Textile Toxicity

68
KAISA KLEMOLA Textile Toxicity Cytotoxicity and Spermatozoa Motility Inhibition Resulting from Reactive Dyes and Dyed Fabrics JOKA KUOPIO 2008 KUOPION YLIOPISTON JULKAISUJA C. LUONNONTIETEET JA YMPÄRISTÖTIETEET 241 KUOPIO UNIVERSITY PUBLICATIONS C. NATURAL AND ENVIRONMENTAL SCIENCES 241 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

Transcript of Textile Toxicity

Page 1: Textile Toxicity

KAISA KLEMOLA

Textile Toxicity

Cytotoxicity and Spermatozoa Motility InhibitionResulting from Reactive Dyes and Dyed Fabrics

JOKAKUOPIO 2008

KUOPION YLIOPISTON JULKAISUJA C. LUONNONTIETEET JA YMPÄRISTÖTIETEET 241KUOPIO UNIVERSITY PUBLICATIONS C. NATURAL AND ENVIRONMENTAL SCIENCES 241

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 17th October 2008, at 12 noon

Department of BiosciencesUniversity of Kuopio

Page 2: Textile Toxicity

Distributor : 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

Series Editors: Professor Pertti Pasanen, Ph.D. Department of Environmental Science

Professor Jari Kaipio, Ph.D. Department of Physics

Author’s address: 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 : [email protected]

Supervisors: Docent Pirjo Lindström-Seppä, Ph.D. Faculty of Medicine University of Kuopio

Professor Jyrki Liesivuori, Ph.D. Department of Pharmacology, Drug Development and Therapeutics University of Turku

Reviewers: Professor Hanna Tähti, Ph.D. Faculty of Medicine, Medical School University of Tampere

Professor Pertti Nousiainen, Ph.D. Department of Materials Science Tampere University of Technology

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

ISBN 978-951-27-0979-3ISBN 978-951-27-1094-2 (PDF)ISSN 1235-0486

KopijyväKuopio 2008Finland

Page 3: Textile Toxicity

ABSTRACT The textile industry utilises chemicals in the production of fi bres, to refi ne 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 fi 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 µg/ml (yellow), 124 µg/ml (red) and 127 µg/ml (blue). After 72 hours exposure, the blue dye was most toxic to the spermatozoa cells. In hepa-1 cells, no statistical signifi cant difference in the toxicity between blue, red and yellow was found, the IC50 values being as follows: 392 µg/ml (yellow), 370 µg/ml (red), 361 µg/ml (blue). The IC20 values were 176 µg/ml (yellow), 108 µg/ml (red), 158 µg/ml (blue). In HaCaT cells, IC50 values were 237 µg/ml (yellow), 155 µg/ml (red), 278 µg/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 µg/ml (yellow), 28 µg/ml (red), 112 µg/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 fi nished cotton fab-rics 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 fl 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 informa-tion which will help in the development of less harmful textile processes and products.

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 978-951-27-0979-3ISBN 978-951-27-1094-2 (PDF)ISSN 1235-0486

Universal Decimal Classifi cation: 667.281, 677.027.423.5 National Library of Medicine Classifi cation: QV 235, QV 602, QV 627, QV 663, WA 465, QY 95Medical 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

Page 4: Textile Toxicity
Page 5: Textile Toxicity

ACKNOWLEDGEMENTS

This study was carried out in the Department of Biosciences, University of Kuopio during 2002-2008. I am deeply indebeted for her kindness, all her advice and support to Docent Pirjo Lindström-Seppä, 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 provi d-ing 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 Tähti and Professor Pertti Nousiainen, the refe rees 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 scientifi c research of textiles and for his pleasant collaboration. I thank Professor Osmo Hänninen 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-Hämäläinen, M.Sc., for discussions, encouragement and her friendship. I owe my warmest thanks to Virve Kärkkäinen, M.Sc., and Mrs. Riitta Venäläi nen 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 fi eld of laboratory work or by providing technical as-sistance. I express my thanks to Mrs. Mirja Rekola, Mr. Jouni Heikkinen, Mr. Tuomo Jalkanen and Mr. Väinö 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 Mähönen. 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 Väinö, for their care, patience and un-derstanding. 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 Andström Fund (International Zonta District 20).

Kuopio, September 2008

Kaisa Klemola

Page 6: Textile Toxicity
Page 7: Textile Toxicity

ABBREVIATIONS

ASA

ATPBSAC of VCICMCCYP1ADDTDMDHEUDMEMDMSODNFEC50ECVAMEPAERODFDAGLPHaCaTHepa-1IARCIC20IC50INVITTOXISOLD50LOAELMAPMEICMEMMTTNOAELOECDPBDEPBSREACH

Syöpäsairauden vaaraa aiheuttaville aineille ja menetelmille ammatissaan altistu-vien rekisteri. Vuosittainen tilasto. Työterveyslaitos, Helsinki. The Finnish Register of occupational exposure to carcinogens. Finnish Institute of Occupa-tional Health.adenosine triphosphatebovine serum albumincoeffi cient of variationColour Indexcarboxymethylcellulosea subfamily of cytochrome P4501,1,1-trichloro-2,2-bis(p-chlorophenyl)ethanedimethylolhydroxyethyleneureaDulbecco`s Modifi ed Eagle`s Mediumdimethylsulphoxide2,4-dinitrophenoleffective concentration for 50% of maximal effectThe European Centre for the Validation of Alternative MethodsEnvironmental Protection Agency7-ethoxyresorufi n O-deethylaseFood and Drug AdministrationGood Laboratory Practicehuman keratinocyte cell linehepa-1 mouse hepatoma cell lineInternational Agency for Research on Cancerinhibitory concentration decreasing response to 80% compared to controlinhibitory concentration decreasing response to 50 % compared to controldata bank on the use of in vitro techniques in toxicology and toxicity testingInternational Standard Organizationlethal dose, required to kill 50% of animals in the acute toxicity testlowest adverse effect levelmitogen-activated protein kinaseThe Multicenter Evaluation of In Vitro CytotoxicityMinimum Essential Medium(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) testno adverse effect levelOrganization for Economic Cooperation and Developmentpolybromide diphenyletherphosphate buffered salineThe Registration, Evaluation and Authorisation of Chemicals

Page 8: Textile Toxicity

SDTHPTHPCTHPSVOCWHOWST-1

standard deviationtetrakis-hydroxymethyl-phosphoniumtetrakis-hydroxymethyl- phosphonium- chloridetetrakis-hydroxymethyl-phosphonium- sulphatevolatile organic compoundsWorld Health OrganizationWater-soluble tetrazolium assay

Page 9: Textile Toxicity

LIST OF ORIGINAL PUBLICATIONS

This thesis is based on following original publications referred to in the text by their Romannumerals I-IV.

Klemola K, Honkalampi-Hämäläinen U, Liesivuori J, Pearson J, Lindström-Seppä P. Evaluating the toxicity of reactive dyes and fabrics with the spermatozoa motility inhibi-tion test. AUTEX Research Journal 2006 6(3), 182-190.Klemola K, Pearson J, von Wright A, Liesivuori J, Lindström-Seppä P. Evaluating the toxicity of reactive dyes and dyed fabrics with the hepa-1 cytotoxicity test. AUTEX Re-search Journal 2007 7(3), 224-230.Klemola K, Pearson J, Lindström-Seppä 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, Lindström-Seppä P. Evaluating the toxicity of fabric extracts using the hepa-1 cytotoxicity test, the HaCaT cytotoxicity test and the spermato-zoa motility inhibition test. The Journal of Textile Institute, in press.

I

II

III

IV

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

Page 10: Textile Toxicity
Page 11: Textile Toxicity

CONTENTS

1. INTRODUCTION

2. REVIEW OF LITERATURE

2.12.1.12.1.2

2.22.2.12.2.22.2.3

2.3

2.42.4.12.4.22.4.3

2.52.5.12.5.22.5.32.5.4

3. OBJECTIVES

4. MATERIALS AND METHODS

4.14.1.14.1.24.1.3

4.2

4.34.3.1

Textile fi bresClassifi cation of textile fi bres Cellulose in cotton

Textile dyesDye moleculeClassifi cation of textile dyesReactive dyes

Finishing of cellulosic textiles

Adverse effects of textile substancesAdverse effects of chemicals caused by the production of cellulosic fi bresAdverse effects of reactive dyesAdverse effects of fi nishing chemicals used for cellulosic textile materials

Toxicity testsMechanisms of toxicityTesting for toxicity The use of cells in vitroEndpoints used in the evaluation of toxicity in in vitro cell tests

Dyes and fabric samplesReactive dyesFabricCommercial fabrics

Origin of the cells

Procedures for sample preparation and toxicity testingProcedure for dyeing the fabrics

13

15

151516

17171920

22

23232425

2626272930

32

33

33333333

33

3434

Page 12: Textile Toxicity

4.3.24.3.34.3.4

4.3.5 5. RESULTS

5.15.25.35.4

6. DISCUSSION

6.16.26.36.46.5

7. CONCLUSIONS

8. REFERENCES

Preparation of fabric extractsThe spermatozoa motility inhibition testCytotoxicity test with hepa-1 mouse hepatoma cells and with human keratinocyte HaCaT cellsStatistical methods

The IC50 and IC20 values for three reactive dyesThe toxicity of the fabric extractsReliability of the resultsStatistical signifi cance

Reactive dyes in the cell testsThe fabric extracts in the cell testsIn vitro cell tests for assaying textile substancesThe reliability of the testsThe possibilities for utilizing cell-based tests for studying textile substances

3434

3536

37

37 39

4041

42

4244454647

49

50

Page 13: Textile Toxicity

13Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

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 fi nishes (Trotman 1984). A sig-nifi cant 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 fi nished materials. Although a reagent itself may be toxic, its presence in the fi nished material may cause no adverse effects. Offi cial patient organizations concerned with asthma and allergies as well as consumer organisa-tions provide some information about the safety of different consumer products (http://www.efanet.org/, http://www.allergia.com, http://www.kuluttajavirasto.fi ). 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, Järvholm 2000). In a study of 72 textile workers in North Carolina, contact dermatitis developed in 24 of them after fi 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. Some textile dyes have been assessed for potential mutagenicity (Przybojewska et al. 1989, Jäger et al. 2004, Schneider et al. 2004, Mathur and Bhatnagar 2007,) and genotoxicity (Sharma and Sobti 2000). For example, a high incidence of bladder cancer was detected in Mataro, Spain among the textile workers using reactive dyes (Gonzales et al. 1988). In a European Union EU-funded re-search project, 281 textile dyes were assessed for potential mutagenic properties using Salmonella typhimurium strains TA98 and TA100. The study revealed positive results for about 28% of the dye products investigated (Schneider et al. 2004). Currently, the EU has set the limiting values for the amounts of carcinogenic aromatic amines (30 ppm) allowed to evaporate from textiles. (2002//61/EY, 2003/3/EY, VNa694/2003). It is well-known that certain textile fi nishing compounds are able to release formaldehyde, which can cause adverse effects (IARC 2004). Finland has set the limiting values (100mg – 300mg/kg) for the amounts of formaldehyde permitted in textiles (KTMa 210/1988). The United Kingdom Health and Safety Executive has 2 ppm workplace exposure limit for formaldehyde. There have been many studies conducted on environmental problems of wastewaters due to the presence of toxic textile chemicals and techniques for decolourisation of dyes and removal of tex-tile chemicals are under development (Choudhary et al. 2004, Khan and Husain 2007, Dincer et al. 2007). However, many textile chemicals are organic compounds and not easily extracted from water. It has been shown that some surface waters in India, e.g. in Jaipur, have a high mutagenic activity due to the presence of chemicals released from the textile industry (Mathur et al. 2005a). There is some information about the possible toxic effects of textile chemicals. A globally used

Page 14: Textile Toxicity

14 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

Kaisa Klemola: Textile Toxicity

Öko-Tex-100 textile standard assesses whether textile products with this eco-label contain harmful amounts of certain compounds, for instance heavy metals (Öko-Tex Standard 100, 1997). Many chemical analyses have been performed on these eco-labelled fabrics. However, this standard does not require any biological tests to evaluate the adverse effects of textile materials. Information about product safety is still limited. In the present study, in vitro cell tests were used to evaluate the potential toxicity of textile dyes and dyed fabrics. Cell tests were selected because of their sensitivity to chemicals. Three different types of cells were used: boar spermatozoa cells, mouse hepatocyte cell line (Hepa-1) and human keratinocyte cell line (HaCaT). Reactive dyes used to dye cotton were used as the test material. Cot-ton was selected because it is the most widely used natural textile fi bre and reactive dyes since they are widely used for dying cotton. These cell tests may be useful in the development of safer working conditions in the textile industry and healthier textile products for consumers.

Page 15: Textile Toxicity

15Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

Review of the literature

2. REVIEW OF THE LITERATURE

2.1 Textile fi bres

2.1.1 Classifi cation of textile fi bres

Textile fi bres contain natural and man-made fi bres. The natural fi bres include cellulose fi bres and protein fi bres. The man-made fi bres consist of raw oil based synthetic fi bres and natural polymers regenerated fi bres. The fi bres can be classifi ed according to their chemistry or according to their origin, the latter being the most commonly used (ISO 6938 1984, ISO 2076 1999, Sundquist 1987, 1988). (Table1: a and b).

About 49 million tonnes of all textile fi bres were produced during 1994 in the whole world about 38 per cent of this being cotton. In fact, cotton is the mostly produced natural fi bre in the world (Worldwide Textile Production 1980-2003). Since the production of cellulose fi bres is so extensive, the need for chemicals for the industrial treatments of the cellulose fi bres is also widespread.

