DD287276A5 - METHOD FOR THE TOXICOLOGICAL IN VITRO INVESTIGATION OF INGREDIENTS OF WAESSER SOLUTIONS OR BZW. OF WATER-PURIFIED PURE SUBSTANCES USING CELL LINES - Google Patents

Abstract

The invention relates to a method for the in vitro toxicological analysis of ingredients of aqueous solutions or of water-soluble pure substances using cell lines. She writes a toxicological in vitro method for determining the effect of water-soluble xenobiotics, z. As of water pollutants or pharmaceuticals, on animal or human cells and to assess the health risk to humans. It is used in environmental toxicology, environmental monitoring and toxicological product testing. According to the invention, the associated problem is solved by incubating cell cultures with the culture media containing the test samples and applying a virus strain which is capable of propagation in these cultures. Under normal conditions, the value of viral proliferation inhibition is determined as an expression of cytotoxicity. {Environmental Toxicology; Environmental monitoring; toxicological product testing; xenobiotics; Water pollutants; toxicological in vitro method; cytotoxicity; Cell cultures; animal or human cells; viruses; Virus propagation}

Description

Field of application of the invention

The invention! can be used in the fields of environmental toxicology, environmental monitoring and product testing. In particular, the method is suitable for investigating and monitoring the quality of water resources (surface, ground and groundwater and bank filtrate) and for assessing the health risks of industrial, agricultural and municipal wastewater to humans. When commissioning or during the operation of sewage treatment plants and drinking water treatment plants can be carried out by means of the method according to the invention a review of the performance parameters from a toxicological point of view werdon. Furthermore, the invention is suitable for the toxicological testing of pharmaceuticals as well as products and intermediates of the chemical industry.

Characteristic of the known state of the art

Toxicological studies of xenobiotics with regard to their effect on humans are increasingly no longer exclusively carried out in rodent animal experiments. The advantages of alternative methods are known (Halle, W .: Fundamentals of cytotoxicity in vitro and its significance for the toxicological testing of antiseptics.) In: A. Kramer, G.Berneciu: W. Weuffen (Hrsgb.): Handbook of Antiseptics 1 / 5. VEB publishing house Volkund health, Berlin 1985, P.84-112) in particular this applies to toxicological investigations in environmental media, especially in water resources, but also for use in product testing (pharmacy, industrial toxicology). However, mainly for the toxicological examination of water samples, methods have hitherto been used which use a wide variety of aquatic organisms (for example water bacteria, algae, small crabs, fish) as test objects. The results obtained give a statement on the possible impairment of the ecological balance with respect to aquatic plants and animals biw. to influence the bacterial self-purification capacity of the water, however, do not allow direct conclusions on the health risk to humans.

In contrast, when using animal cell cultures, a high degree of correlation of the results obtained in the cell culture test could be demonstrated in comparison to the results obtained in conventional animal experiments (patent DD 241750 C2). For the test underlying the aforementioned patent, a calf cake cell was used for the determination of general cytotoxicity in vitro. Low-serum medium was used. By means of a specific evaluation mode, a good correlation between the measured variables obtained in vitro (IC ₆₀ - 50% inhibition concentration / in moles per liter of medium) and the data available in vitro (LD ₆ C = 50% lethal dose / in moles per kilogram Body mass of the experimental animal), but the cell culture test was more sensitive by two orders of magnitude relative to the units of measure indicated.

Significant disadvantages of the variant of the cell culture proliferation test used in the cited patent (see, for example, Halle, W., SB Savoly, G. Herder, W.-E. Siems and T. Rösner: On the Effect of Corpus Iuteum Preparations on the Proliferation Kinetics of endothelial cells from calf aorta in cell culture, Act blol med med. germ. 4) are:

- There are still possibilities of an increase in sensitivity, which is important for the planned use of the method (finding low foxic substances, especially in environmental media).

- For an assessment of the health risk to humans, the use of an animal cell culture is not yet optimal.

- In the evaluation of the results, a characterization of the concentration of the constituents of a water sample by the molarity is not possible, since this is a substance mixture of unknown composition.

