Cytotoxicity Evaluation Using in vitro System as a Criteria of Rational Choice of Tear Substitutes

Purpose: to evaluate the cytotoxic effect of a wide range of lubricant eye drops on the epithelial cells of the cornea in vitro.

Materials and methods. The objects of the study were 11 moisture eye drops with various preservatives and 9 moisture eye drops without preservatives. As a test-system permanent transformed cell  lines of corneal epithelial (HCEC) were used. The cytotoxicity of the  “artificial tears” was assessed by the viability of the cells, cultured in substratum containing the solutions of the evaluated eye drops.

Results. The most toxic for corneal epithelial cells among the  moisture eye drops with preservatives were Lacrisifi, Slezin,  Hypromelose-P and Ophtolique®. Close to them in toxicity were  Cationorm®, Artelac® Balance and Optiv®. Eye drops Styllavit,  Systane® Ultra and Blink® contacts showed moderate toxicity.  Vismed® Light from the group of eye lubricants with preservatives  did not exert a cytotoxic effect on the cells in vitro, as well as the  non-preserved eye lubricants Hyabak®, Thealoz®, Thealoz Duo®  and EvoTears™. Cytotoxic effect on corneal epithelial cells was  detected for Hylozar Comod® and Hylo® Fresh eye drops, despite  the absence of preservatives in them.

Conclusion. The study showed a principal possibility of using in vitro systems for comparative evaluation of the cytotoxic effects of various medicines with a purpose of a rational choice of moisture eye drop.

1. Baudouin C., Labbe A., Liang H., Pauly A., Brignole-Baudouin F. Preservatives in eye drops: the good, the bad and the ugly. Prog Retin Eye Res. 2010;9:312–34. DOI: 10.1016/j.preteyeres.2010.03.001

2. Whitson J., Petroll W. Corneal epithelial cell viability following exposure to ophthalmic solutions containing preservatives and/or antihypertensive agents. Adv Ther. 2012;29:874–88. DOI: 10.1007/s12325-012-0057-1

3. Tu E. Balancing antimicrobial efficacy and toxicity of currently available topical ophthalmic preservatives. Saudi J Ophthalmol. 2014;28(3):182–7. DOI: 10.1016/j.sjopt.2014.06.006

4. Elder D., Crowley P. Antimicrobial Preservatives Part One: Choosing a Preservative System. American Pharmaceutical Review. 2012, available from http://www.americanpharmaceuticalreview.com/Featured-Articles/38886-Antimicrobial-Preservatives-Part-One-Choosing-a-Preservative-System/

5. Freeman P., Kahook M. Preservatives in Topical Ophthalmic Medications: Historical and Clinical Perspectives. Expert Review of Ophthalmology 2009;4(1):59–64.

6. Ammar D., Noecker R., Kahook M.. Effects of benzalkonium chloride-preserved, polyquad- preserved, and sofZia-preserved topical glaucoma medications on human ocular epithelial cells. Adv Ther. 2010 Nov; 27(11):837–45. DOI: 10.1007/s12325-010-0070-1.

7. Schiffman R., Christianson M., Jacobsen G., Hirsch J., Reis B. Reliability and validity of the ocular surface disease index. Arch Ophthalmol. 2000;118:615–21.

8. Management and therapy of dry eye disease: report of the Management and Therapy Subcommittee of the International Dry Eye WorkShop (2007). Ocul Surf. 2007 Apr;5(2):163–78.

9. Данченко Е.О. Оценка цитотоксичности фармацевтических субстанций с использованием клеточных культур. Иммунология, аллергология, инфектология. 2012;2:22–31. [Danchenko E.O. Evaluation of cytotoxicity of pharmaceutical substances using cell cultures. Immunopathology, allergology, infectology=Immunopatologija, Allergologija, Infektologija. 2012;2:22–31. (In Russ.)]