Table 1: a and b. The classifi cation of the textile fi bres (modifi ed from ISO 6938 1984, ISO 2076 1999);

a= natural fi bres, b= man-made fi bres.

mineral

plant fibres animal fibres fibres

seed fibres bast fibres fruit fibres leaf fibres wool, fur

cotton fl ax coconut abaca alpaca silk asbestos

kapok hemp sisal angora

jute henequen camel’s hair

ramie cashmere

lama

mohair

vicuna

wool

a.

b.

man-made fi bres

regenated fi bres synthetic fi bres inorganic fi bres

viscose polyester glass

modal polyamide metallic

lyocell acrylic ceramic

acetate modacrylic

triacetate elasthane

Page 16: Textile Toxicity

16 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

Kaisa Klemola: Textile Toxicity

2.1.2 Cellulose in cotton

Cotton cellulose is a linear, cellulose polymer, the repeat unit (monomer) being cellobiose which consists of two glucose units (Figure 1). The degree of polymerisation (DP) is about 5000 and the polymer is about 5000 nm long and about 0,8 nm thick. The DP in fl ax is higher and lower in vis-cose. The important reactive groups on cellulose are the highly polar hydroxyl (–OH) and methylol (–CH

2OH) groups: reactive dye molecules react with these groups. Hydrogen bonds are formed be-

tween the polar groups on adjacent polymer chains in crystalline areas of the fi bres. Van der Waals` bonds are also present but compared with hydrogen bonding, they are of little signifi cance.

Figure 1. Cotton cellulose consists of cellobiose units.

The cotton fi bre is hygroscopic owing to the polar –OH groups in its polymers. This enables cotton to avidly absorb the polar dye and pigment molecules. However, water and dye molecules can only enter the polymer system in its amorphous regions since the inter-polymer spaces in the crystalline regions are too small to accommodate these molecules (Gohl & Vilensky 1983, Gordon 2006). In cotton, the polymer system is about 65 to 70% crystalline and, correspondingly, about 30-35% amor-phous (Figure 2). Cellulose fi bres are weakened and destroyed by strong acids. Acidic conditions hydrolyse the polymer at the glucoside oxygen atom, which links the two glucose units to form the cellobiose unit. Cellulose fi bres are relatively resistant to alkalis. Oxidising bleaches leave the fi bre polymer system largely intact if the process is done carefully.

Figure 2. Amorphous and crystalline regions of the polymer system (Gohl & Vilensky 1983).

Page 17: Textile Toxicity

17Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

Review of the literature

2.2 Textile dyes

2.2.1 Dye molecules Organic molecules become coloured, and are thus useful dye molecules, if they contain at least one of the following radicals called chromophores (which provide colour) and auxochromes (which intensify and deepen the colour) which can selectively absorb and refl ect incident light (Tables 2-3), (Gohl & Vilensky 1983, Broadbent 2001). Chromophores are unsaturated organic radicals. Their specifi c state of unsaturation enables them to absorb and refl ect incident electromagnetic radiation within a very narrow band of visible light. Loosely held electrons in the conjugated system of the chromophores are able to absorb certain in-cident light waves (Gohl & Vilensky 1983). The auxochromes infl uence the orbitals of the loosely held electrons of the chromophores, which causes these electrons to absorb and refl ect incident light energy only of specifi c wavelengths. This also intensifi es and deepens the hue of the dye molecule. Auxochromes also increase the overall polarity of the dye molecule and make it more readily soluble in water and more readily attracted to the fi bre polymer (Figure 3) which improves the colour- fast-ness properties of the dyed fi bre (Gohl & Vilensky 1983).

Table 2. Chromophores, (Gohl & Vilensky 1983). *In the formula of NO2 the bond should be drawn as -O-,

not as .

Page 18: Textile Toxicity

18 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

Kaisa Klemola: Textile Toxicity

Table 3 Auxochromes (Gohl & Vilensky 1983).

Figure 3. Structural formula of a textile dye molecule. C.I. Acidic Blue 86, 44075 – an acid dye.

(Gohl and Vilensky 1983).

Page 19: Textile Toxicity

19Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

Review of the literature

The majority of dyes can be regarded as resonance hybrids with the colours obtained depending on the energy states of the orbitals. Lengthening the conjugated chains increases the number of double bonds and decreases the energy gaps between the π-orbitals. Therefore, the longer the conjugated chain, the less energy will be required to excite the electrons and the greater will be the wavelength of the absorbed light. This can be seen in the properties of carbocyanine dye (Trotman 1984) (Figure 4). As the number (n) of double bonds increases, there is a clearly defi ned shift of the light absorbed towards red, with a corresponding relative increase in the proportion of blue refl ected (Trotman 1984).

2.2.2 Classifi cation of textile dyes

There are over 13,000 different compounds classifi ed as dyes in the Colour Index (CI) 2001. About 8000 compounds of them are textile dyes and they give rise to about 40000 commercial names which are used as textile dyes. The CI classifi es the dyes according to their application class and to their chemical structure (Table 5). For the textile dyer, the classifi cation according to application class is more signifi cant. The classes are: acidic dyes, azoic (naphthol) dyes, metal-complex dyes, developed dyes, disperse dyes, mordant dyes, reactive dyes, direct dyes, cationic (basic) dyes, sul-phur dyes and vat dyes as well as pigments and fl uorescent brighteners (Sundquist 1985, modifi ed from Aalto et al. 1994, Talvenmaa 1998, Colour Index 2001). Each dye has a fi ve-digit CI-number.

Figure 4. Carbocyanine dye. When n = 0 (yellow), n=1 (red), n=2 (greenish blue), n=3 (blue)

(Trotman 1984).

Page 20: Textile Toxicity

20 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

Kaisa Klemola: Textile Toxicity

Synthetic textile dyestuffs contain many different chemicals with different applications, for instance to increase shelf life, to improve water solubility and to reduce dusting. Thus commercial dyestuffs contain many other molecules in addition to dye molecules themselves (Talvenmaa 1997, Broadbent 2001).

2.2.3 Reactive dyes

Reactive dyes are named according to their chemical reactivity with fi bre polymers. These dyes are widely used in dyeing cotton and other cellulose-based fi bres. Around 120,000 tonnes are produced per year accounting for over 60% of all dyes for cellulosic fi bres (Holme 2004). Reactive dyes form a covalent bond between the dye molecule and the fi bre polymer (Figures 5-7). This produces a stable covalent bond which results in excellent colour fastness. This property as well as the simplic-ity of the dyeing process, means that reactive dyes, despite their relatively high cost, are widely used on cellulosic fi bres.

Figure 5. The vinyl sulphate radical of a reactive dye molecule and its bonding to the cellulose polymer.

(Gohl & Vilensky 1983)

nitroso dyes indamine- and indophenol dyes

nitro dyes azine dyes

azoic dyes oxanthine dyes

azoic developed dyes thiazine

stilbene dyes sulfur dyes

carotenoid dyes amino developed dyes

diphenylmethane dyes hydroxyketone dyes

triarylmethane dyes anthracinone dyes

xanthene dyes indigo dyes

arcidine dyes phtalocyanine dyes

cinoline dyes organic natural dyes

methine and polymethine dyes oxidation developed dyes

thiazole dyes inorganic pigments

Table 5. Textile dyes according to their chemistry. (Colour Index 2001, modifi ed from Aalto et al. 1994)

Page 21: Textile Toxicity

21Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

Review of the literature

Figure 7. A monochlorotriazinyl reactive dye. (Trotman 1984)

Figure 6. A dichlorotriazinyl reactive dye and its bonding to the cellulose polymer. (Gohl & Vilensky 1983)

Page 22: Textile Toxicity

22 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

Kaisa Klemola: Textile Toxicity

Typically reactive dyes are applied using salt and subsequently fi xation is brought about by the ad-dition of soda ash to the dye bath. The reaction between the fi bre and the dye molecule takes place under alkaline conditions. The dye can also undergo hydrolysis with water and this decreases the colour yield of the dye as well as lessening the colour fastness due to reduced number of covalent bonds within the fi bres (Gohl & Vilensky 1983, Trotman 1984, Broadbent 2001).

2.3 Finishing of cellulosic textiles

Cellulose fi bres ignite and burn easily and therefore it has been important to develop fl ame-retar-dant treatments for those materials. Many of these compounds are based on water-soluble inorganic salts that are easily removed by water, rain and perspiration: they provide only temporary protection. Boron (polybromide diphenylether PBDE) and phosphorous compounds are widely used. Durable fl ame retardancy is typically obtained by the use of organophosphorous compounds (Lewin and Sello 1983). Tetrakis-hydroxymethyl-phosphonium-salts such as Proban (Rhodia; - tetrakis-hydroxymethyl phosphonium chloride (THPC)-urea) are used as durable fl ame retardants for cotton, cellulose and cellulose-blend clothing fabrics. Pyrovatex CP (Ciba Speciality Chemicals) is a phosphonoalkyl-amide used to confer durable fl ame retardancy on cellulosic fabrics being used in furnishings (Gohl & Vilensky 1991, WHO 2000, Schindler and Hauser 2004). There are numerous chemicals used to make fabrics water repellent. These include aluminium and zirconium soaps, waxes, wax like substances, metal complexes, and pyridinium- and methylol- com-pounds. The use of methylol stearamide with partially formed urea-formaldehyde provides a good water-repellent fi nishes with adequate fastness properties. Fabrics can also be made water-repellent by incorporation of thermo-setting silicone resins. Water-repellency combined with oil- and soil-repellency can also be obtained by the application of fl uoropolymers such as Scotchguard (3M) and Tefl on (Du Pont) (Kissa 1984, Schindler and Hauser 2004). Modern developments include the use of nanotechnology. Cotton textiles can be protected against attack by micro-organisms with quaternary ammonium com-pounds, such as cetyl trimethyl ammonium chloride being used to achieve this goal. Chemical modi-fi cation of the cellulose polymers is possible: reaction of fi bres with acrylonitrile produces durable protection (Gohl and Vilensky 1983, Schindler and Hauser 2004). Chun & Gamble (2007) reported the use of silver nanoparticles, graft polymerisation of N-halamide monomers, chloromelamine de-rivatives and cross-linked chitosan to produce durable effects. A number of durable easy-care fi nishes have been developed to reduce creasing of cellulose fi bres with most of these based on dimethyloldihydroxy-ethyleneurea (DMDHEU) (Priha 1988, Schindler and Hauser 2004). Many fi nishing formulations require the use of catalysts, residues of which may be found in treated fabrics, and non-ionic softeners.

Page 23: Textile Toxicity

23Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

Review of the literature

2.4 Adverse effects of textile substances

Asthma, rhinitis and dermatitis are three of the most common adverse effects evoked by textile pro-cessing (Eskelson and Goodman 1963, James 1985, Docker et al. 1987, Jahkola et al. 1987, Jolanki et al. 1999, Piipari and Keskinen 2003). Textile manufacture is a global activity and it is diffi cult for consumers to obtain information about the production and chemicals of textile products. It is known that harmful chemicals may have been used, but potential adverse effects of textile products are not widely appreciated. However, informa-tion about the chemicals of textile products can be important, especially for sensitized consumers. The Austrian Textile Research Institute and the German Hohenstein Research Institute jointly developed the Öko-tex-100 standard and eco-label (www.oeko-Tex.com) which is now used globally to indicate that the textile product has been tested for the presence of harmful substances. However, the overall toxic effects of end products are not included and information about overall toxicity is not available. 2.4.1 Adverse effects of chemicals caused by the production of cellulosic fi bres

Many chemicals are used in the cultivation of the cotton plant, for instance chlorinated phenols, zinc and copper salts and residues may still be present in cotton-based textiles (Suojanen 1995, Öko-Tex Standard 100, 1997). Some fertilizers contain high concentrations of cadmium (about 138 mg/kg) in phosphoric fertilizers (Malm & Louekari 2000) and some insecticides e.g. methoxyethylmercurium (Komulainen et al. 1992). Synthetic pyrethroids cause irritation effects (Priha 1988). Serious effects on the central nervous system have been detected following the use of organophosphate insecticides (Minton et al. 1988, Marrs 1993, Lopez-Carillo and Lopez-Cervantes 1993, Stevens et al. 1995). It has been stated that women working with organophosphates are at risk of developing non-Hodgkins lymphoma (Zahm et al. 1993). It has been found that patients who were exposed to organophos-phates and carbamates had low levels of cholinesterase activity in their blood (De Peyester 1993). For products with the Öko-Tex Standard 100 label, limiting values have been set for the amounts of recognized harmful chemicals allowed to be present (Öko-Tex Standard 100 1997). Much work has been done in Western Europe and Scandinavia to ensure that textile products do not contain chemicals that might be harmful to the consumer and as consequence many textile chemicals have been banned. However, 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) was found in England and Sweden not only in raw fabrics but also in clothes (Ahonen 1994, Suojanen 1995). Although most of the harmful chemicals are washed away during industrial processing, some may still remain in the textile end products. It has been recognized for over 200 years that prolonged exposure to cotton dust can produce bys-sinosis. This results in the weakening of the lungs and chronic pulmonary infl ammation (Duffell 1985, Sigsgaard 1992, Beckett et al. 1994, Hayes et al. 1994, Christiani & Wang 2003). It has also been claimed to be a risk factor in the development of nose- and skin cancer (Lund 1991, Luce et al. 1992). There is less large-scale use of chemicals in the production of other natural cellulosic fi bres. How-

Page 24: Textile Toxicity

24 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

Kaisa Klemola: Textile Toxicity

ever, the production of regenerated cellulosics such as viscose, utilises harmful chemicals and these can cause problems. For example, adverse effects on the eyes and respiration have been detected in persons who have been exposed to carbon disulfi de which is a known neurotoxic compound (Aa-serud et al. 1990, Vanhoorne et al. 1991). The most widely used chemicals after fi bre formation are surfactants and auxiliaries, which may act as irritants. Acidic detergents can cause eczema and contact dermatitis. Enzymes, bleaches and brighteners can evoke respiratory allergic reactions. Aromatic, and chlorine-containing organic solvents are known irritants. Other auxiliaries, including strong acids, mineral oils and salts can cause irritation and allergic reactions (Estlander and Jolanki 1980, Virtanen and Hannuksela 1999). In conclusion, the production of cellulosic fi bres utilises chemicals that can evoke adverse effects including allergic reactions and irritation to the skin and the respiratory system. Some of these harm-ful substances may remain in the textile end products. However, there is minimal information for consumers about these adverse effects. Although cellulosic fi bre processing has been extensively studied, the actual end products remain to be more thoroughly investigated.

