DD256760B5 - Method for testing the dermal toxicity of substances or substance mixtures and for selection of skin-compatible active substances - Google Patents

Description

Characteristic of the known state of the art

It is known that chemical substances or mixtures of substances which come into contact with the human skin and the mucous membranes close to the skin can cause skin diseases. Frequently, weak irritants only damage after prolonged exposure and repeated exposure, often with additional factors. The growing environmental awareness in all industrialized countries is in line with the demand for a dermato-toxicological test for the approval of new active ingredients in the agrochemical industry, for household chemicals, dyes, cosmetics, disinfectants and for all chemical-technical products that may come into contact with human skin. The requirement for statements on the effectiveness of the skin applies, as is known, to finished preparations as well as to intermediates arising in the technological process.

It is known that for the examination of the skin and mucous membrane compatibility of substances there are no generally valid test specifications which, in terms of the export of such products, also meet international requirements. The examination of the primary irritant effect of substances on the skin is known to be very tedious and expensive. The toxicological tests are carried out in stages on experimental animals of lower and higher genera, z. As in mice, rats, guinea pigs, rabbits and subsequently on humans themselves. It is trying to expand the animal studies so that subsequent tests in humans with the greatest possible safety can be minimized.

Despite the high testing effort, the results of the animal experiments can not be satisfied in every case. It sometimes happens that dermatological concerns only become apparent after prolonged use of substances in humans themselves. There is some risk in the transmission of animal results to humans.

In addition, a larger number of animals must be included in corresponding experiments in order to exclude the uncertainty factor by the individuality of the experimental objects. Since the animals have to be killed after completion of the dermatological examinations for the purpose of a detailed examination, such methods are also increasingly criticized by animal welfare associations.

Object of the invention

The aim of the invention is to develop a method which allows a rapid, safe and reproducible statement regarding the skin compatibility of substances without animal experiments.

Explanation of the essence of the invention

It was therefore the object to develop a method for testing the dermal toxicity of substances or mixtures of substances and for the selection of skin-friendly active ingredients that uses the modern histological-morphological examination technique.

According to the invention the object is achieved in that as a test specimen in plastic surgery of women of mature age healthy resected vaginal skin sections of at least two patients with the respective substances to be tested in contact and then made SEM images.

Surgery whose goal is an anterior or posterior vaginal surgery is known to have been performed for many decades in gynecological clinics. The preferably resulting healthy skin is discarded.

It has been found that the resected virgin sheath skin is particularly suitable for obtaining information about the skin compatibility of substances.

By avoiding elaborate and lengthy animal experiments, the method according to the invention makes it possible to quickly and easily obtain such statements and, if appropriate, to establish tolerability limits for substances; Skin-compatible substances can be selected for specific areas of application.

An additional advantage associated with the method according to the invention can also be seen in the fact that it opens up the possibility of determining the effectiveness of the substances investigated against pathogenic pathogens. In special cases, one can then use transmission electron microscopy.

- 2 - embodiments

By way of example, the method according to the invention will be explained in more detail and its meaningfulness will be illustrated. The vaginal skin sections resected during plastic surgery are each divided into up to four equally sized pieces. To test the dermal toxicity of certain substances, they are allowed to act in a defined concentration for 24 hours on at least two different skin samples. These skin samples, which were treated with the substance to be tested, and in each case two untreated controls from the same skin sections are then imaged by electron microscopy, compared with one another and the degree of skin damage assessed

 The preparation of the test samples for electron microscopy is carried out in a known manner:

a) Preparation for Scanning Electron Microscopy

The lying in ethanol skin sections of about 8 mm ² size are dried for 45 minutes at 2O ⁰ C and a final pressure of about 5Pa. Then they are fixed with adhesive on the object holder of the scanning electron microscope and steamed in a high-vacuum vapor deposition system with carbon and gold as the conductive layers. The sample surfaces are magnified 10,000 times in the scanning electron microscope.

b) preparation for transmission electron microscopy

Drying of the skin sections of approximately 2 × 2 mm size intended for transmission electron microscopy is carried out in the same manner as for the examination in the scanning electron microscope. After drying, the tissue samples are introduced into a embedding agent which polymerizes at 48 ^° C. within 3 to 4 days. The embedding is done so that cuts can be made parallel to the surface.

The ultrathin sections (thickness 150-200nm, section area about 0.2 χ 0.4mm) are made with an ultramicrotome.

The transferred to support networks sections are nachkontrastiert 2 hours in saturated Uranylacetatlösung and magnified 22,000 times magnified in a transmission electron microscope.