10. Еропкин М.Ю., Еропкина Е.М. Клеточные культуры как модельная система исследования токсичности и скрининга цитопротекторных препаратов. Санкт-Петербург: Морсар, 2003. [Eropkin M.Ju., Eropkina E.M. The cell cultures as a model system toxicity studies and screening of cytoprotective drugs. St. Petersburg: Morsar AV, 2003. (In Russ.)]

11. Аникина Л.В., Пухов С.А., Дубровская Е.С., Афанасьева С.В., Клочков С.Г. Сравнительное определение жизнеспособности клеток с помощью МТТ и ресазурина. Фундаментальные исследования. 2014;12:1423–1427. [Anikina L.V., Puhov S.A., Dubrovskaja E.S., Afanas’eva S.V., Klochkov S.G. Comparative determination of cell viability using the MTT and Resazurin. Fundamental research=Fundamental’nye issledovaniya. 2014;12:1423–1427. (In Russ.)]

12. Urcan E., Haertel U., Styllou M., Hickel R., Scherthan H., Reichl F. Real-time xCELLigence impedance analysis of the cytotoxicity of dental composite components on human gingival fibroblasts. Dent Mater. 2010;26:1:51–8. DOI: 10.1016/j.dental.2009.08.007

13. Александрова О.И., Околов И.Н., Тахтаев Ю.В., Хорольская Ю.И., Хинтуба Т.С., Блинова М.И. Сравнительная оценка цитотоксичности антимикробных глазных капель. Офтальмологические ведомости. 2015;8(1):89–97. [Aleksandrova O.I., Okolov I.N., Tahtaev Ju.V., Horol’skaja Ju.I., Hintuba T.S., Blinova M.I. Competitive evaluation of cytotoxicity of antibacterial eye drops. Sravnitel’naja ocenka citotoksichnosti antimikrobnyh glaznyh kapel’. Oftal’mologicheskie vedomosti. 2015;8(1):89–97. (In Russ.)]

14. Александрова О.И., Хорольская Ю.И., Майчук Д.Ю., Блинова М.И. Исследование общей цитотоксичности антибиотиков аминогликозидного и фторхинолонового ряда на клеточных культурах. Вестник офтальмологии. 2015;5:39–48. [Aleksandrova O.I., Horol’skaja Ju.I., Majchuk D.Ju., Blinova M.I. Study of the general cytotoxicity of antibiotics of aminoglycoside fluoroquinolone groups on cell cultures. Annals of Ophthalmology=Vestnik oftal’mologii 2015;5:39– 48. (In Russ.)] DOI: 10.17116/oftalma2015131543-53

15. Александрова О.И., Околов И.Н., Хорольская Ю.И., Блинова М.И., Чураков Т.K. Оценка влияния бензалкония хлорида на цитотоксичность глазных капель Неттацин и Тобрекс в условиях in vitro. Cовременные технологии в офтальмологии. 2016;3:163–166. [Aleksandrova O.I., Okolov I.N., Horol’skaja Ju.I., Blinova M.I., Churakov T.K. Evaluation of BAK influence on cytotoxicity of eye drops Nettacin and Tobrex in vitro. Ocenka vlijanija benzalkonija hlorida na citotoksichnost’ glaznyh kapel’ Nettacin i Tobreks v uslovijah in vitro. Sovremennye tehnologii v oftal’mologii, 2016;3:163–166. (In Russ.)]

16. Александрова О.И., Околов И.Н., Хорольская Ю.И., Панова И.Е., Блинова М.И. Возможности клеточных технологий для рациональной фармакотерапии глазных патологий. Cовременные технологии в офтальмологии. 2017;7:5–7. [Aleksandrova O.I., Okolov I.N., Horol’skaja Ju.I., Panova I.E., Blinova M.I. Possibilities of cell technologies for rational pharmacotherapy of eye pathologies. Vozmozhnosti kletochnyh tehnologij dlja racional’noj farmakoterapii glaznyh patologij. Sovremennye tehnologii v oftal’mologii. 2017;7:5–7. (In Russ.)]