2.4.2 Adverse effects of reactive dyes

Symptoms of asthma, rhinitis and dermatitis have been frequently detected in workers exposed to reactive dyes (Hatch 1984, Thoren et al. 1986, Nilsson et al.1993, Manzini et al. 1996, Park et al. 2006, 2007). Dyes containing anthraquinone or azo structures are known to cause contact dermati-tis (Estlander 1988, Wilkinson and McGechaen 1996). The result of a clinical and immunological investigation of respiratory disease indicated that about 15% of 400 workers handling reactive dyes experienced work-related respiratory and nasal symptoms (Docker et al. 1987). Many studies have also found statistically signifi cant relationships between reactive dyes and increasing immunoglobu-lin blood values in workers who have been contact with these dyes (Alanko et al. 1978, Topping et al. 1989, Park et al. 1991). However, it should be noted that dermatological problems associated with dyes in textiles are relatively rare (Maurer et al. 1995), although Hatch et al. (2003) have noted such effects and have given the name ‘colored clothing allergic contact dermatitis (ACD)’ to these symptoms. Moreau & Goossens (2005) reported similar effects and stated that reactive dyes should be classifi ed as potential allergens, even their presence in clothes. The cause of skin reactions is diffi cult to trace because the dye usually acts as a delayed sensitizer and as such does not cause an immediate response (Hatch and Maibach 1995, Wang et al. 2002). It has been assumed that since the properties that enable the dyes to react with textile fi bres also allow them to bind to body protein, the health hazard resulting from exposure to such substances is signifi -cant (Keneklis 1981). Since reactive dyes are chemically very active, they can cause harmful effects, especially when in their powdered form. It was noticed as early as 1981 that some sulphonyl ethyl sulphate derivatives were carcinogenic (Keneklis 1981). The international register of cancer-causing chemicals includes many textile dyes (including those based on benzidine and o-toluidine) and their raw chemicals for synthesis (IARC 1987, TMp 838/1993, Aalto et al. 1994). However, in Finland, the register of

Page 25: Textile Toxicity

25Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

Review of the literature

individuals exposed to carcinogens contains little information about textile industry workers (Kaup-pinen 1999, Vuorela 2003). The UK Health and Safety Executive warns industrial workers who have contact with reactive dyes that they may become sensitized, stressing the potential for respiratory sensitization. In many European countries, the use of textile dyes releasing certain aromatic amines at concentrations above 30 ppm is forbidden (VNa 694/2003, 2002/61/EY and 2003/3/EY). If one wishes to detect any adverse effects of textile dyes, then it is important to conduct tests for mutagenicity and genotoxicity (Przybojewska et al. 1989, Schneider et al. 2004, Mathur et al. 2005a, Mathur et al. 2005b, Dogan et al. 2005), carcinogenicity (De Roos et al. 2005) and teratogenicity (Birhanli and Ozmen 2005). All these studies have revealed adverse effects of dyes, although not all the dyes tested were reactive dyes. Due to the problems associated with textile dyes, the dyeing process is under constant development, with increasing attention being paid to the ecological effects of these chemicals.

2.4.3 Adverse effects of fi nishing chemicals used for cellulosic textile materials

Many fi nished fabrics may release formaldehyde. This problem is associated especially with the permanent press, fl ame proofi ng and antimicrobial treatments. Formaldehyde has been demonstrated to cause harmful effects (James 1985, Priha et al. 1986, 1988, Priha 1992,1995, Priha et al. 1996, Jahkola et al. 1987, WHO 1989, Garcia et al. 1995, IARC 2004, Carlson et al. 2004). Specifi c effects include irritation to skin, eyes and the respiratory tract (James 1995) and it may also cause asthma (Piipari and Keskinen 2003). In Finland, limiting values have been set for the amount of formal-dehyde allowed in fabrics (Priha 1995; Jolanki et al. 1999; Suomen säädöskokoelma 210/88). The effects of formaldehyde in textile work places have been widely studied (Nousiainen 1979, 1982, 1983,1984:a and b, Roberts and Rossano 1984, Priha et al. 1996, Scheman et al. 1998). Since high concentrations of formaldehyde may cause cancer, the international register of cancer causing chemi-cals classifi es formaldehyde to The Group 1: carcinogenic to humans (IARC 2004). With regard to fl ame retarding agents, polybromide biphenyls have been found to cause adverse effects and have been removed from sale (WHO 1997). Polybromide diphenylethers (PBDE) may form polychloride dioxins and furans in a fi re and their use is now greatly restricted. PBDE and polychlorinated PCBs can interact and enhance developmental neurobehavioral effects when the ex-posure occurs during a critical stage of neonatal brain development (Eriksson et al. 2006). Neonatal exposure to brominated fl ame retardant, 2,2´,4,4`,5-pentabromodiphenyl ether has been shown to cause altered susceptibility in the cholinergic neurotransmitter system in the adult mouse (Vikberg et al. 2002). In addition, it has been noted that PBDE disrupts spontaneous behaviour, impairs learn-ing and memory in adult mice (Vikberg et al. 2003). An increase in the amounts of PBDE has been found in the environment and this chemical is even present in mother`s milk. However, in the USA, the measured amounts of brominated fl ame reagents in the environment have been claimed to pose no threat to the health of children (Hays and Pyatt 2006). According to IARC (1998) it is not certain whether the widely used phosphonium salt as fl ame retardants are carcinogens. Tetrakis-hydroxymethyl-phosphoniumchloride (THPC) was tested and treated fabrics did not cause skin irritation to humans. THPC was not shown to be carcinogenic in

Page 26: Textile Toxicity

26 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

Kaisa Klemola: Textile Toxicity

rats and mice in a 2-year bioassay. However, dermal studies with rabbits have shown that THP salts are promoters but not initiators of skin cancer (WHO 2000). Antimicrobial treatments are not widely used on clothing: their use is limited mainly to tent fabrics and woollen carpets. Many antimicrobial agents are relatively toxic and have been found to sensitize humans (Kanerva 1998, Kalimo and Lahti1999, Yazdankhah et al. 2006). Other fi nishing agents: water repellents, stain repellents, dirt repellents and antistatic agents have seldom caused skin or other health problems (Priha et al. 1988). However, these reagents are also available in aerosol forms where they are combined with carbohydrates and fl uorochemicals, and in these situations they have caused pulmonary infl ammation (Wright and Lee 1986, von Essen 1996, Vernez et al. 2006). These aerosol products are available for home use by consumers. Indigo-dyed denim fabrics were shown to be mutagenic, showing the importance of analysing the fi nished fabrics in addition to the pure chemicals. Since the mutagenicity of indigo was low, the genotoxicity of denim extracts must have been due either to some unknown chemicals or to some unknown reactions (Rannung et al. 1992). Knasmuller et al. (1993) showed that 18 of 196 fabric samples examined were mutagenic, 16 of them only after metabolic activation. Though many azo dyes have been shown to be mutagenic (Chung and Cerniglia 1992, Kaur 1993), it seems that azo-dyed fabrics are not toxic (Kaur et al. 1993). Nonetheless, when azo-dyed fabrics for clothing were tested with the Ames bacterial assay, almost 20% of the silk fabrics and over 10% of the cotton fab-rics gave positive results in this test for mutagenesis (Pfi tzenmeier 1990). The textile industry utilises different washing, dyeing and fi nishing chemicals which can contain nonylphenol and nonylphenolethoxylates. These chemicals and their metabolites are strongly cor-rosive and have been found to be toxic to the aqueous environment and to humans. These chemicals have been claimed to cause hormonal changes and therefore the European Union has recommended that their use should be limited (2001/838/EY, VNa 596/2004). The causes of toxicity of a fabric can be diffi cult to trace since they may be due to the combined ef-fects of several of the chemicals present in the textile. Although the problems caused by many textile chemicals are recognized, potential problems in fabrics have not been widely studied and, as with the production of cellulosic fi bres, there is limited information available to consumers.

2.5 Toxicity tests

2.5.1 Mechanisms of toxicity

The toxicity is of a compound becomes apparent when a toxicant is delivered to its target and reacts with it and the resultant cellular dysfunction manifests itself in toxicity. Sometimes a xenobiotic does not react with a specifi c target molecule but rather adversely infl uences the biological (micro) environment causing molecular, organellar, cellular or organ dysfunction and leads to deleterious effects. (Gregus and Klaassen 2001) The most complex path to toxicity involves four steps. First, the toxicant is delivered to its target or targets, after which the ultimate toxicant interacts with endogenous target molecules, triggering perturbations in cell function and/or structure, which initiate repair mechanisms at the molecular, cel-

Page 27: Textile Toxicity

27Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

Review of the literature

lular and/or tissue level. When the perturbations induced by the toxicant exceed the repair capacity or when repair becomes malfunctional, then toxicity occurs. The examples of chemically induced toxicities followed by this four-step course are tissue necroses, cancer and fi broses. (Gregus and Klaassen 2001) One chemical may yield several ultimate toxicants, one ultimate toxicant may react with several types of target molecules, and the reaction with one type of target molecule may have a number of different consequences. Thus, the toxicity of a chemical may involve several mechanisms which can interact with and infl uence each other in an intricate manner. (Gregus and Klaassen 2001) A number of xenobiotics such as heavy-metal ions, strong acids and bases, nicotine, and ethylene oxide are directly toxic, whereas the toxicity of other compounds is largely due to metabolites. Bio-transformation of a compound to a harmful compound is called metabolic activation. However, the conversion of a bioactive parent compound to a less bioactive or inactive metabolite(s) that is/are effi ciently eliminated is mostly usual. This conversion is called metabolic inactivation, or detoxifi ca-tion. (Parkinson 2001) Non-polar xenobiotics accumulate into lipid-containing tissues or are metabolised to a more water soluble compounds. The fi rst step is xenobiotic metabolism, the so-called phase I reactions that consisting of non-synthetic reactions like oxidation, reduction and hydrolysis. Phase II reactions in the second phase are conjugation reactions with compounds having hydroxyl -OH, amine –NH2 or carboxylic –COOH groups. The functional groups may be present in the parent compound or may have been formed during phase I reactions that lead to toxicity. (Bend and James 1978, Sijm and Op-perhuizen 1989, Parkinson 2001) Benzene, similar to other aromatic compounds, is oxidised into a variety of reactive metabolites which are normally more toxic than the original compounds. For instance, benzene can be oxidized to a variety of quinines and semiquinones that can cause hematopoietic toxicities and leukaemia. Benzene and many other volatile organic compounds (VOCs) are converted via multiple metabolic pathways to products with varying toxicities. Some of these competing pathways are considered as bioactivation, others as detoxifi cation pathways. A variety of factors (for example differences between species, functions of enzymes) can infl uence the prominence of the different pathways and hence alter toxicity outcomes. When different cell signalling pathways become disrupted, the cell has typically become exposed to some toxic substance (Alberts et al. 2002, Gregus and Klaassen 2001).

