Textile Chemicals in EA

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Textile ToxicityCytotoxicity and Spermatozoa Motility Inhibition Resulting from Reactive Dyes and Dyed Fabrics

Doctoral dissertation To be presented by permission of the Faculty of Natural and Environmental Sciences of the University of Kuopio for public examination in Auditorium ML1, Medistudia building, University of Kuopio on Friday 17 th October 2008, at 12 noon

Department of Biosciences University of Kuopio


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Kuopio University Library P.O. Box 1627 FI-70211 KUOPIO FINLAND Tel. +358 40 355 3430 Fax +358 17 163 410 http://www.uku.fi/kirjasto/julkaisutoiminta/julkmyyn.html Professor Pertti Pasanen, Ph.D. Department of Environmental Science Professor Jari Kaipio, Ph.D. Department of Physics

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Savonia University of Applied Sciences Kuopio Academy of Design P.O. Box 98 FI-70101 KUOPIO FINLAND Tel. +358 17 308 111 Fax +358 17 308 222 E-mail: kaisa.klemola@designkuopio.fi Docent Pirjo Lindstrm-Sepp, Ph.D. Faculty of Medicine University of Kuopio Professor Jyrki Liesivuori, Ph.D. Department of Pharmacology, Drug Development and Therapeutics University of Turku



Professor Hanna Thti, Ph.D. Faculty of Medicine, Medical School University of Tampere Professor Pertti Nousiainen, Ph.D. Department of Materials Science Tampere University of Technology


Docent Eero Priha, Ph.D. Finnish Institute of Occupational Health Tampere

ISBN 978-951-27-0979-3 ISBN 978-951-27-1094-2 (PDF) ISSN 1235-0486 Kopijyv Kuopio 2008 Finland

Klemola, Kaisa. Textile toxicity: Cytotoxicity and spermatozoa motility inhibition resulting from reactive dyes and dyed fabrics. Kuopio University Publications C. Natural and Environmental Sciences 241. 2008. 67 p. ISBN 978-951-27-0979-3 ISBN 978-951-27-1094-2 (PDF) ISSN 1235-0486 ABSTRACT The textile industry utilises chemicals in the production of bres, to rene materials in different processes and to produce better quality textile products. Although the chemical itself may be toxic, there is limited data relating to the toxicity of the nal textile product. This information is of clear importance for consumers. The aim of this study was to investigate the toxicity of textile substances by using cell tests in vitro. These tests have been found to be useful when materials containing unknown chemicals need to be evaluated. Boar semen, mouse hepatoma cell line (hepa-1) and a human keratinocyte cell line (HaCaT cells) were exposed to different concentrations of three reactive dyes (Reactive Yellow 176, Reactive Red 241 and Reactive Blue 221) and to the extracts of cotton fabrics dyed with these dyes. The viability of the cell cultures was evaluated. The concentrations IC50 and IC20 to decrease cell protein concentrations in Hepa-1 and HaCaT cell cultures were calculated. These values represent the concentration of the test sample where the protein content in the wells is 50% (IC50) or 80 % (IC20) compared to that of non-exposed cells. The IC20 values were taken to represent the limit of toxicity for fabric extracts. The IC50 and IC20 values were estimated when the dyes were studied. The spermatozoa motility inhibition test was considered to show evidence of toxicity, if at least 25% of the cells were not motile by microscopic observation (50% was set as maximal value of viability). Thus in the spermatozoa test only IC50 value was estimated. After 24 hours exposure of spermatozoa cells to reactive dyes, the IC50 values were 135 g/ml (yellow), 124 g/ml (red) and 127 g/ml (blue). After 72 hours exposure, the blue dye was most toxic to the spermatozoa cells. In hepa-1 cells, no statistical signicant difference in the toxicity between blue, red and yellow was found, the IC50 values being as follows: 392 g/ml (yellow), 370 g/ml (red), 361 g/ml (blue). The IC20 values were 176 g/ml (yellow), 108 g/ml (red), 158 g/ml (blue). In HaCaT cells, IC50 values were 237 g/ml (yellow), 155 g/ml (red), 278 g/ml (blue). HaCaT cells exhibited toxicity with low concentrations of the dyes, with the red dye being the most toxic. The IC20 values in the HaCaT cell line were 78 g/ml (yellow), 28 g/ml (red), 112 g/ml (blue). However, the dyed fabrics were not toxic to all studied cells. The fabric extracts were not toxic to hepa-1 and HaCaT cells since the measured protein content was over 80% of control. In the spermatozoa test compared to control, more than 50% of the test spermatozoa cells showed motility. In addition to reactive dyes and dyed fabrics, the effects of industrial dyed and nished cotton fabrics were investigated in cell tests. All of the studied raw fabric materials (untreated) were non- toxic. The reactive dyed and press shrunk fabric was not toxic. The ame retarded cotton fabric caused little toxicity to the spermatozoa cells. Most of the knitted cotton fabrics were toxic to hepa-1 and HaCaT cells with the exception that the yellow fabric extract was not toxic to HaCaT cells neither was the red fabric extract toxic to the hepa-1 cells. The other knitted fabric extracts affected the viability of the cells less than 80% compared to control. These results show that cell tests are suitable for studies into the toxicity of textile dyes and fabrics but different cell models should be used in these evaluations. The in vitro bioassays provide information which will help in the development of less harmful textile processes and products.Universal Decimal Classication: 667.281, 677.027.423.5 National Library of Medicine Classication: QV 235, QV 602, QV 627, QV 663, WA 465, QY 95 Medical Subject Headings: Textiles/toxicity; Cotton Fiber; Coloring Agents/toxicity; Azo Compounds/toxicity; Flame Retardants/toxicity; Spermatozoa; Sperm Motility; Toxicity Tests; Cell Line; Cells, Cultured; Cell Survival; Inhibitory Concentration 50; Biological Assay; In Vitro