A selection of the active ingredients or active substance preparations included in the dermal toxicity test is reproduced in Table I.

Table I: Compilation of the active substances included in the dermal toxicity test

Test sample No. Active substance

1 2-hydroxybutyraldehyde

2!, 'U-trichloro-methyl-pentene-ol

3 trisodium phosphonoformate hexahydrate

4 poly (dimethyldiallylammonium) chloride

5 peracetic acid

6 Ujosan (iodine / peracetic acid)

7 Camposan (2-chloroethylphosphonic acid 50%)

The scoring of the scanning electron micrographs takes place according to the following key:

Credit note findings

0 = no damage to the cells compared to untreated sample

1 = damage to the cells low

2 = damage to the cells strong

3 = detachment of several cell layers

4 & severe damage and destruction of the tissue

The scanning electron micrographs (Annex) corresponding to the credit score show:

Credit note findings

0 the surface was not changed; the superimposed cells belong to the physiological cytolysis and are also detectable on the untreated preparation;

1 the superficial cells show a marked morphological change due to the action of the test substance.

The individual cells show some shrinkage and also some cytolysis;

2 the superficial cell layers are missing or are still present as debris.

One clearly recognizes structures that no longer belong to the superficial cell layer;

3 the upper and middle cell layers are partly missing. There is abundant cellular degeneration, although cell fragments are still clearly visible;

4 The cell sections are completely degraded except for residues. One already recognizes the vascular system and connective tissue. The destruction of the multilayered tissue has been almost completely successful.

The comparative image shows untreated vaginal skin (only fixation process). The surface shows no morphological changes.

Table II shows the degree of damage to the skin by different substances or substance mixtures as a function of the concentration. Thus, the method according to the invention makes it possible to select skin-compatible substances or to determine maximum use concentrations of these active ingredients if any skin damage is to be ruled out.

TABLE II Examination of the dermal toxicity of various substances or substance mixtures by the method according to the invention

Test sample no. concentration Bonuses corresponding to the degree of injury 1 1 5 10 25 1 2 3 4 2 50 100 2 (K) 3 3 2 4 4 (ointment) 2 3 (K) 0 4 1 5 25 0 0 2 5 0.5 5 100 2 2 4 6 1 5 50 1 1 7 0.5 5 100 1 2 3-4

* The numbers of the test samples correspond to the information in Table I; K means crystal precipitation

In Table III, the method according to the invention is compared with the known rabbit test.

Examination of the primary irritant effect of a substance involves the determination of the degree of irritation of uninjured and injured skin using the index of primary skin irritation according to the method of Draize [Dermal toxicity, Publ. In Appraisol of the safety of chemicals in foods, drugs and cosmetics, Wyd. Assoc. of Food and Drug Officials of the United States, 1959] on rabbit skin.

Per substance and concentration 6 albino rabbits were used. After 1.3 and 7 days, all skin changes were scored (erythema, swelling and encrustations).

Draize uses the following classification:

0 = not irritating to the skin

0-2 = slightly irritating to the skin

2-5 = moderately irritating to the skin

5 = strongly irritating to the skin

These classification numbers result from the average of all points obtained from the scores after 1.3 and 7 days for injured and uninjured skin. However, the method does not allow to classify specimens with a weak irritation exactly.

Table III: Assessment of the dermal toxicity of various substances *

a) according to the inventive method and

b) after the rabbit test after Draize

Test sample no. Concentration [% by mass] Bonus notes according to the invention to Draize 1 «- CM 1 2 3 4 1-2 3 3 2 50 100 2 3 CO CO 3 2 4 4 (ointment) 2 3 0 0 0 1-2

Test sample no. Concentration • [% by weight] Bonus notes according to the invention to Draize 4 1 5 25 0 0 2 0 0 1-2 6 1 5 50 1 1 0 0 0 7 0.5 5 100 2 2 3-4 0 2 3-4

* The numbers of the test samples correspond to the data in Table I.

Claims (1)

Claim: A method for testing the dermal toxicity of substances or substance mixtures and for the selection of skin-friendly active ingredients, in particular for products of the agrochemical industry, pharmacy, dyes, disinfectants, health care and household chemicals, characterized in that as test specimens incurred in plastic surgery of women of adult-onset age healthy resected vaginal skin sections of at least two patients are used and brought into contact with the respective substances to be tested, and that for the evaluation SEM images are made. Field of application of the invention The process according to the invention can be used to quickly select those from a variety of active ingredients, in particular for products of the agrochemical industry, pharmacy, disinfectants, dyes, health care products and household chemicals, which are skin-friendly.