2.5.2 Testing for toxicity

Toxicity tests are not designed to demonstrate that a chemical is safe but to characterize the toxic effects it can produce. Currently there is a new set of regulations and toxicity tests that have to be performed on chemicals intended for commercial use (www.ymparisto.fi -REACH). During the past years, some regulations have been introduced, for instance, the Food and Drug Administration (FDA), Environmental Protection Agency (EPA) and Organization for Economic Cooperation and Development (OECD) have issued good laboratory practise standards (GLP). These guidelines are expected to be followed when toxicity tests are conducted in support of the introduction of a chemical

Page 28: Textile Toxicity

28 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

Kaisa Klemola: Textile Toxicity

to the market. (Eaton and Klaassen 2001) The typical in vivo acute toxicity test (LD50) is estimated by determining the number of animals that die in a 14-day period after treatment of a single dosage of the chemical. Clinical chemistry and histopathology are performed after 14 days of exposure. However, this test has been removed from the guidelines (in 2002), and replaced by three ethically more acceptable refi nement tests, which use much less animals and in which the lethal dose is not determined any more (Worth and Balls 2002). Subacute toxicity tests are performed to obtain information on the toxicity of a chemical after repeat-ed administration and as an aid in deciding how to conduct subchronic studies. Long-term or chronic exposure studies are performed similarly to subchronic studies except that the period of exposure is longer than three months, usually from six months to two years. (Eaton and Klaassen 2001) Numerous in vivo and in vitro procedures have been devised to test chemicals for their ability to cause mutations. Some genetic alterations can be visualized with the light microscope. The test for mutagens that has been most widely used in toxicology is the Salmonella/microsome test developed by Ames et al. (1975). In vitro tests for mutagens are in widespread use. In addition, many in vitro tests based on cell cultures have been developed, each with its own particular advantage in the area of toxicological research. In addition to ethical benefi ts, these tests are cheaper and quicker to carry out and generate smaller quantities of toxic waste than other tests (Baksi and Frazier 1990). Cytotoxicity can include changes in the integrity of membranes and cytoskeleton, cellular metabolism, energy metabolism and synthesis and degradation of cellular constituents, ion transport and cell division. In addition, compounds can be selectively cytotoxic or cause cytotoxicity by interference with cell-specifi c func-tions (Seibert et al. 1992). The most important results obtained from toxicity tests are the values of LOAEL (the lowest dose that causes adverse effects) and NOAEL (the dose that does not cause any adverse effect) which set the limiting values of toxicity. The toxic value from an in vitro test is typically stated as the effective concentration EC50 which represents the dose that causes 50% of cells to die. Another value often used in in vitro studies is the IC50 value, e.g. when the inhibition of enzyme activity is measured as an endpoint to indicate the viability of the cells in culture. In the present study, IC50 values are used to describe the inhibition of the viability of the cells in culture and the inhibition of the movements of spermatozoa. In the area of toxicology, LD50 is no longer in use. NOAEL is the most important value and is help-ful when the limiting values for chemicals need to be set. At present, in all chemical safety testing animal experiments must be applied. In the OECD and EU guidelines there are in vitro replacement tests only for skin absorption, skin corrosion, phototoxicity, severe eye irritation and mutagenicity. In addition, several tests have been validated by the European Centre for the Validation of Alterna-tive Methods ECVAM, but they have not yet been accepted for regulatory purposes (http://ecvam.jrc/it). Nonetheless, in vitro tests can provide mechanistic data and also these kinds of tests are useful for screening of chemicals. The safety data sheets still include results of LD50 assays. (Eaton and Klaassen 2001, Liesivuori, oral communication). It is known that in vivo animal tests do not always predict toxicity in humans. However, it is often possible to calculate relatively safe doses for humans. It has also become increasingly evident that

Page 29: Textile Toxicity

29Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

Review of the literature

chemicals causing carcinogenicity in animals are not necessarily carcinogenic to humans (Grisham 1997, Dybing and Sanner 1999, Hengstler et al. 1999). However, for regulatory and risk assessment purposes, positive carcinogenicity tests in animals are usually interpreted as being indicative of po-tential human carcinogenicity (Eaton and Klaassen 2001).

2.5.3 The use of cells in vitro

A large range of cell lines is available for studying toxicity: human and animal cells, lines from pathological cases, tumour lines, normal lines and transformed lines. These cell lines have many uses e.g. when studying the effects of various anticancer drugs during the early phase of the drug development. Retinal pigment epithelial cell lines have been exposed to anti-oestrogenic drugs in studies of eye toxicity (Toimela et al. 1995, Mäenpää et al. 2004). The effects of hyperoxia have been studied with cervical cancer cells (Campian et al. 2004) and the effects of radiation with malig-nant pleural mesothelioma cell lines (Häkkinen et al. 1996). Cells can be useful for analysing different kinds of materials to reveal acute toxicity. In addition, it is possible to study the overall toxicity of materials with unknown chemical compositions. Informa-tion about the toxicity of individual chemicals may be available, but the combined effects are diffi cult to study and are not often available in the literature. In different studies, boar spermatozoa cells, hepa-1 mouse hepatoma cells and human keratinocyte HaCaT cells have been found to be useful. Boar spermatozoa cells have a simple metabolism compared to somatic cells. They are completely dependent on their surrounding environment for nutrients and they are also not able to detoxify toxic end products due to the low concentrations of detoxifying enzymes in the cell cytosol. Many physi-ological processes in spermatozoa are controlled by membrane potentials and ion fl uxes (Mann and Lutwak-Mann 1982). The motility of semen can be measured in several ways e.g. both hyperacti-vation and inhibition. Inhibition of motility is a consequence of membrane depolarisation (Gao et al. 1997). Hyperactivation of motility is associated with membrane hyperpolarisation (Zeng et al. 1995). Bacterial toxins have been found to affect the cell membrane and the mitochondrial mem-brane potential in the sperm cells at very low concentrations (Hoonstra et al. 2004). Bovine spermatozoa have been used in studying cytotoxicity (Seibert et al. 1989, 1992, Seibert and Gosch 1990, INVITTOX Protocol 21 1991). The spermatozoa in vitro test has been found to be useful when detecting the presence of hazardous substances in indoor building materials subjected to moisture damage and containing complex microbial communities of bacteria and fungi (Andersson 1999). In addition, paper materials have been studied (Severin et al. 2005). Hepa-1 mouse hepatoma cells (Hepa-1c1c7) are a commonly used cell line to assess the potential toxicity of dioxin and dioxin-like compounds, materials such as laboratory animal beddings and feeds, fl y ash samples and paper products (Kopponen et al. 1991, 1992a,b, 1993, 1994a,b,c, 1994, Kärenlampi and Törrönen, 1990, Törrönen et al. 1989, 1991, 1994, Severin et al. 2005). Kopponen et al. (1997) conducted some preliminary studies with textile dyes and fabrics using the hepa-1 cy-totoxicity test. Since 1989, the Hepa-1 cytotoxicity test has been included as a part of the Multicenter Evaluation

Page 30: Textile Toxicity

30 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

Kaisa Klemola: Textile Toxicity

of In Vitro Cytotoxicity (MEIC) programme, in which the overall toxic potencies of a wide range of chemicals relevant to human toxicity have been studied (Clemedson et al. 1996, INVITTOX Protocol 112 1995). The programme revealed that the various cell lines and growth end point measurements gave similar cytotoxicity results in most cases. The Hepa-1 cytotoxicity test has given satisfactory values for practically all chemicals tested and these mouse cells are, in general, better indicators of human toxicity than rat cells. Human keratinocyte HaCaT cells possess some metabolic activity, but are not as versatile as hepa-1 cells. HaCaT cells are human skin cells and for this reason they have been considered as relevant when human toxicity has been studied. HaCaT cells have been widely used for instance in evaluat-ing skin irritation (Wilhelm et al. 1994), skin cancer (Merryman et al. 1999), genotoxicity and mu-tagenicity of textile dyes (Wollin et al. 2004) and the cytotoxicity caused by potential contact with nickel and chromium (Little et al. 1996). In particular, HaCaT cells have been used for investigating adverse effects of UV-radiation (Isoherranen et al. 1999, O`Reilly and Mothersill 1997). The cells have also been reported as being useful in clarifying cell signalling pathways (Assefa et al. 1997; Shimizu et al. 1999).

2.5.4 Endpoints used in evaluation of toxicity in in vitro cell tests

The total protein content of the cells is a widely measured and validated endpoint (INVITTOX Protocol 112 1995). In this test, some dead cells may also be measured and may cause variation in the viability results. However, the content of the total protein provides valuable information about viability. The neutral red (NR) cytotoxicity test determines the number of living cells in the culture. In li-ving cells, neutral red penetrates cell membranes and accumulates in lysosomes and can be measured photometrically (INVITTOX, Protocol 64). The MTT-test (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) measures the ac-tivity of mitochondrial succinate-tetrazolium reductase system of cells (Kwang-Mahn et al. 2005) Immunotoxicological activity has been tested by exposing mouse macrophages and measuring the endpoints by the MTT-test when indoor air of moisture-damaged buildings has been studied ( Hut-tunen et al. 2008). Apoptosis and cell viability have also been measured with the MTT-test when anticancer drugs have been evaluated (Giovagnini et al. 2008). In addition, in the studies where the skin models have been validated, the MTT-test has been used (Kidd et al. 2007). A water-soluble tetrazolium assay, the WST-1 test, which is similar to the MTT test, has been performed to measure the mitochondrial synthesis and cellular proliferation rate (Kwang-Mahn et al. 2005). For example, the test has been used when the proliferation rate of human osteoblast-like cells has been studied (Weibrich et al. 2002). The rate of apoptosis has been measured when the antidepressant drug, desipramine has been evaluated in human PC3 prostate cancer cells (Chang et al. 2008). Mitochondrial viability and apoptosis induced by aluminium, mercuric mercury and meth-ylmercury have been assessed with the WST-1 test (Toimela and Tähti 2004). In addition, pyrethroid compounds in neural cell cultures have been examined with the WST-1 test (Kakko et al. 2004). In that study, the energy of the exposed cells was also investigated by measuring their ATP content.

Page 31: Textile Toxicity

31Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

Review of the literature

Lactate dehydrogenase leakage is coupled to energy production of the cells. The enzyme catalyzes the fi nal reaction of anaerobic glycolysis, the reduction of pyruvate to lactate (Campbell and Far-rell 2006). LDH has been used as an indicator of cellular damage of membrane when the effects of mercury, methylmercury and aluminium on gial fi brillary acidic protein expression in rat cerebellar astrocyte cultures have been studied (Toimela and Tähti 1995). If one wishes to understand the mechanisms of toxic effects, several types of cells are available for use and several endpoints are available for measurement.

Page 32: Textile Toxicity

32 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

Kaisa Klemola: Textile Toxicity

3. OBJECTIVES

There is limited information about the toxicity of textile substances when they are present in fi n-ished textile articles. The aim of this study was to evaluate the toxicity of textile reactive dyes and fabric extracts with cell tests in vitro. The detailed aims were as follows:

To assess the usefulness of two cell lines and bovine spermatozoa for the determination of possible acute toxicity of textile reactive dyes (I-III).

To examine whether dyed materials differ in their toxicity from the pure reactive dyes. The aim was to study the overall toxicity of the dyes in fabrics (I-III).

To evaluate the toxicity of common commercially available fabrics. The aim was to deter-mine if the materials after fi nishing and dyeing differ in their toxicity compared to the raw materials (IV).

To defi ne which tests are useful in providing information about the toxicity of reactive dyes. In addition, the purpose was to study whether data from cell tests could provide useful infor-mation about potential toxicity or safety of textile products. It was also of interest to examine if these tests could be further developed into routine tests for use in the textile industry.

1.

2.

3.

4.

Page 33: Textile Toxicity

33Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

Materials and methods

4. MATERIALS AND METHODS

4.1 Dyes and fabric samples

4.1.1 Reactive dyes

The three reactive dyes studied were monchlorotriazinyl dyes commonly used in cloth-making and by workers in the crafts industry. They are also the basic three dyes used when mixing different colour combinations. Colour Index numbers for the dyes were: Reactive Red 241, Reactive Yellow 176 and Reactive Blue 221 (Drimarene red CL-5B, Drimarene yellow CL-2R and Drimarene blue CL-2RL respectively). The dyes were obtained from Clariant Ltd. The samples of the dye powder were dissolved in the appropriate cell medium solution.

4.1.2 Fabric

A typical sheeting fabric was used in this study: plain weave bleached cotton (white), obtained from a commercial fabric shop. It was observed that the material contained some brightener. One sample was self dyed with an unknown reactive brilliant yellow dye.

4.1.3 Commercial fabrics

In this study, those fabrics dyed in industrial processes are called commercial fabrics. Two qualities of 100% cotton fabrics for working clothes were investigated. One fabric was vat dyed and treated with fl ame retardant: blue, twill woven (175g/m2). The other fabric was reactive dyed and press shrunk: brown and blue, twill woven (260g/m2). In addition, there were two different types of knitted fabrics: 100% cotton 160g/m2 and 50% /50% cotton/modal 145g/m2. Knitted fabrics were reactive dyed with yellow, red and blue dyes respectively. The fabrics were fi nished in textile factories in Finland. More detailed information about these fabrics was not available. All commercial fabrics were available to study in the form of raw fabric material. However, the chemical content of the raw fabrics was not known.

4.2 Origin of the cells

Boar semen for testing was available from the Insemination Center of Pieksämäki, Finland. Hepa-1 mouse hepatoma cells (the wild type Hepa-1c1c7) were obtained from The Department of Physiology, University of Kuopio, Finland. The cells were originally obtained from Dr. D.W. Ne-bert, Department of Environmental Health, University of Cincinnati, Ohio, USA in 1982. Human keratinocyte HaCaT cells were obtained as a gift from the Department of Anatomy, Uni-versity of Kuopio, Finland.

Page 34: Textile Toxicity

34 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

Kaisa Klemola: Textile Toxicity

4.3 Procedures for sample preparation and toxicity testing

4.3.1 Procedure for dyeing the fabrics

Fabric samples (10g) were washed gently without soap. The amount of dye used was 3% on each fabric. The dye bath contained 400 ml water with 50g Na

2SO

4/l H

2O and 20g

Na2CO

3/l H

2O. Dyeing continued for one hour at 55ºC. Na

2CO

3 was added to the dye bath ten min-

utes after the beginning of the dyeing process to adjust the pH. After dyeing, the fabrics were spooled in cool and warm water baths and kept in pure boiling water for 10 minutes. The dyeing process in the present study was based on the common procedures used in industry and by crafts workers. A total of 3% dye represents a strong colour on fabrics.

4.3.2 Preparation of fabric extracts

The fabric pieces (1cm x 1cm) were extracted with sterilized water (1g fabric/20ml H2O) in labora-

tory test tubes. The tubes were shaken at room temperature for two hours and incubated at 37ºC for 18 hours. The samples were shaken thoroughly before centrifugation for 5min at 4500 rpm. The fab-ric extracts were sterile fi ltered through polyester and cell growth medium compounds were added to the extracts before exposure to the cells. The fabric extracts contained the same concentration of cell growth compounds as in the controls of pure growth medium. There were 3-4 parallel samples. However, there was only one extract sample for each commercial fabric extract.

