Justification of an alternative method for determination of toxicity of perfume and beauty products by in vitro method on a short-term suspension cell culture-cattle spermatozoa (literature review)

  • Authors: T.F. Kharchenko, V.M. Levitskaya, O.A. Kharchenko, G.V. Golovaschenko, S.O. Khomak, S.S. Isaeva
  • UDC: 615.9:591.1:576.322
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State Enterprise “L.I. Medved’s Research Center of Preventive Toxicology, Food and Chemical Safety, Ministry of Health of Ukraine”, Kyiv, Ukraine

Resume. Introduction. Based on the literature data and directive documentation, the main reasons for the use of alternative methods for determining the toxic effects of perfume and beauty products are set out.
Methods — alternative methods based on toxicity assessment using short-term suspension culture of cells of cattle spermatozoa. The toxicological prognosis of the alternative method according to the results of studies is close to the significance of the toxicological prognosis obtained in the experiment with the use of laboratory animals. Mobility is an integral parameter of the physiological, biochemical and morphological status of the short-term suspension culture of mammal cells cattle spermatozoa.
Conclusion. The argument for the implementation of alternative methods for determining the toxic effect of perfume and beauty products (PBPs) is aimed at the humane attitude towards animals, as well as reducing costs and reducing the time spent on the study.
Key words: toxicity, perfume and beauty products, alternative methods, spermatozoa, cattle.

Abroad, the main motive for using alternative methods in toxicology is ethical one. It is lobbied by influential social forces. The toxicological and hygienic significance of the alternative method is determined by the selected test — object and its test function. It should be noted that mobile cells (cattle) are an appropriate test — object for the development of alternative methods. The proof is that the test object is suitable for toxicological studies with a high correlation of the results of studies compared with the results of studies using animals with classical toxicological methods [1].

Extract toxicity is assessed by changing the test function (in the experiment and in the control), which allows to predict the effect of extract on the whole body and culture of mammalian cells. If the extract belongs to a specific group of products (e.g. shampoos), then it is worth determining the minimum dilution (or normative) in which the extract can be recognised as either toxic or non-toxic. The high feasibility of the alternative method and the low cost of one study determine the necessary conditions for the significant distribution of the method with a short-term suspension culture of cells.

The express method for assessing the toxicity of extracts from beauty products is based on the analysis of the dependence between cattle spermatozoa mobility and time. Testing of beauty products for cytotoxic action is carried out in accordance with the requirements of DSTU EN ISO 10993-5: 2015 “Cytotoxicity test”. Currently, the main documents regulating the circulation of beauty products in Ukraine are DSanPiN “Sanitary rules and standards of safety of products of perfume and beauty industry. State Sanitary Rules and Standards”, DSTU 2472-94 “Perfume and beauty products”. Terms and definitions, DSTU 4186: 2003 “Liquid oral hygiene products”, DSTU 4315: 2004, “Beauty products for the cleansing of skin and hair”, DSTU 4093-2002 “Cosmetic lotions and tonics, and interstate standards, which set requirements to beauty products according to Regulation No. 1223/2009 of the European Parliament and Council “On Beauty Products”.

Alternative methods based on the assessment of toxicity using a short-term suspension culture of cells of bull spermatozoa have been already developed for the following perfume and beauty products: hair and body shampoos, liquid toilet soap, foam for baths, shower gel, deodorants and depilators in aerosol packaging, eau de toilette, eau de parfum, perfumes, colognes, alcohol-based lotions (Guidelines No. 29 ФЦ/394 as of January 29, 2002). Developed express method for the assessment of the general toxic and skin irritating action of PBPs in vitro allows conducting toxicological studies without animals using mammalian cells as test objects. It should be noted that the toxicological prognosis of the specified alternative method according to the results of studies is close to the significance of the toxicological prognosis obtained in the experiment with the use of laboratory animals. If according to the results of studies by the alternative method the index of toxicity is within 70 to 120 %, then, according to the authors of publications [2, 3], the study lot of products does not cause general toxic or skin irritating action and meets hygienic safety requirements. Assessment of the results is carried out by comparing the obtained value of the toxicity index for the experimental sample with the permissible interval of the values of the index of toxicity (70–120 %). Determination of the total mobile activity of the spermatozoa is carried out in the isotonic filtrate (in accordance with the requirements of the Guidelines No. 29 ФЦ/394 as of January 29, 2002).

According to the results of studies of the MIEC international program, studies were conducted on the selection of biological models. Investigators selected mobile cattle cells, which are very sensitive to the action of toxic substances and which are evolutionarily adapted to existence outside the body. The integral parameters of their physiological, biochemical and morphological status is mobility. For the creation of alternative methods based on the assessment of cytotoxicity, developers use a short-term suspension culture of mammalian cells — cattle spermatozoa [4, 5].