Object of the invention

The aim of the invention is a toxicological in vitro method that in the study of low-toxic aqueous solutions with respect to the health risk for humans has a higher significance.

Explanation of the essence of the invention

The object of the invention is to increase the sensitivity of the test over the known prior art and to determine the relative toxicity of dissolved mixtures (based on their concentration). According to the invention the object is achieved in that dilute a culture medium concentrate to the normal concentration with the aqueous solutions to be examined after sterile filtration or antibiotic and Antimykotikazugabe and any necessary correction of pH and a review of the salt concentration (compliance with isotonicity)! or that the medium components are weighed into the prepared sample as dry substances. The resulting test media and control media prepared with bidistilled water are contacted with cultures of a preferably human cell line and incubated. In order to increase the sensitivity of the cytotoxicity test to the known level, the zeMspezifische ability for virus replication is used as an indicator of cell toxicity as an original inventive solution, so that in addition to the known inhibition of cell proliferation, the impairment of the propagation of a subsequently applied to the damaged culture virus strain to effect comes.

The proliferation of cells is a complex process that provides targets for a variety of damaging noxious agents. Similarly, in the proliferation of viruses, especially those that proliferate in the nucleus, the performance of all cell compartments is required, which in turn gives toxic substances influence possibilities. In determining the viral growth inhibition versus an uncontrolled control at the end of the incubation period, the product is detected from both of these effects. As a measure of the cytotoxic influences is the inhibition of virus multiplication H _y according to the relationship

H _v = IUO -

calculated.

The desired effect is achieved through the use of the human amniotic FL cell line and det>

on dL-er cell line propagating virus strain adenovirus 5.

By carrying out positive controls, a normalization of the results can be made, so that the results

different trial days to the exclusion of systematic errors are well comparable.

The classification of the relative toxic effects of substance mixtures is carried out according to the invention on the basis of

quantity-equivalent sum parameters (CSVc ₁ , TOC), which makes a meaningful comparison between such samples of different origins possible.

Advantages of the method according to the invention are:

- Increased sensitivity by detection of cell proliferation and virus proliferation inhibition

- Better predictability of human health risk through the use of a human instead of an animal cell line

- Possibility not to have to carry out the evaluation immediately, since the test or control cultures can be stored frozen after the incubation period until the quantitative determination of the virus concentration τ presence of proven and rational virological determination methods (micro methods)

- Possibility to determine the concentration-independent relative cytotoxicity also for solutions of unknown composition and those of several different substances by using the values for the CSVo (chemical oxygen consumption / chromate) or the TOC (total organic carbon) instead of the molarity.

embodiments

1. Toxicological examination of a Waiserprohe

In preparation for the test, the medium components (10% Medium 199 according to Parker: 90% Laktalbuminhydrolysst) are dry weighed into the water sample to be examined, so that the proportion of the sample to the total liquid is 100%. The medium must contain only 1 to 2% serum and must be buffered with the Hanks salt mixture when incubated closed, or with the Earle salt solution in open culture in a CO ₂ atmosphere.

Another variant is that a medium concentrate (water content a 20% of the normal content) is diluted so with the water sample to be examined that the total water content required for cell cultivation is maintained. The content of the water sample in the total liquid is then: s 80%. In order to prevent microbial contamination of the cell culture medium, the water sample is first sterile filtered by means of a suitable filter or admixed with a sufficient amount of antibiotics and antimycotics. In addition, for more saline samples, the increase in osmotic pressure must be checked and, if necessary, corrected. For both variants, a control medium is prepared in parallel - using bidistilled water instead of the water sample.

If necessary, ζ. For example, in highly toxic water samples and for the exact determination of the IC ₆₀ (ie the 50% inhibition concentration of a sample in the cell culture) dilutions (minimum 3 steps) of bidistilled water must be applied from the water samples.

Subsequently, the human amnion-derived FL cell line is transformed by the known methods into a suspension of single cells having a high cell count per unit volume. 1 part of this suspension is mixed with 9 parts of the medium containing the water sample to be examined or mixed with control medium and introduced into the culture vessels. The final concentration of cells in the medium to be reached depends on the time regime of the experiment and other culture conditions. Another variant consists of exchanging the growth medium for the young to be examined water sample containing »medium or control medium in young, already grown cell cultures (about 24 hours old), which are still at the beginning of the logarithmic growth phase. As with the first variant, several parallel batches (optimally 5 to V) must be applied per sample and dilution or per control culture.