4.3.3 The spermatozoa motility inhibition test

The spermatozoa test based on that used by Andersson (1999) and it was modifi ed for use with textile samples. Valinomycin dissolved in dimethylsulphoxide (DMSO) in several concentrations was used as the positive control: 2ng, 4ng, 8ng and 16ng/2ml respectively. Plain semen and water were used as negative controls to determine the level of normal values. A solution of valinomycin in DMSO with no semen was also tested. The samples were added to semen, using 2ml of semen with 40µl of the dye samples or fabric extracts. All samples were compared to the control sample of plain semen. Exposure continued for 24 hours and/or 72 hours at room temperature. The tubes were inverted once a day. Before analysis, the tubes were gently mixed manually. All processing was carried out under sterile conditions. After 24 hours and/or 72 hours of exposure, sperm motility was measured and compared to the motility of sperm in the plain semen controls. The effects of water and DMSO were also tested. After gently mixing the tubes, 200µl samples were taken to small plastic tubes for incubation at 37ºC for fi ve minutes before microscopic analysis. The temperature of the pre-warmed objective glasses used in the microscopic analyses was also 37ºC. The samples were gently mixed and the amount of li ving spermatozoa cells was qualitatively observed by light microscopy (100 – 400 x magnifi cation). The results refl ected the motility capacity percentages of living cells. The optimal value of the plain

Page 35: Textile Toxicity

35Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

Materials and methods

control sample was set at 50% because the semen always contains dead and damaged cells. In the evaluation, the following observations of sperm activity were conducted: speed of movement, abil-ity to move forward, damage that can be detected by microscopy, rotation, vibration and dead cells. The toxicity limit was considered to be 25% of unexposed cells: levels of 25% and lower represent toxicity. The limiting value of 25% therefore represents the IC50 (inhibitory concentration) which means the concentration where 50% of the original living spermatozoa (optimal value of control was set 50%) were dead, nonmotile, weakly moving or rotating at the end of the exposure time. The toxic effects on spermatozoa were qualitatively evaluated in the fi eld of vision. The crude categories were: 50% - cells move forward, have strong vibration and high activity; 40 - 45% - cells move forward, have strong vibration but less activity than controls; 30 - 35% - some vibration re-maining, many dead cells, most of the cells are rotating, only some are moving forward very slowly; 20 – 25% - most of the cells are dead, some are rotating; 10 - 15% - only some cells are moving slowly, most of the cells dead and 5%- isolated cells are vibrating slowly. The results were recorded within these 5% boundaries.

4.3.4 Cytotoxicity test with hepa-1 mouse hepatoma cells and human keratinocyte HaCaT cells

In order to assess the potential toxicity of dyes and fabric extracts, the hepa-1 cell cytotoxicity test was used (modifi ed from INVITTOX [data bank on the use of in vitro techniques in toxicology and toxicity testing] protocol number 112). The same modifi ed method was used for human kera-tinocyte HaCaT cells. The cells were grown as a monolayer at 37ºC in an atmosphere of 5% CO

2;

hepa-1 cells were grown in -MEM (Minimum Essential Medium) and HaCaT-cells were grown in DMEM (Dulbecco’s Modifi ed Eagle’s Medium). Both medium solutions were supplemented with 1% glutamine, 10% foetal calf serum and 1% penicillin/streptomycin solution. The test was carried out in 96-well plastic microplates seeded with a 200µl cell suspension per well (5x104 cells/ml). After growing for 24 hours, the culture was about 60% confl uent. The cells were exposed to the dye samples or to the samples of the sterilized fi ltered (0.2µm pore size) fabric extracts. Non-exposed cells with medium were used as a negative control. All results were compared to these controls. 2,4-dinitrophenol was used as a positive control at three concentrations: 0.5 mg 2,4-dinitrophenol/ml DMSO was used as one control and diluted to concentrations of 0.05mg/ml and 0.005mg/ml medium respectively. After 72 hours of exposure, the cells were washed twice with PBS-buffer (phosphate buffered saline, pH 7.2). Before the addition of sodium phosphate buffer it was possible to observe the viability of the cells by light microscopy to obtain preliminary information. Subsequently, 50µl of sodium phosphate buffer (0.05mM, pH 8.0) was added to each well before freezing the plates for at least 15 min at -70ºC. After breaking the frozen cells, the plates were thawed for 15 min and cell growth was detected by assaying the total protein content in the cultures (Kennedy et al 1993, 1995). 150µl of sodium phosphate-buffer was added to the wells followed by 50µl of cold fl uorescamine (1.08mM in acetonitrile). The plates were allowed to stand at room temperature for 15 min before being stirred in a microtitration plate shaker for one minute. The total protein content in each well was measured using a plate reading fl uorometer at a wavelength of 405/460nm. The protein (bovine serum albumin BSA) standard curves were measured in each bioassay. All processing, except for the

Page 36: Textile Toxicity

36 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

Kaisa Klemola: Textile Toxicity

protein assays, was carried out under sterile conditions. The inhibitory concentration value, the IC50 value is the concentration of the sample in which the wells with exposed cells have only 50% of the protein content compared to that of the non-exposed cells (100%): the level of the protein content is a refl ection of the viability of the cells. The inhibitory concentration IC20 denotes the sample concentration where the protein content is 80% compared to the protein content of non-exposed cells. In such a case, the studied dye or fabric extract has inhibited the cell proliferation by 20 %. The IC20 value was considered to represent the value of LOAEL (lowest observed adverse effect level) which is the lowest concentration value of the sample showing adverse effects. The sample was considered to have low toxicity if the protein content was less than 80%, but more than 50% compared to that of non-exposed cells. A reduction of more than 50% in the protein content would represent a clearly toxic effect.

4.3.5 Statistical methods

The coeffi cients of variation (C of V % were used as evidence of reliability with lower values than 10%) and standard deviations (SDs) were calculated for all measurements to evaluate the reliability. The number of parallel samples was in the range of 3-11 when the spermatozoa cells were used as the test cells. In the hepa-1 and HaCaT cell tests, the number of the parallel samples was 3-4. The number of the samples represented the number of independent measurements and the number of plates. Every plate contained 3-4 parallel dye or fabric extract samples. Statistical signifi cance of differences between three reactive dyes was performed for each cell test by variance analyse, ANOVA. Values of p < 0.05 were considered as statistically signifi cant.

Page 37: Textile Toxicity

37Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

Results

5. RESULTS

5.1 The IC50 and IC20 values for three reactive dyes

The three reactive dyes studied were Reactive Red 241, Reactive Yellow 176 and Reactive Blue 221. The dyes were assayed with boar spermatozoa cells, hepa-1 mouse hepatoma cell line and hu-man keratinocyte cell line, HaCaT-cells. The inhibitory concentrations measured as IC50 and IC20 values were mathematically calculated from the functions which were drawn from the mean values of different dye concentrations. Cell growth was assessed as the amount of cellular protein after the exposure (Figures 8-10).

Figure 8. IC50 value for the yellow dye when boar spermatozoa cells were used, exposed for 24 hours

(I); *n=3-10;SD = ±( 2-5,2); *The number of the samples (n) represents the number of the plots at the

various concentrations of the dye on the curve.

Figure 9. IC50 and IC20 values for the yellow dye when hepa-1 cells were used, exposed for 72 hours (II);

n = 2-7, SD = ±(2,1-9).

Page 38: Textile Toxicity

38 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

Kaisa Klemola: Textile Toxicity

Figure 10. IC50 and IC20 values for the yellow dye when HaCaT-cells were used, exposed for 72 hours

(III); n = 2-6; SD = ±(3,5-9,8).

According to the IC50 mean values, hepa-1 cells tolerated the highest dye concentrations. The spermatozoa cells were most sensitive and displayed toxicity with the lowest dye concentrations. Of the three cells used, HaCaT cells were of intermediate sensitivity also showing somewhat different responses to the different dyes. The results for all three dyes studied showed the same trends (I-III: Table 6). The IC20 mean values showed that with the reactive dyes studied, the hepa-1 cells had the high-est tolerance (II-III: Table 6). The red dye was the most toxic to HaCaT cells and this value was even lower than the IC50 value for spermatozoa cells after the same 72 hours of exposure (Table 6). However, after 72 hours of exposure, the blue dye evoked such high toxicity to the spermatozoa cells that it was not possible to determine the IC50 value (I. Table 6). However, in HaCaT cells the blue dye was less toxic than the yellow dye (I-III: Table 6). Thus, the relative toxicity of the dye studied depended on which cells were used.

Table 6. Summary of the mean concentration values of IC50 and IC20 (µg/ml) for the dyes studied.

HaCat Hepa-1 spermatozoa

72 h 72 h 24 h 72 h

µg/ml (n) µg/ml (n) µg/ml (n) µg/ml (n)

IC50 Reactive Red 241 155 (5) 370 (4-6) 124 (4-9) 46 (3-9)

IC50 Reactive Yellow 176 237 (6) 392 (4-7) 135 (4-9) 60 (4-9)

IC50 Reactive Blue 221 278 (2-4) 361 (2-4) 127 (4-10) - (3-9)

IC20 Reactive Red 241 28 (5) 108 (4-6)

IC20 Reactive Yellow 176 78 (6) 176 (4-7)

IC20 Reactive Blue 221 112 (2-4) 158 (2-4)

Page 39: Textile Toxicity

39Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

Results

5.2 The toxicity of the fabric extracts

In the present study, the plain cotton fabric was dyed with the same reactive dyes as above: Reactive Red 241, Reactive Yellow 176 and Reactive Blue 221. In addition, a commercially obtained fabric dyed with an unknown brilliant yellow reactive dye was studied. The plain weave bleached cotton fabric samples which were used as controls were toxic when studied with hepa-1 and HaCaT cells. The protein contents of the samples measured as an indication of cell growth were found to be 35 - 50% of the non-exposed cells (100%) when studied with hepa-1 and HaCaT cells. However, the dyed fabrics were not toxic. In addition to the dyed fabrics, a com-mercial fl ame retarded fabric was studied. It was not toxic to these cell lines (IV: Figure 11).

Figure 11. The cytotoxicity of the dyed plain weave and commercial fl ame retardant fabrics. (IV).

Thus, according to the spermatozoa motility inhibition test and the cytotoxicity tests with hepa-1 and HaCaT cells, none of the untreated fabrics studied were considered toxic. Over 50% of the spermatozoa cells retained motility (n=3-6). The protein contents in the extract samples indicated cell growth levels of over 80% compared to the non-exposed cells (100%) when hepa-1 and HaCaT cells were used (n=3: IV). The same fabrics after industrial dyeing and fi nishing were then analyzed. The spermatozoa cells did not show toxicity with the fabric extracts apart from the fl ame retardant fabric, which displayed low toxicity (n=3-11: IV). The fabric extracts from the commercial woven fabrics dyed with reac-tive dyes resulted in decrease in measured protein contents to less than 20% compared to the non-exposed cells when hepa-1 and HaCaT cells were used (IV). The extracts from the two types of untreated knitted fabrics were not toxic when assayed with the three different cell tests. The studied knitted fabrics did not display any toxicity in the spermatozoa inhibition test after commercial dyeing and fi nishing (n= 3-11, Figure 12: IV). However, after in-dustrial dyeing and fi nishing, fabric extracts were toxic to the hepa-1 and HaCaT cells (n=3: Figure 12) though the yellow and red cotton fabrics were exceptions (IV). The yellow cotton was not toxic

Page 40: Textile Toxicity

40 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

Kaisa Klemola: Textile Toxicity

in the HaCaT- and the red cotton was not toxic in the hepa-1-cytotoxicity tests (IV). When the blue knitted fabrics were studied in hepa-1 cells the extracts evoked strong toxic effects compared to the situation with the same extracts in HaCaT cells. In addition, the blue knitted fabrics were most toxic to the hepa-1 cells (IV).

5.3 Reliability of the results

The commercial fabrics in the study were industrially dyed and fi nished. These woven fabrics were not toxic when tested with hepa-1 and HaCaT cells. Before and after dyeing and fi nishing, the coeffi cients of variation (C of V) were 3 – 13% (n=3) (IV). The industrial untreated knitted fabrics showed C of V values of 4-13%. When the materials were industrially dyed and fi nished, most knitted fabrics were toxic to hepa-1 mouse cells and on HaCaT cells (n=3-4). The C of V values ranged from 2 –24% (IV). The spermatozoa motility test showed only the fl ame retardant fabric to be toxic. The industrial fabrics had C of V values of 0 – 18 % (n=3-11) (IV).

Figure 12. The percentage of protein in cell exposure by knitted fabric extracts compared to the non-

exposed cells when hepa-1 and HaCaT cells were used (n=3: IV). * CO=cotton, CMD=modal

Table 7. Coeffi cient of variation values (C of V %) for dye samples and fabric extracts. The fabrics were

dyed with Reactive Red 241, Reactive Yellow 176 and Reactive Blue 221 (I-III).

toxic results non-toxic results

spermatozoa (dye) after 24h exposure 31-69 0-18

spermatozoa (dye) after 72h exposure 0-58 9-37

hepa-1 (dye) 9-46 <10

HaCat (dye) 7-21 2-14

spermatozoa (fabric) after 24h exposure 10-16

spermatozoa (fabric) after 72h exposure 8-35

hepa-1 (fabric) 10-17

HaCat (fabric) 9-13

Page 41: Textile Toxicity

41Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

Results

For assays using spermatozoa cells, valinomycin was used as a positive control. The values cov-ered the toxic and non-toxic areas. Two concentrations, 2ng/2ml and 4ng/2ml of valinomycin had no adverse effects after 24 hours exposure but 8 ng/2ml and 16 ng/2ml concentrations did cause toxicity. DMSO was not toxic at the concentrations used in the tests (I, IV). In the HaCaT and hepa-1 assays, non-exposed cells with medium were used as negative controls and all results were compared to them. As a positive control, 2,4-dinitrophenol (DNF) was used giv-ing C of V values of 2 – 25%. High C of V values were noted in the toxic range. (II-IV). DNF was used as a positive control and the mean value of the protein content measured from cell suspensions after exposure at low concentrations was 80% of the protein content of the non-exposed cells (II-IV). The low control concentration of DNF (0,05mg/ml) resulted in 60% protein content (C of V 11-18%), while the highest control concentration of DNF caused severe inhibition of cell growth, only 10 % remaining of protein content of the control (C of V 2-25%: II-IV).

