection.

Record the test substance from feces, urine, exhaled air and other excretion rate and total excretion of the test substance in animals provide material balance data.