In both variants, this is followed by an approximately 24-hour incubation. Thereafter, under sterile conditions, the medium is removed from cultures and stored separately for each batch. Subsequently, a precisely defined amount of the suspension of a virus strain is introduced into each culture, which cell culture is capable of replication (e.g., adenovirus 5 using the FL cell line). After an inoculation time of 2-4 hours (depending on cell line and virus strain) the stored medium is reapplied. The amount of virus particles must be such that after completion of the subsequent incubation period in the control cultures maximum virus replication has occurred. The duration of the incubation until the test evaluation is determined by the propagation characteristics of the virus strain used in the associated cell line (3 days for the strain adenovirus 5 in the FL cell line). After completion of the incubation period, all cultures are disrupted for complete virus release by a (at least three) freeze-thawing process. Thereafter, the virus concentration in each parallol mixture is determined by the methods known per se (Chang, SL, G.Berg, KA Busch, RE Stevenson, NA Clarke and PW Kabler: Application of the "Probable Number" method for estimating of animal viruses by the tissue technique, Virology β [1958] pp. 27-42)

The inhibition of the virus multiplication H, in a test batch in relation to the control batch, results from the following relationship:

f * 1

H = 100 - * - * 100 I K J

Hv = inhibition of virus multiplication V = arithmetic mean of all parallels of an experimental approach R = arithmetic mean of all parallels of the control approach For the necessary random-critical investigations, a parameter-independent significance test (eg rank sum test) is used Commitment. With an error probability of S 5%, the observed differences between R and V become significant

considered.

In any case, to rule out systematic errors for a reliable result reproduction when the test is repeated

To carry a positive control, which is treated analogously to experimental and control approaches. As positive controls, known water pollutants (eg phenol) in pure substance and defined concentration can serve, the

Virus proliferation inhibition was determined in preliminary experiments. In the manner of a standard, these positive controls are in

each test used to standardize the results.

To determine the relative toxicity of a tested water sample (determination of 50% concentration IC _M )

In contrast to the method known per se (patent DD 241750 C2), it is necessary to refer to such sum parameters as the chemical oxygen consumption / chromate (CSVc ₁ ) or the total organic carbon (TOC) in order to determine the amount of water constituents the reference to the molarity is not possible because of the presence of an unknown substance mixture.

2. Toxicological investigation of water pollutants In pure substance, Pharmazeutlka u.a. chemical products or intermediates

In the toxicological examination of products of the pharmaceutical and chemical industries, including water pollutants, the process described in Example 1 is used modified. The pure substances to be investigated are weighed into the finished medium as dry substances in the first process step. All further steps are carried out as indicated under 1.. However, the final calculation of relative toxicity (as IC _M ) is made with reference to molarity. From the normalized values for the tested pure substances a catalog of the cytotoxicity is created.

Claims (5)

1. A method for in vitro toxicological analysis of ingredients of aqueous solutions or of water-soluble pure substances using cell lines, characterized in that the culture media prepared with the samples to be tested act on animal or human cell lines, soften after an incubation cell-specific V'ren whose proliferation inhibition is determined in a further incubation period compared to a control batch as a measure of the cytotoxicity of the samples examined and their health risk to humans. 2. The method according to claim 1, characterized in that are used as the cell line derived from the human amnion FL cell line and strain of the proliferating on this cell line strain adenovirus 5. 3. Ancestors according to claim 1, characterized in that the Vermehrungshemmung according to the relationship f * Ί H = 1.00 - -ξ- * 100 t κ J is determined. 4. The method according to claim 1, characterized in that by carrying a substance of known cytotoxicity, which acts as a positive control in the function of a standard, a normalization in the mathematical sense of the values of Vermehrungshemmung is realized. 5. The method according to claim 1 and 3, characterized in that are used for samples with several different ingredients or unknown composition as a reference value for the relative cytotoxicity not the molarity, but the values for the CSV _Cr or the TOC.