5.4 Statistical signifi cance

In the spermatozoa 24-h test, the difference in the results between the three studied reactive dyes was not statistically signifi cant; for the high concentration of the dyes (196µg/ml), p = 0.763; for the low concentration (39µg/ml), p = 0.122. Hepa-1 cell test showed statistical signifi cant differences compared to control at a concentration of 150µg/ml, p = 0.022, but not at the high concentration of the dyes (600µg/ml), p = 0.225. With HaCaT cells, the differences between the three studied dyes were statistically signifi cant; for the concentration of 100µg/ml, p = 0.003; for 190 µg/ml, p = 0.002; for 380 µg/ml, p = 0.000.

Page 42: Textile Toxicity

42 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

Kaisa Klemola: Textile Toxicity

6. DISCUSSION

6.1 Reactive dyes in the cell tests

When the reactive dyes were studied in vitro (Reactive Red 241, Reactive Yellow 176 and Reac-tive Blue 221), toxicity was detected with the spermatozoa cells at low dye concentrations. Sper-matozoa are dependent on their surrounding environment for nutrients and also for removal of toxic end products since they have low levels of detoxifying enzymes in their cytosol. The physiological processes in spermatozoa are controlled by membrane potentials and ion fl uxes (Mann and Lutwak-Mann 1982) and therefore the spermatozoa cells may be useful for indicating acute toxicity: they can be assumed to give relevant information about toxicity (Seibert 1992, Hoonstra 2004). The cell lines have been widely used and different tests have been developed as an alternative for animal tests (http://www.ecvam.jrc.it). The IC50 and IC20 values for the above mentioned reactive dyes were higher with hepa-1 cells than with HaCaT cells. This can be explained by the ability of hepa-1 hepatoma mouse cells to metabolise xenobiotics (INVITTOX Protocol 112 1995). Induction of CYP1A1 (detected as arylhydrocarbon hydroxylase and 7-ethoxyresorufi n o-deethylase, EROD activity) has been shown to be useful for the assessment of the biological potencies of pure chemicals like PCBs and for the estimation of dioxin-like compounds in extracts of environmental samples (Kennedy et al. 1993, Kennedy et al. 1995). Hepa-1 cells have been widely used as a model for studying the induction mechanism of CYP1A1 (Kärenlampi and Törrönen 1990, Kopponen et al. 1994a). Though metabolism of xenobiotics usually facilitates the excretion of chemicals, certain steps in the biotransformation pathway can be responsible for the activation of foreign chemicals to reactive intermediates that ultimately result in toxicity, carcinogenicity and other adverse effects. This has been clearly detected in aquatic species (Varanasi et al. 1987, Stegeman and Lech 1991). In this thesis, no enzyme activities were measured as the aim was simply to assess cell viability to reveal acute toxicity of the studied substances. Human keratinocytes have some ability to metabolise xenobiotics. However, the phase I metabo-lism in the skin is usually only a small fraction of that found in the liver. The enzyme cytochrome P4501A1 is inducible in the epidermis by agents that are inducers also in other tissues. Thus expo-sure to this kind of agent could infl uence skin biotransformation and even sensitize epidermal cells to other agents that are not good inducers themselves, a phenomenon observable in cell culture (Walsh et al. 1995). In addition, the enzymes participating in phase II metabolism are expressed in the skin. In general, this activity occurs at a much lower rate than that observed in the liver, but exceptions are evident, as in the case of quinone reductase (Khan et al. 1987). In this study, the aim was to obtain information about acute toxicity and therefore only the viability of the human keratinocyte cells was evaluated. The cytotoxicity tests with hepa-1 and HaCaT cells provided information about the tox-icity of the substances studied, not information about their exact metabolic effects. When the dyes were studied, it seemed that hepa-1 cells tolerated higher dye concentrations than HaCaT cells, as refl ected by the higher IC50 and IC20 values with hepa-1 cells than with HaCaT cells. This may be attributable to differences in the metabolic abilities of the cell lines. Since the various cells used in the present study possess different capabilities it is not unexpected

Page 43: Textile Toxicity

43Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

Discussion

that they exhibit some differences in the results obtained. However, when the dyes were studied similarities were noted in the end points and the results supported each other. This was evaluated via the determination of the IC20 and IC50 values. It is possible to determine which concentration of the particular dye evokes the fi rst level of adverse effects and when the material is clearly toxic to the cells. Kopponen et al. (1997) studied reactive textile dyes using hepa-1 cells, but these dyes were not the same as those used in the present study. The IC50 values were in the range 53 –825µg/ml. In the Safety Data Sheets, these dyes had LD50 values of 2000 – 9000mg/kg. In the present study, all IC50 values with hepa-1 cells were in the range 158 - 392µg/ml (I-III). The Chemical Safety Data Sheets for the reactive dyes in this study indicate the blue and red dyes were more toxic than the yellow dye. In the Safety Data Sheet, the LD50 value (rats, orally) for the yellow dye is 5000mg/kg, and for the red and blue dyes 2000mg/kg. According to toxicity tests con-ducted with activated sludge, the toxicity of the blue dye measured as EC50 (the molar concentration of an agonist, which produces 50% of the maximum possible response for that agonist) was greater than 100µg/ml. In the present study, the IC50 values for the blue dye were 127µg/ml (spermatozoa cells, exposed for 24 hours), 361µg/ml (hepa-1 cells, exposed for 72 hours), 278µg/ml (HaCaT cells, exposed for 72 hours). When the spermatozoa cells were exposed to the blue dye for 72 hours, all cells displayed no motility. The IC20 values in the present study for the blue dye were between 112-158µg/ml with HaCaT and hepa-1 cells. According to the OECD 209 method (an acute toxicity test for testing sludge), the red and yellow dyes had IC50 values higher than 1000µg/ml. According to the OECD 203 method (an acute fi sh toxicity test), the LC50 values were claimed to be in excess of 100µg/ml. In the present study, the IC50 mean values from the spermatozoa test and hepa-1 test for the yellow dye pointed to higher values than those obtained for the red and the blue dyes. The IC20 mean values with HaCaT cells were lower than 100 µg/ml for the red and the yellow dyes (exposed for 72 hours). In the present study, the IC20 mean values from the HaCaT test for the red and the yellow dyes indicated that these compounds may be more toxic than the corresponding values for toxicity published in the Chemical Safety Data Sheets. The dyestuffs used in the present study are, in fact, mixtures of different chemicals (I-IV). They contain, for instance, carboxymethylcellulose CMC, calcium stearate and other chemicals, but it is not possible to obtain information from the manufacturers about what other chemicals are present. It is possible that not only the dye molecules, but also the other chemicals may evoke adverse effects. In addition, it is possible that the dye formulations which were studied are not consistent. The dyes are monochlorotriazinyl dyes e.g. containing a sulphonyl-group, chlorine, fl uorine and nitrogen, and the blue dye contains copper, but the precise chemical structures of the dye molecules are not avail-able. This information would be useful in assessing the toxicity of dye molecules. The discussion of the results of this study relates to the toxicity of a mixture of different chemicals and not to the pure molecules. This study was not designed to evaluate the effects of a pure dye. The pH of the dyes is in the range 4,5 – 6,5 and it is evident that these pH values do not cause toxic effects to the cells. When hepa-1 and HaCaT cells were used, the growth medium was buffered (pH 7,1-7,2) and the samples diluted. The sample concentrations during the tests were low (less than 400µg/ml). When the spermatozoa cells were used, the concentrations of the samples were lower

Page 44: Textile Toxicity

44 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

Kaisa Klemola: Textile Toxicity

than those incubated with the hepa-1 and HaCaT cells. In the textile dyeing process, the dye bath is alkaline because reactive dyes demand a high pH for good reactivity. It can therefore be assumed that the dyes may be more toxic during the dyeing pro-cess than during the conditions of the present series of studies.

6.2 The fabric extracts in the cell tests

Reactive dyes bind to the fi bre covalently (Trotman 1984). The binding is very stable and dur-ing the process, the dye molecule loses its reactivity. Although the extracts of some fabric samples contained colour, the extracts were not toxic. This is because dye molecules are easily hydrolysed in water. In the present study, the plain cotton fabric (bleached) became less toxic after the dyeing process. It can be assumed that washing and dyeing must have removed harmful chemicals (I-IV). Although reactive dyes themselves cause adverse effects like asthma, rhinitis and dermatitis (Estlander 1988, Nilsson et al. 1993, Hatch and Maibach 1995, Manzini et al. 1996, Wilkinson and McGechaen 1996), after reacting with fi bre molecules, the dyes are stable and should not be toxic within the fabric. However, any unbounded dye remaining in the fabric could cause allergic reactions. Since reactive dyes and their hydrolysis products are water-soluble, unbounded dye can be washed off. Therefore, washing of newly dyed products is recommended (Moreau and Goossens 2005). However, more information is needed about the overall toxicity of reactive dyes and dyed fabrics. The fabrics to be dyed in the present study were more toxic in the HaCaT cell tests than they were in the hepa-1 cells. The fabric itself, before it was dyed, was toxic. The protein contents of the samples taken as indications of cell growth were in the range 35-50% of control. However, after dyeing, the material was no longer toxic with cells demonstrating over 80% protein content of control. This in-dicates that for these reactive dyes studied, the end products of the dyeing process do not cause toxic effects in cell tests: i.e. the fabric became non-toxic. In addition, all data obtained in the spermatozoa motility tests confi rmed these non-toxic responses (IV). The commercial untreated industrial woven and knitted fabrics were not toxic (the protein content of the cells did not decline to any signifi cant degree, i.e. less than 20 %). After the industrial dyeing and fi nishing processes, the fl ame retardant fabric displayed low toxicity when incubated with the spermatozoa cells. The other commercial woven fabrics were not toxic. However, after industrial dyeing and fi nishing, the knitted fabrics evoked adverse effects in hepa-1 and HaCaT cells (protein contents of cells were considerably below 80% of control with the exception of the yellow cotton in HaCat cells and the red cotton in hepa cells), evidence that some harmful chemicals were present. The knitted fabrics were coloured with reactive dyes. According to the present study, these agents should not produce adverse effects in the fabrics. It has been observed that some fabric softeners may cause adverse effects in fi sh (Wester and Roghain 1992). It is also well known that free form-aldehyde in textiles can cause toxicity (Priha 1992, 1995, Priha et al. 1988, 1996). However, in the present study there was no information available about what kind of different chemicals were used when the knitted fabric was manufactured. It is usual to treat knitted fabrics with softeners, so it can be assumed that the softeners may have caused some toxic effects. In addition, it is possible that the

Page 45: Textile Toxicity

45Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

Discussion

studied material contains chemicals for binding the extra dye to the fi bre to improve the wet-fastness. In addition, residues of harmful nonylphenols (surfactants; 2001/838/EY) have been found. Howev-er, in the present study with the knitted fabrics, it remained unclear which chemical or combinations of chemicals produced the adverse effects on the cells. In this study, it was interesting to note that hepa-1 mouse cells were more sensitive to toxic knitted fabrics than human keratinocyte HaCaT cells. This was opposite to the anticipated results. The knit-ted fabrics produced a particularly clear toxic effect on hepa-1 cells, although these cells would have been expected to be resistant since they possess better metabolic capabilities than HaCaT cells. 6.3 In vitro cell tests for assaying textile substances

The cells in culture usually grow well, and in the present tests the quality of the cells was standard-ized. However, there were differences in the quality of the spermatozoa cells used in the present assays and this affected their motility. In some assays, a substantial number of cells were broken, while in others, a signifi cant proportion had good motility. In the cell viability experiments, it was noted that the cell growth at the edges of the plates was often less impressive than in the middle. However, the exposure results in every cell culture assay as in the spermatozoa motility assay were compared to those of the non-exposed cells, avoiding possible errors due to differences in the qual-ity of the cells. Since the evaluation of the spermatozoa motility inhibition is subjective, this test is a qualitative test, and variations may occur in the results. However, in the present study, several researchers studied the same samples and the spread of their results was within 5%. The results of the spermatozoa test are read within 5% bands and this can have an extra effect on the variation in-herent in the results. However, despite these limitations, the test can be used reliably for screening samples and when combined with other tests: the inhibition of boar spermatozoa motility has proved valuable when evaluating the toxicity of food constituents (Salkinoja-Salonen et al. 1999). In addi-tion, the test has demonstrated its value as a cell toxicity assay in detecting hazardous substances in products without the need for whole-animal exposure or foetal calf serum for cell cultures (Hoornstra et al. 2004). The present study indicated that the spermatozoa test is also valuable for studying the potential toxicity of textile substances. However, further development will be necessary when fabric extracts are going to be studied. When hepa-1 mouse cell line and human keratinocyte HaCaT cell line were used, the total protein contents were measured to indicate the viability and proliferation capacity of the cells. However, this is not a very exact method for assessing the viability of the cells, since the protein content of some dead cells may be also measured even though the washing procedure should remove the dead (unattached) cells. There is also the possibility that some living cells are lost before protein measure-ments during the washing procedures of the plates. However, protein measurement is a widely used procedure and it does provide some information about the toxicity of the studied material. The hepa-1 cell test has been found to be sensitive and reliable (INVITTOX Protocol 112 1995, Kopponen et al. 1992, Törrönen et al. 1994, Kopponen et al. 1997). In the present study, these cell cultures were sensitive to the toxic effects of the compounds in the textiles under study. The NR test is useful if one wishes more exact information about the cells (INVITTOX Protocol 64

Page 46: Textile Toxicity

46 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

Kaisa Klemola: Textile Toxicity

1992). Mitochondrial activity can be studied by MTT and WST-1 assays (Toimela and Tähti 2004, Kwang-Mahn et al. 2005). In addition, cell energy and membrane effects have been studied by ATP and LDH measurements (Toimela and Tähti 1995, Kakko et al. 2004). However, in the present study, the methods to study overall toxicity of textile substances were the focus of interest and more detailed information about mechanism of toxicity in the cells was not the target of the study. The fabric sample extracts were fi ltered through polyester. Polyester cannot bind covalently to dye molecules. Some extracts were coloured, since they contained free dye molecules that had passed through the fi lter. However, the results did not show any adverse effects. The dye molecules are hydrolysable and hence lose their reactivity in aqueous solutions (Trotman 1984). The fabric extracts were not concentrated. The concentrations of the samples were the same for the hepa-1 and HaCaT cell assays, but the spermatozoa cell concentration was lower, as this was a requirement of the assay method. The fabric extracts were not concentrated because that process may have produced chemical reactions during the procedure, possibly affecting their toxicity. The extraction method for the spermatozoa motility test needs further development. The fabrics may contain compounds that are lipid soluble. The physical property that commonly enables xenobiotics to be absorbed through the cell membrane is the lipophilicity (Gregus and Klaas-sen 2001). Therefore, in the future, it will be important also to use lipophilic solvents when extract-ing fabric materials.

