RU1789559C - Method of water-soluble substances toxicity determination - Google Patents

Abstract

Usage: toxicologists, environmental protection, ecologists, chemical and pharmacological industry, sanitary and hygienic supervision. SUMMARY OF THE INVENTION: Hela cultured mammalian cells are placed in a liquid culture medium (20-30 cells / ml). A 1: 50-1: 100 (in May.%) Solution of the toxicant is added thereto. Samples are incubated until macrocolonies appear. 2 tab.

Description

The invention relates to methods for determining the biological activity of compounds in the aqueous phase and can be used in toxicology, environmental protection, ecology, chemical-pharmacological industry, sanitary-hygienic studies in scientific and practical institutions of sanitary-hygienic supervision.

A known method for determining the toxicity of liquids by treating a micro-and macroalgae with a toxicant is calculated by calculating the ratio of living and dead cells to necrotic sites. A known method for assessing the toxicity of water-soluble compounds by treatment with a toxicant of cultured mammals, followed by incubation and determination of reproductive cell death.

The purpose of the invention is to simplify, increase the efficiency and accuracy of the Method

determination of the toxicity of water soluble

substance test. r --- --...... ..........

The method is as follows. In vttro cultured Hela mammalian cells are placed in a liquid nutrient medium at a steady concentration (20-30 ml / ml) in control and experimental samples. The choice of such a density is due to the fact that an increase in Pl6Ti1bet1y PT5 and b11T to reduce the accuracy due to the rt / ibn; with a decrease in density, there is also a decrease in the accuracy of the determination by reducing the efficiency of sowing. Then, to a test tube with inoculated ones, a toxicant solution (0.1-0.05 ml of a test substance solution, living at a concentration such that at

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said dilution operation creates the selected working concentration). Salt solution used to prepare toxicant solutions was added to control tubes in the same ratio. After that, the samples are incubated in hermetically sealed vessels at 37.0-37.5 ° C for the time required for the appearance of macrocolonies, and the toxic effect is judged, compounds with a statistically significant deviation of the number of colonies in the presence of a toxic agent compared with the control a sample.

The proposed method for determining the toxicity of water-soluble substances has obvious advantages over the known one, the sensitivity of the method is increased by 30 times. The materials presented in the table illustrate the achievement of the goal,

The proposed method is more economical, since there is no need to use expensive equipment to create a sterile gas environment of constant composition and humidity in a thermostatic. the chamber is simpler because it does not contain the operations necessary to create different concentrations of cells in the experimental and control samples, as well as operations associated with the introduction of a toxicant in physiological saline, and then replacing the solution with the culture medium, because the proposed method has a toxicant the entire incubation period is introduced directly until the appearance of visible macrocolonies.

Example 1. To prepare a cell culture, they take as a monolayer in a glass mattress with a capacity of 0.5 l in an environment of the following composition: medium 199-45%, medium Needle 48%, cattle serum without preservative 7%, streptomycin 250 units / ml On the third day of cultivation, the cells were removed from the glass with a 0.25% trypsin solution, resuspended in nutrient medium, lumps of cells were broken by pipetting, the number of cells per 1 ml was counted, and a single cell suspension was prepared in a culture medium with a density of 20. cells / ml , 5 ml of a suspension of cells, i.e., 100 cells per sample, are inoculated in 10 chambers, 5 of them serve as a control, 5 x mg g of chlorophos are added in 5. Cells were cultured at 37 ° C until macrocolonies were visible to the naked eye, then the number of colonies grown in control and experimental chambers was counted. The average number of colonies in control tubes is 98, + 1.16, the average number of colonies in experimental chambers is 82.5 + 1.5, i.e., cell survival under the action of chlorophos in concentration

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thirty

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tion 5 x mg% is equal to 84.2% of the control. A lower limit of chlorophos determination of 5 x 10 mg% is obtained.

The proposed method, in comparison with the known one, has an increased sensitivity while simplifying the determination of the toxicity of water-soluble substances.

Example 2. Carried out as example 1, but mg% chlorophos was added to the test chambers, control and experimental samples were cultured for about 8 days. The average number of colonies in the control tubes was 98.0 ± 1.6, in the experimental ones 48.2 ± 1.8, i.e., the test rate was 46.1% of the control, which indicates the high toxicity of this concentration of chlorophos on the reproductive capacity of mammalian cells ,

Example 3. Carry out, as example 1, but 5-methoxitritamine at a concentration of 1 x mg% was added to the test tubes. The average number of colonies in control tubes was 91.1 ± 2.2 tons, in experimental ones - 42.2 ± 1.4 tons, i.e., survival was 46.1%, and a highly toxic compound.

Example 4. Carried out as example 1, but 2% ammonia-2-thiazoline mg% was added to the test tubes and a 49% survival rate was obtained compared to the control.

Example 5. Carried out as example 1, but the toxicant is not added to the test chambers, but the medium is replaced with fresh medium of the same composition. After the formation of visible macrocolonies, they are counted in experimental and control chambers. In the control, the average number of colonies is 82.0 ± 6.3; in the experiments, 74.8 ± 5.9, i.e., 91.2% of the control. Thus, the medium change procedure leads to a decrease in the number of colonies of the order of 10%.

PRI me R 6. Carry out, as the winner 1, only a suspension of single cells is taken at a concentration of 30 cells / ml. Cell survival under the action of chlorophos at a concentration of 5x10 mg-% is 84.1 ± 0.1, which indicates a high convergence of the results in the range of cells at a concentration of 20-30 cells / ml (comparison with example 1). Data on efficiency and seeding in these experiments are given in. table.

From the data presented in the table, it follows that it is the concentration of 20-30 cells / ml that gives the acceptable seeding efficiency and clearly illustrates the advantages of the proposed method in comparison with the prototype.

The temperature of the cultivar for mammalian cells is acceptable within the given limits. Cultivation time

cells depends on the toxicant, therefore, the appearance of visible macrocolonies is noted (about 8-10 days from the day of sowing, but not more than

12 days, because after this the colonies slide off the glass due to a change in pH and cannot be counted).

Claims (1)

The claims A method for determining the toxicity of water-soluble substances, involving the cultivation of mammalian cells, introducing a suspension of cells into thermostatic test and control chambers for the period necessary for cell attachment, introducing the test solution into the test chambers and incubating it, followed by estimating the number of macrocolonies in experimental and control chambers and assignment of the test solution with a significant decrease in the number of macrocolonies in the experimental chambers, characterized in that, in order to simplify and improve the accuracy of the method, Hela human cells are cultured, hermetically closed vessels are used as thermostatic chambers, the cell suspension is introduced in concentration 20-30 cells / ml, and incubation is carried out until the appearance of visible microcolonies. Table 1 table 2