ACKNOWLEDGEMENTS This study was carried out in the Department of Biosciences, University of Kuopio during 20022008. I am deeply indebeted for her kindness, all her advice and support to Docent Pirjo Lindstrm-Sepp, the principal supervisor of my work. My sincere thanks are also due to my supervisor Professor Jyrki Liesivuori, for his advice and encouragement. I owe my thanks to Professor Atte von Wright, Head of the Department of Biosciences, for providing the facilities and position for my work in his department. I am delighted to have had the change to enjoy such a pleasant working atmosphere. I wish to express my gratitude to Professor Hanna Thti and Professor Pertti Nousiainen, the referees of this thesis, for their constructive comments on my work. I am deeply grateful to my co-author Professor John Pearson. I greatly appreciate his efforts in scientic research of textiles and for his pleasant collaboration. I thank Professor Osmo Hnninen for giving encouragement and his belief to me. I express my sincere thanks to Ewen MacDonald, Ph.D., for revising the language of this thesis. I am particularly grateful to Ulla Honkalampi-Hmlinen, M.Sc., for discussions, encouragement and her friendship. I owe my warmest thanks to Virve Krkkinen, M.Sc., and Mrs. Riitta Venlinen for guiding me with cell cultures. I wish to thank all those persons who have made this series of studies possible by helping me either in the eld of laboratory work or by providing technical assistance. I express my thanks to Mrs. Mirja Rekola, Mr. Jouni Heikkinen, Mr. Tuomo Jalkanen and Mr. Vin Klemola. I thank warmly my colleagues in the Kuopio Academy of Design, Mrs. Marke Iivarinen, Mrs. Riitta Junnila-Savolainen, Mrs. Helena Kauttonen, Mrs. Eeva Kontturi and Mrs. Raili Mhnen. During this work, their patience and friendship has been valuable. The encouragement and support of my friends and relatives are deeply appreciated. My warmest thanks belong to my family, my husband Paavo and our son Vin, for their care, patience and understanding. This work was conducted mainly with the support of Finnish Concordia Fund, Magnus Ehrnrooth Foundation and Juho Vainio Foundation. This work was also supported by a grant from the Lisa Andstrm Fund (International Zonta District 20). Kuopio, September 2008 Kaisa Klemola

ABBREVIATIONS ASA Sypsairauden vaaraa aiheuttaville aineille ja menetelmille ammatissaan altistuvien rekisteri. Vuosittainen tilasto. Tyterveyslaitos, Helsinki. The Finnish Register of occupational exposure to carcinogens. Finnish Institute of Occupational Health. adenosine triphosphate bovine serum albumin coefcient of variation Colour Index carboxymethylcellulose a subfamily of cytochrome P450 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane dimethylolhydroxyethyleneurea Dulbecco`s Modied Eagle`s Medium dimethylsulphoxide 2,4-dinitrophenol effective concentration for 50% of maximal effect The European Centre for the Validation of Alternative Methods Environmental Protection Agency 7-ethoxyresorun O-deethylase Food and Drug Administration Good Laboratory Practice human keratinocyte cell line hepa-1 mouse hepatoma cell line International Agency for Research on Cancer inhibitory concentration decreasing response to 80% compared to control inhibitory concentration decreasing response to 50 % compared to control data bank on the use of in vitro techniques in toxicology and toxicity testing International Standard Organization lethal dose, required to kill 50% of animals in the acute toxicity test lowest adverse effect level mitogen-activated protein kinase The Multicenter Evaluation of In Vitro Cytotoxicity Minimum Essential Medium (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) test no adverse effect level Organization for Economic Cooperation and Development polybromide diphenylether phosphate buffered saline The Registration, Evaluation and Authorisation of Chemicals



standard deviation tetrakis-hydroxymethyl-phosphonium tetrakis-hydroxymethyl- phosphonium- chloride tetrakis-hydroxymethyl-phosphonium- sulphate volatile organic compounds World Health Organization Water-soluble tetrazolium assay