6.4 The reliability of the tests

The coeffi cient of variation was typically high when cell toxicity was present. This is understand-able when living material is being analysed. However, the limiting values for toxicity and non-toxic-ity were clearly identifi ed. When the three reactive dyes were compared to each other, the results of the spermatozoa test (ex-posed for 24 h) did not reach statistical signifi cance (p > 0.05). Statistical signifi cance in hepa-1 cells was observed when the concentrations of the dyes were low. In HaCaT cells, all concentrations of the tested dyes resulted in statistically signifi cant changes. It is clear that there is a need to develop further the spermatozoa test, for instance by studying a fuller range of fabric extracts. It has to be emphasised that the spermatozoa motility test is a qualita-tive test. In addition, the quality of the spermatozoa cells can lead to variations in the fi nal results. The spermatozoa motility inhibition test is only suitable for crude screening of chemical samples. According to the C of V values in this study (0-18%), it seems that the optimal exposure time is 24 hours since this results in the lowest C of V values. In the hepa-1 and HaCaT tests, further development may lead to better reproducibility; e.g. it may be useful to expose cells in the medium that contains a lower concentration of serum. The high con-centrations of the serum used may cause mistakes since it may be able to bind certain molecules. All three methods must therefore be developed and validated further if they are to be used routinely. For the standardization of the tests, the range of substances should be extended and the validation of the test should be made in co-operation with other laboratories. However, the present study shows that these tests are promising novel methods for studying the toxicity of components on textiles.

Page 47: Textile Toxicity

47Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

Discussion

6.4 The possibilities for utilizing cell-based tests for studying textile substances

Improvements have occurred not only in the working conditions in the textile industry but also in the environmental-friendliness of industrial dyeing-fi nishing processes (Nousiainen 1997). In addi-tion, the ecology of laundry services (Kalliala 1997) has been studied. Some information about the impacts of waste waters from textile processing is available. However, more advanced studies in the area of toxicology are needed, especially using a range of cell tests, since there is a scarcity of re-search providing information for the consumer about the potential toxicity of textile end-products. The Öko-Tex-100 textile standard assesses the amounts of certain harmful compounds in textile products related to prescribed limits, for instance heavy metals (Öko-Tex Standard 100 1997). How-ever, this standard does not require that common biological tests have to be carried out to detect any adverse effects of the products, nor have the combined effects of different chemicals been studied. In addition, fabrics may contain chemicals that are not assessed in the Öko-Tex-100 standard, so the cell tests represented here are useful in confi rming the safety of the product. Safety Data Sheets show information about the toxicity of individual chemicals. The EU new chemical legislation (REACH, The Registration, Evaluation and Authorisation of Chemicals) de-mands that the safety of 30000 industrial chemicals should be evaluated by the year 2018. The evaluations of 20000 of these chemicals have been planned to be performed without animal experi-ments by using all information available and also by increasing the use of validated alternative tests. The cell tests can provide extra information to the standard tests. It is also recommended that more tests that do not involve the use of animals should be devised, although subchronic and chronic ef-fects with animals may still needed in the foreseeable future. Cell tests can be used not only for pure chemicals but also for testing materials containing different chemical components. In vitro cytotoxicity tests provide preliminary information about acute toxicity and other param-eters. One of the most sensitive biochemical responses is the induction of specifi c cytochrome P450 (CYP) enzymes. It would be interesting to study further in more detail the specifi c effects of textile dyes and fabric extracts, for instance their effects on mitogen activated protein kinase pathways and other cell signalling pathways in human keratinocytes. The effects of organochlorine pesticides have been studied using HaCaT cells (Ledirac et al. 2005) and it would be useful to use HaCaT cells in toxicity tests with different textile chemicals. Another approach would be to use macrophages to evaluate the effects of the compounds on immunological activity. In addition, it would be of inter-est to measure the energy metabolism of the cells, e.g. the ATP balance in cells exposed to different textile chemicals. Cell proliferation studied by MTT and WST-1 assays may be useful when more detailed information about the effects of textile substances needs to be obtained. In the European Centre for the Validation of Alternative Methods, ECVAM, skin models have been validated and endorsed as suitable for testing skin corrosion and irritation in vitro. Two skin models have been validated: the EPISKIN model and the EpiDerm model (EU, ECVAM 2007, Grindon et al. 2007, El Ghalbzouri et al. 2008). The EPISKIN model is a three-dimensional human skin model, formed from a bovine collagen matrix, the surface with a type of human collagen (Valerie et al. 2005). The EpiDerm model consists of a multi-layered, differentiated, in vitro model of the human epidermis (Valerie et al. 2005). Textile materials are often in contact with the skin. Thus, in the fu-

Page 48: Textile Toxicity

48 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

Kaisa Klemola: Textile Toxicity

ture studies of textile substances it is recommended that these skin models should be used. In vitro cytotoxicity tests can also be used if wastewaters need to be studied. There have been many investigations for developing cleaning techniques in wastewaters emerging from the textile industry: e.g. the ability of microorganisms to carry out dye decolorization (Banat et al. 1996, Marquez and Costa 1996, Khan and Husain 2007, Asgher et al. 2007), aerobic/anaerobic treatments (Liakou et al. 1997, Ciardelli et al. 2001, Frijters et al. 2006), ion exchange methods (Lin and Chen 1997) and coal-absorption (Santhy and Selvapathy 2005, Dincer et al. 2007). The toxicity of wastewaters has been evaluated with different techniques, for instance, with the luminescent bacterium Vibrio Fischeri (Wang et al. 2002). The cell tests used in the present study represent assays that could also provide information about the toxicity of wastewater. In vitro cell tests are useful when one wishes to study overall cell toxicity. The manufacturing of textiles is a global activity and often virtually nothing is known about the chemicals used. The data is also diffi cult to obtain since in most cases this information is regarded as a trade secret. However, some residues of the chemicals used may remain in the textile products causing health problems for some consumers. Cell tests are effective if materials with unknown chemical contents need to be studied and thus they are suitable for studying textile materials. The toxicity of the textile material which was found in this study may be attributable to some individual chemical or to the combined effects of several chemicals. In quality control laboratories, biological cell tests can provide simple and rapid methods to evaluate the potential toxicity of textile products. In the future, it could be possible to develop a new labelling scheme for textile materials, related to the Öko-Tex standard 100, based on measured biological effects. For consumers, such labelling could provide information about the safety of the product. In addition, cell tests could be useful in developing ecologically sound textile processes, resulting in a labelling scheme analogous to the Öko-Tex standard 100 used today by some textile manufacturers.

Page 49: Textile Toxicity

49Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

Conclusions

7. CONCLUSIONS

In the present series of studies, three in vitro cell tests were used to assess the acute toxicity or potential adverse effects of textile reactive dyes and reactive dyed fabrics. All cell tests showed clearly that the three reactive dyes (Reactive Red 241, Reactive Yellow 176, Reactive Blue 221) were toxic, but extracts from reactive dyed fabrics were not toxic even when they had been coloured with a dye which on its own was toxic. The dye in the extracts had been appar-ently hydrolysed and therefore had not the same level of reactivity as the intact dye. After dyeing with reactive dyes, a previously toxic textile material was no longer toxic apparently because during the dyeing process, harmful chemicals must have been removed or the material had become inactivated in some other way, for instance by the chemicals losing their reactivity. Before industrial fi nishing, the commercial knitted fabrics studied did not evoke any adverse effects in the cells. After dyeing and fi nishing, some commercial fabrics did cause toxicity. Since these fabrics had been dyed with reactive dyes (which are not toxic in the dyed fabric) it is hypothesized that the toxicity is caused by some unknown chemical(s) used in fi nishing. In the hepa-1 and HaCaT cell tests, the same sample concentrations were used, whereas the concen-tration was lower in the spermatozoa test. This latter test needs further refi nement to be of practical use for analysing fabrics. The spermatozoa inhibition test is still a qualitative, rapid test and may be used for screening of samples. Human keratinocyte HaCaT cells were more sensitive than hepatoma hepa-1 liver cells when incubated with the dyes and reactive dyed fabric extracts were. This might be attributable to the fact that liver cells have a good capability to metabolise different substances. There were no major differences in the toxicities of the three studied reactive dyes when the dye concentration was low with hepa-1 cell line. However, all tested concentrations of these dyes evoked statistically signifi cant effects on the viability of HaCaT cells. These facts support the need for using a combination of dif-ferent cell lines in parallel to provide comprehensive information about toxicity. The in vitro cell tests can clearly reveal acute toxicity. However, caution is required not to overes-timate the capabilities of these cells to display all responses which can be extrapolated to humans. These tests were found to be suitable when materials such as textiles with unknown components need to be analysed and this study shows that they may be valuable in forming the basis for develop-ing routine tests for use by the textile industry.

Page 50: Textile Toxicity

50 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

Kaisa Klemola: Textile Toxicity

Aalto A, Priha E, Schimberg R, Uitti J, Vuorinen R. Tekstiilivärien terveysvaikutukset. Työterveys-

laitos- Työministeriö, Helsinki 1994. 52 s.

Aaserud O, Hommeren Oj, Tvedt B, Nakstad P,Mowe G, Efskind J, Russee D, Jörgensen EB,

Nyberg-Hansen R, Rootwelt K, Gjerstad L. Carbon disulfi de exposure and neurotoxic sequelae

among viscose rayon workers. Am J Ind Med 18: 25-37, 1990.

Ahonen E. Ympäristömerkit tulossa tekstiilituotteille. Tekstiililehti 1: 28-30, 1994.

Alanko K, Keskinen H, Björksten F, Ojanen S. Immediate-type hypersensitivity to reactive dyes.

Clin Allergy 8(1): 25-32, 1978.

Alberts B., Johnson A., Lewis J., Raff M., Roberts K., Walter P. Molecular Biology of The Cell.

Garland Publishing, 3th Edt, New York, 2002, 1616 p.

http://www.allergia.com. Allergia- ja astmaliitto. Allergy and Asthma Federation; 15.10.2007

Ames BN, McCann J, Yamasaki E. Methods for detecting carcinogens and mutagens with

salmonella/mammalian-microsome mutagenicity test. Mutat 31: 347-364, 1975.

Andersson M. Bacterial Diversity and Toxicity in Air, Indoor Environment and Foods. Gummerus CO

Ltd, Saarijärvi, Finland,1999.

Anonymous. Tetrakis( hydroxymethyl)phosphonium salts. Environmental Health Criteria,

218: 61-108, 2000.

ASA. Syöpäsairauden vaaraa aiheuttaville aineille ja menetelmille ammatissaan altistuvien

rekisteri. Vuosittainen tilasto. Työteveyslaitos. Helsinki.

Asgher M, Bhatti HN, Shah SA, Asad MJ, Legge RL. Decolorization potential of mixed microbial

consortia for reactive and disperse dyestuffs. Biodegradation 18(3): 311-316, 2007.

Assefa Z, Garmyn M, Bouillon R, Merlevede W, Vandenheede JR, Agostinis P. Differential

stimulation of ERK and JNK activities by ultraviolet B irradiation and epidermal growth factor in

human keratinocytes. J Invest Dermatol 108(6): 886-891, 1997.

Baksi SM, Frazier JM. Isolated fi sh hepatocytes – model system for toxicology research. Aquat

Toxicol 16: 229-256, 1990.

8. References

Page 51: Textile Toxicity

51Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

References

Banat IM, Nigam P, Singh D, Marchant R. Microbial decolorization of textile-dye-containing effl uents.

Bioresource Technol 58(3): 217-227, 1996.

Beckett WS, Pope CA, Xu XP, Christiani DC. Women`s respiratory health in the cotton textile

industry: an analysis of respiratory symptoms in 973 non-smoking female workers. Occup Environ

Med 51: 14-18, 1994.