It should be noted that for this alternative method, accessible, cheap, standard biological materials are used. To develop an algorithm that would indicate the toxic effect of the extract and could be a prediction of its action on a whole body. In the experiment, the investigators analysed two data groups at the same time and calculated the mathematical dependence, according to which the correlation between these groups would be maximal. The assessment of the toxicity of perfume and beauty products in vitro shows that the creation of alternative methods is based on the use of a short-term suspension culture of mammalian cells.

In 2010, the European Union adopted the Directive 2010/63/EU for the protection of animals used for scientific purposes. The purpose of this directive was to strengthen legislation and improve the welfare of animals used for experiments and testing of the safety of pharmacological, beauty and other products, as well as the implementation of the 3Rs (REDUCE, REFINE, REPLACE) principles in the EU legislation and conditions of use of animals. Directive 2010/63/EU became effective since January 01, 2013. Testing of beauty products on animals in the European Union has been prohibited since 2004, and in 2009, also on their ingredients for complying with the requirements of Regulation (EU) No. 1223/2009.  Actions of scientists are aimed at replacing experimental studies with alternative methods, which will facilitate the emergence of new normative acts and an ideological basis for them. In 1959, ECVAM, the European Centre for Validation of Alternative Methods, was established. Article 7 of Directive 2010/63/EU categorically prohibits the use of laboratory animals for testing if there is another scientifically validated and approved method that provides the same level of information as an animal experiment [6]. A complete statutory ban on the testing of cosmetic ingredients in animals may lead to the fact that the products tested in animals may be outlawed. And manufacturers will be forced to remove an illegal component from the product or replace it [7]. The specified prohibition may also result in the restriction of innovative products (in particular products with UV filters, or anti-carries pastes).

The International Animal Protection Coalition adopted an international standard defining the main criteria for using the “Not tested on animals” sign. The disadvantage of alternative methods is that tissue culture or cell lines of the human skin can not always replace a whole body. When working with a small piece of skin, it is impossible to predict what the systemic reactions will be.

It is known that human skin consists of many types of cells and cell structures. Cell reactions in the whole body and in culture are not identical. This is a preliminary assessment which can help to make positive predictions that there is no obvious safety for the body. It should be noted that the bioethical expert evaluation of scientific projects, which provides for the use of animals, has not yet become a mandatory requirement in our country [8]. To a certain extent, this is due to the fact that Ukraine has no legislation to protect laboratory animals. Currently, Bioethics Committee and Commission at the Praesidium of the National Academy of Sciences, Academy of Medical Sciences of Ukraine carry out activities aimed at ensuring the regulation of animal experiments in accordance with the principles of bioethics. There is also a need for consensus between government regulators and scientific institutions regarding proper procedures, as well as monitoring compliance of protocols to established standards.

The aim of this work is to prepare and accelerate the adoption of legislative acts, as Ukraine cannot remain aside from the world bioethical actions, and the accumulated experience will contribute to the adoption of more improved laws and regulations.



1. Fedoseeva T. A. Evaluation of the toxicity of synthetic detergents by in vitro method on short-term suspension culture – bull spermatozoa / T. A. Fedoseeva, T. A. Menshykova // Toxicological bulletin-2004. –No. 6. – P. 17–19.

2. Eskov A. P. Author’s Certificate No. 168 635 52 for the invention “Method for evaluating the toxicity of a polymeric material” / A. P Eskov, R. I. Kaiumov // 1991. - Bulletin P.39–68

3. Eskov A. P. Toxicological tests. Alternative methods / A. P. Eskov, R. I. Kaiumov, A. E. Sokolov // Toxicological bulletin. – 2003. – No. 5. – P. 25.

4. Lappo V. H. Evaluation of the local irritant action of medical devices in vitro on the short-term suspension culture of mobile cells – bull spermatozoa / V. H. Lappo, N. M. Petrova, R. I. Kaiumov // Theses of the 2nd congress of toxicologists of Russia, M: MU. – No. 1.1.578–96. – Ministry of Health of the Russian Federation, M., 2003.

5. Toxicological evaluation of individual chemical compounds and mixtures of complex composition using mobile cell test object / [Eskov A. P., Kaiumov R. I., Ariefieva V. P., Lappo Yu. S.]  // Bulletin of experimental biology and medicine 2003. – No. 1. –P.48–50; 2003. – No. 5. –P. -25.

6. Biotesting of products of polymer and other materials. M. 1.1.037-95. M: State Communication Vigilance of RF, 1996.

7. Alternative methods and test systems: Monograp / Trakhtenderh I. M., Kovalenko V. M., Kokshareva N. V. [et al] – K.: Avicena. – 2008. –272 p.

8. Instructions on toxicological evaluation of polymer materials used in the food industry / No. 2395 – 81 – M, – 1984. – P. 17.


Надійшла до редакції 5.06.2018 р.