LIST OF ORIGINAL PUBLICATIONS This thesis is based on following original publications referred to in the text by their Roman numerals I-IV. I Klemola K, Honkalampi-Hmlinen U, Liesivuori J, Pearson J, Lindstrm-Sepp P. Evaluating the toxicity of reactive dyes and fabrics with the spermatozoa motility inhibition test. AUTEX Research Journal 2006 6(3), 182-190. Klemola K, Pearson J, von Wright A, Liesivuori J, Lindstrm-Sepp P. Evaluating the toxicity of reactive dyes and dyed fabrics with the hepa-1 cytotoxicity test. AUTEX Research Journal 2007 7(3), 224-230. Klemola K, Pearson J, Lindstrm-Sepp P. Evaluating the toxicity of reactive dyes and dyed fabrics with the HaCaT cytotoxicity test. AUTEX Research Journal 2007 7(3), 217-223. Klemola K, Pearson J, Liesivuori J, Lindstrm-Sepp P. Evaluating the toxicity of fabric extracts using the hepa-1 cytotoxicity test, the HaCaT cytotoxicity test and the spermatozoa motility inhibition test. The Journal of Textile Institute, in press.




The original papers in this thesis have been reproduced with the permission of the publishers.

CONTENTS 1. INTRODUCTION 2. REVIEW OF LITERATURE 2.1 2.1.1 2.1.2 2.2 2.2.1 2.2.2 2.2.3 2.3 2.4 2.4.1 2.4.2 2.4.3 2.5 2.5.1 2.5.2 2.5.3 2.5.4 Textile bres Classication of textile bres Cellulose in cotton Textile dyes Dye molecule Classication of textile dyes Reactive dyes Finishing of cellulosic textiles Adverse effects of textile substances Adverse effects of chemicals caused by the production of cellulosic bres Adverse effects of reactive dyes Adverse effects of nishing chemicals used for cellulosic textile materials Toxicity tests Mechanisms of toxicity Testing for toxicity The use of cells in vitro Endpoints used in the evaluation of toxicity in in vitro cell tests 13 15 15 15 16 17 17 19 20 22 23 23 24 25 26 26 27 29 30 32 33 33 33 33 33 33 34 34

3. OBJECTIVES 4. MATERIALS AND METHODS 4.1 4.1.1 4.1.2 4.1.3 4.2 4.3 4.3.1 Dyes and fabric samples Reactive dyes Fabric Commercial fabrics Origin of the cells Procedures for sample preparation and toxicity testing Procedure for dyeing the fabrics

4.3.2 4.3.3 4.3.4 4.3.5

Preparation of fabric extracts The spermatozoa motility inhibition test Cytotoxicity test with hepa-1 mouse hepatoma cells and with human keratinocyte HaCaT cells Statistical methods

34 34 35 36 37 37 39 40 41 42 42 44 45 46 47 49 50

5. RESULTS 5.1 5.2 5.3 5.4 The IC50 and IC20 values for three reactive dyes The toxicity of the fabric extracts Reliability of the results Statistical signicance

6. DISCUSSION 6.1 6.2 6.3 6.4 6.5 Reactive dyes in the cell tests The fabric extracts in the cell tests In vitro cell tests for assaying textile substances The reliability of the tests The possibilities for utilizing cell-based tests for studying textile substances


Introduction 1. INTRODUCTION The manufacture and processing of textiles utilises many different chemical reagents, such as acids, bases, water softeners, salts, organic solvents, dyes and a range of nishes (Trotman 1984). A signicant number of these are harmful to the environment, to the people working in textile processing and potentially to consumers. There is some information available about the toxic and other effects of the individual reagents on textile workers. However, there is limited information about the overall toxicity of dyed and nished materials. Although a reagent itself may be toxic, its presence in the nished material may cause no adverse effects. Ofcial patient organizations concerned with asthma and allergies as well as consumer organisations provide some information about the safety of different consumer products (http://www.efanet. org/, http://www.allergia.com, http://www.kuluttajavirasto.). However, there is little information available about possible toxic effects of textile products, although the toxic effects of many of the reagents used in their manufacture are known. Allergic reactions and irritation to the skin and respiratory tract have been found to be the most common occupational diseases in workers in the textile industry (Hatch 1984, Nilsson et al. 1993, Zuskin et al. 1996, 1998, Niven et al. 1997, Jrvholm 2000). In a study of 72 textile workers in North Carolina, contact dermatitis developed in 24 of them after ve years exposure to textile chemicals (Soni and Sheretz 1996). In contrast, in Finland, a mere 26 work-related diseases were recorded in the 25,000 workers in the textile industry during 2001 (Karjalainen 2002, ASA): this may be due to good working conditions....