17. Александрова О.И., Околов И.Н., Хорольская Ю.И., Панова И.Е., Блинова М.И. Оценка цитотоксичности слезозаместительных препаратов с использованием системы in vitro. Офтальмология. 2017;14(1):59–64). [Aleksandrova O.I., Okolov I.N., Horol’skaja Ju.I., Panova I.E., Blinova M.I. Cytotoxicity evaluation of tear substitutes using in vitro system. Ocenka citotoksichnosti slezozamestitel’nyh preparatov s ispol’zovaniem sistemy in vitro. Оphthalmology in Russia=Oftal’mologiya. 2017;14(1):59–64). DOI: 10.18008/1816-5095- 2017-1-59-66. (In Russ.).] DOI: http://dx.doi.org/10.18008/1816-5095-2017-1-59-66

18. Александрова О.И., Околов И.Н., Хорольская Ю.И., Панова И.Е., Блинова М.И. Влияние нестероидных противовоспалительных глазных капель на клетки эпителия роговицы и конъюнктивы человека в условиях in vitro. Офтальмология. 2017;15(3):251– 259. [Aleksandrova O.I., Okolov I.N., Horol’skaja Ju.I., Panova I.E., Blinova M.I. Influence of NSAID eye drops on corneal epithelial cells in vitro. Оphthalmology in Russia=Oftal’mologiya. 2017;15(3):251– 259. (In Russ.)] DOI: 10.18008/1816-5095-2017-3-251-259

19. Cancer cell culture: methods and protocols / Ser. Methods in Molecular Medicine. Vol. 88. (Ed. S.P. Langdon). Totowa, NJ: Humana Press, 2003. 165–169 p.,

20. Kahook M. Preservatives in Topical Ophthalmic Medications: Historical and Clinical Perspectives. Expert Review of Ophthalmology. 2009;4(1):59–64.

21. Epstein S., Ahdoot M., Marcus E., Asbell P. Comparative toxicity of preservatives on immortalized corneal and conjunctival epithelial cells. J Ocul Pharmacol Ther 2009;25(2):113–9.

22. Noecker R. Effects of common ophthalmic preservatives on ocular health. Adv Ther. 2001 Sep-Oct; 18(5):205–15.

23. Schrage N., Frentz M., Spoeler F. The Ex Vivo Eye Irritation Test (EVEIT) in evaluation of artificial tears: Purite-preserved versus unpreserved eye drops. Graefes Arch Clin Exp Ophthalmol. 2012 Sep; 250(9):1333–40. DOI: 10.1007/s00417-012-1999-3

24. Samar K. Basak Preservatives and Ocular Surface Diseases. Kerala Journal of Ophthalmology. 2016;18(4):311–6.

25. Schrage N., Frentz M., Reim M. Changing the composition of buffered eye-drops prevents undesired side effects. Br J Ophthalmol. 2010;94:1519–22. DOI: 10.1136/bjo.2009.177386

26. Mueller-Lierheim. Traenenersatz- und Kontaktlinsenbenetzungsloesungen. In: Köln Biermann, ed. Aktuelle Kontaktologie. 2015:8–15.

27. Houlsby R., Ghajar M., Chavez G. Antimicrobial activity of boratebuffered solutions. Antimicrob Agents Chemother. 1986;29:803–6.

28. Lehmann D., Cavet M., Richardson M. Nonclinical safety evaluation of boric acid and a novel borate-buffered contact lens multi-purpose solution, Biotrue™ multipurpose solution. Cont Lens Anterior Eye. 2010 Dec; 33 Suppl 1:S24–32. DOI: 10.1016/j.clae.2010.06.010

29. Graupner O., Hausmann C. The alternation of the pH in the anterior chamber of the rabbits eye burned with smallest volumes of high concentrated acid and base [in German]. Albrecht Von Graefes Arch Klin Exp Ophthalmol. 1968;176:48–53.