Bend JR, James MO. Xenobiotic metabolism in marine and freshwater species. In: Biochemical and

biophysical perspectives in marine biology. Malins OC and Sargent JR. Academic Press. Vol 4, pp

125-188, New York 1978.

Birhanli A, Ozmen,M. Evaluation of toxicity and teratogencity of six commercial textile dyes using the

frog embryo teratogenesis assay – Xenopus. Drug Chem Toxicol 28(1): 51-65, 2005.

Broadbent A.D., Basic principles of textile coloration. Woodhead Publishing Ltd, Cambridge 2001,

592 p.

Campian EC, Li J, Gao X, Qian M, Joenje H, Eaton JW. Oxygen toxicity and mitochondrial function.

Toxicologist Mar 78(1-S):307, 2004.

Carlson RM, Smith MC, Nedorost ST. Diagnosis and treatment of dermatitis due to formaldehyde

resins in clothing. Dermatitis 15: 169-175, 2004.

Chang HC, Huang CC, Huang CJ, Cheng JS, Liu SI, Tsai JY, Chang HT, Huang JK, Chou CT, Jan

CR. Desipramine-induced apoptosis in human PC3 prostate cancer cells: Activation of JNK kinase

and caspase-3 pathways and protective role of [Ca(2+)](i) elevation. Toxicology, May 27, 2008.

The Chemical Safety data Sheets 2001/58/EY: Drimarene yellow CL-2R, Drimarene blue CL-2RL,

Drimarene red CL-5B (Colour Index numbers CI: RY176, RB221, RR241).

Choudhary E, Capalash N, Sharma P. Genotoxicity of degradation products of textile dyes evaluated

with rec-assay after PhotoFenton and ligninase treatment. J Environ Pathol Toxicol Oncol 23(4):

279-285, 2004.

Christiani DC and Wang X-R. Respiratory effects of long term exposure to cotton dust. Curr Opin in

Pulm Med 9:151-155, 2003.

Chun GTW, Gamble GR. Polymerisation of N-halamide monomers, chloromelamine derivatives and

cross-linked chitosan to produce durable effects. J Cotton Sci 11: 154-158, 2007.

Page 52: Textile Toxicity

52 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

Kaisa Klemola: Textile Toxicity

Chung K-T, Cerniglia CE. Mutagenicity of azo dyes: Structure- activity relationships. Mutat Res 277:

201-220, 1992.

Ciardelli G, Capannelli G, Bottino A. Ozone treatment of textile wastewaters for reuse. Water Sci

Technol 4485: 61-67, 2001.

Clemedson C. McFarlane-Abdulla E, Andersson M, Barile FA, Calleja MC, Chesné C, Clothier R,

Cottin M, Curren R, Dierickx P, Ferro M, Fiskesjö , Garza-Ocanas L, Gómez-Lechón MJ, Gülden

M, Isomaa, B, Janus J, Judge P, Kahru A, Kemp RB, Kerszman G, Kristen, U, Kunimoto M, Kären-

lampi S, Lavrijsen K, Lewan L, Lilius H, Malmsten A, Ohno T, Persoone G, Pettersson R, Roguet R,

Romert L, Sandberg, M, Sawyer T, Seibert H, Shrivastava R, Sjöström M, Stammati A, Tanaka N,

Torres Alanis, O, Voss J-U, Wakuri S, Walum E, Wang X, Zucco F and Ekwall B. MEIC evaluation

of acute systemic toxicity: Part II. In vitro results from 68 assays used to test the fi rst 30 reference

chemicals and a comparative cytotoxicity analysis. Altern Lab Anim 24: 273-311, 1996.

Colour Index, vol. 1-5, 3th Ed. The Society of Dyers and Colourists, American Association of Textile

Chemists and Colourists, Bradford 2001.

De Peyester A, Willis WO, Molgaard CA, MacKendrick TM, Walker C. Cholinesterase and self-

reported pesticides exposure among pregnant women. Arch Environ Health 48:348-351, 1993.

De Roos AJ, Ray RM, Gao DL, Wernli KJ, Fitzgibbons ED, Ziding F, Astrakianakis G, Thoma DB,

Checkoway H. Colorectal Cancer Incidence Among Female Textile Workers in Shanghai, China:

A Case -cohort Analysis of Occupational Exposures. Cancer Causes Control 16(10): 1177-1188,

2005.

Dimethyldioctadecylammonium chloride (DODMAC). European Union risk assessment report 14:

118, 2002.

Dincer AR, Günes Y, Karakaya N. Coal-based ash (CBBA) waste material as adsorbent for removal

of textile dyestuffs from aqueous solution. J Hazard Mater 141(3): 529-535, 2007.

Docker A, Wattie JM, Topping MD, Luczynska CM, Newman Taylor AJ, Pickering CAC, Thomas

P, Gompertz D. Clinical and immunological investigations of respiratory diseases in workers using

reactive dyes. Br J Ind Med 44(8): 534-541, 1987.

Dogan EE. Yesilada E, Ozata L,Yologlu S. Genotoxicity testing of four textile dyes in two crosses of

Drosophila using wing somatic mutation and recombination test. Drug Chem Toxicol 28 (3): 289-301,

2005.

Page 53: Textile Toxicity

53Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

References

Duffel M. Toxic Effects of Organic Solvents. – In the book: Williams PL and Burson JL. Industrial toxi-

cology. Safety and Health Applications in the Workplace. Van Nostrand Reinhold. New York, 1985,

pp.162-196, 502 p.

Dybing E, Sanner T. Species differences in chemical carcinogenesis of the thyroid gland, kidney and

urinary bladder. IARC, Sci Publ 147: 15-32, 1999.

Eaton DL, Klaassen CD. Principles of toxicology. In: Casarett & Doull`s Toxicology: the basic science

of poisons, The McGraw – Hill Companies, 6th Edt., USA, 2001, pp 26-32, 1280 p.

http://www.ecvam.jrc.it; 15.07.2008.

El Ghalbzouri A, Siamari R, Willemze R, Ponec M. Leiden reconstructed human epidermal model as

a tool for the evaluation of the skin corrosion and irritation potential according to the ECVAM guide-

lines. Toxicol In Vitro 22(5): 1311-20, 2008.

http://efanet.org/, The European Federation of Asthma and Allergy Associations; 10.01.2008.

Eriksson P, Fischer C, Fredriksson A. Polybrominated diphenyl ethers, a group of brominated fl ame

retardants, can interact with polychlorinated biphenyls in enhancing developmental neurobehavioral

defects. Toxicol Sci 94(2): 302-309, 2006.

Eskelson YD. and Goodman LS. Contact dermatitis from “Scotchguard”, a stain repellent for fabrics.

J Am Med Assoc 183: 136-139, 1963.

Estlander T, Jolanki R. Tekstiili- ja vaatetusteollisuus – kemialliset aineet ja ihottumat. Työterveystlai-

tos, Helsinki 1980. 75 s.

Estlander T. Allergic dermatoses and respiratory diseases from reactive dyes. Contact Dermatitis

18(5): 290 – 297, 1988.

European Comission, ECVAM. Statement on the validity of in–vitro tests for skin irritation. 2007.

2001/838/EY European Commission Directive relating to restrictions on the marketing and use of

nonylphenol,nonylphenol ethoxylate and cement. Euroopan unionin direktiivi nonyylifenolin ja nonyy-

lifenolietoksyla markkinoille luovuttamisen ja käytön rajoittamisesta.

2002/61/EY European Commission Directive relating to restrictions on the marketing and use of

”blue colorant”. Euroopan Unionin direktiivi tiettyjä atsoväriaineita ja niitä sisältäviä tuotteita koske-

vista kielloista ja rajoituksista.

Page 54: Textile Toxicity

54 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

Kaisa Klemola: Textile Toxicity

2003/3/EY European Commission Directive relating to restrictions on the marketing and use of

”blue colorant”. Euroopan Unionin direktiivi tiettyjä atsoväriaineita ja niitä sisältäviä tuotteita

koskevista kielloista ja rajoituksista.

Final report on the safety assessment of acrylates copolymer and 33 related ingredients.

Int J Toxicol 21, suppl.3, 1- 50, 2002.

Frijters CT, Vos RH, Scheffer G, Mulder R. Decolorizing and detoxifying textile wastewater, con-

taining both soluble and insoluble dyes, in a full scale combined anaerobic/aerobic system. Water

Research 40(6): 1249-1257, 2006.

Gao D, Mazur P,Crittser JK. Fundamental cryobiology of mammalian spermatozoa. In Reproductive

Tissue Banking Scientifi c Principles. Edited by A. Karow and J. Critser, p. 263-312. Academic Press

London, 1997.

Garcia B, Ortiz de Frutos FJ. and Iglesias Diez L. Occupational allergic contact dermatitis due to

formaldehyde and textile fi nish resins. Contact Dermatitis 33(2), 139-140, 1995.

Giovagnini L, Sitran S, Montopoli M, Caparrotta L, Corsini M, Rosani C, Zanello P, Dou QP, Fregona

D. Chemical and biological profi les of novel copper(II)compexes containing S-donor ligands for the

treatment of cancer. Inorg Chem 47(14): 6336-43, 2008.

Gohl EPG and Vilensky LD. Textile science. Longman Cheshire Pty Ltd, Melbourne, 1983. 218 p.

Gonzales CA, Riboli E, Lopez-Abente G. Bladder Cancer among Workers in the Textile Industry:

Results of a Spanish Case-Control Study. Am J Ind Med 14(6): 673-680, 1988.

Gordon S, Hsieh Y L.,Cotton: Science and technology. Woodhead Textiles Series No.59, Woodhead

Publishing Ltd, Cambridge 2006, 568 p.

Gregus Z and Klaassen CD. Mechanisms of toxicity. In: Casarett & Doull`s Toxicology: the basic

science of poisons, The McGraw – Hill Companies, 6th Edt., USA, 2001, pp 35-78.

Grindon C, Combes R, Cronin MT, Roberts DW, Garrod JF. Integrated decision-tree testing strate-

gies for skin corrosion and irritation with respect to the requirements of EU REACH legislation. Altern

Lab Anim 35(6) 673-82, 2007.

Grisham JW. Interspecies comparison of liver carcinogenesis. Implications for cancer risk assess-

ment. Carsinogenesis 18: 59-81, 1997.

Page 55: Textile Toxicity

55Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

References

Hatch KL. Chemicals and textiles, Part 1. Dermatological problems related to fi ber content and dyes.

Textile Res J 54(10): 664-682, 1984.

Hatch KL. and Maibach HI. Textile chemical fi nish dermatitis. Contact Dermatitis 14(1): 1-13, 1986.

Hatch KL, Maibach HI. Textile dye deramtitis. J Am Acad Dermatol 32(4), 631-639, 1995.

Hatch KL, Motschi H, Maibach HI. Textile dye and colored textile allergic contact dermatitis. Exog

Dermatol 2:206-209, 2003.

Hayes GB, Ye T-T, Lu P-L, Dai H-L, Christiani DC. Respiratory disease in cotton textile workers:

epidemiologic assessment of small airway function. Environ Res 66: 31-43, 1994.

Hays SM, Pyatt DW. Risk assessment for children exposed to decabromodiphenyl(oxide)ether(Deca)

in the United States. Integr Environ Assess Manag 2(1): 2-12, 2006.

Hengstler JG, Van der Burg B, Steinberg P. Interspecies differences in cancer susceptibility and

toxicity. Drug Metab Rev 31: 917-970, 1999.

Holme I. Reactive dyes-A celebration of 50 years of innovation. Colourist 2, 2004.

Hoornstra D, Andersson MA, Johansson T, Pirhonen T, Hatakka M, Salkinoja-Salonen MS. Mito-

chondrial toxicity detected in a health product with a boar spermatozoan bioassay. Alternatives to

laboratory animals: Altern Lab Anim 32(4): 407-416, 2004.

Huttunen K, Rintala H, Hirvonen MR, Vepsäläinen A, Hyvärinen A, Meklin T, Toivola M, Nevalainen

A. Indoor air particles and bioaerosols before and after renovation of moisture-damaged buildings:

the effect on biological activity and microbial fl ora. Environ Res 107(3): 291-8, 2008.

Häkkinen AM, Laasonen A, Linnainmaa K, Mattson K, Pyrhönen S. Radiosensitivity of mesothelioma

cell lines. Acta Oncol 35(4): 451-456, 1996.

IARC. Benzidine- based dyes. IARC Monographs on evaluation of carcinogenic risk to humans. Vol

29, IARC, Lyon 1987.

IARC. Formaldehyde, 2-butoxyethanol and 1-tert-butoxy-2-propanol. IARC Monographs on the

evaluation of carcinogenic risk to humans. Vol.88. IARC, Lyon 2004.

IARC. Re-evaluation of some organic chemicals. Hydrazine and hydrogen peroxide. IARC

Monographs on the evaluation of carcinogenic risk to humans. Vol.71. IARC, Lyon 1998.

(http://monographs.iarc.fr/htdocs/monographs/vol148/48-05.htm)

Page 56: Textile Toxicity

56 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

Kaisa Klemola: Textile Toxicity

INVITTOX Protocol number 21. Bovine spermatozoa cytotoxicity test. The cytotoxic effect of test

compounds on bovine spermatozoa is determined by the measurement of spermatozoa motility and

velocity using videomicrography and automatic computer analysis, and ATP contents. 1991.

INVITTOX Protocol number 112. CYP1A1-inducing potency and cytotoxicity test in the Hepa-1

mouse hepatoma cell line. 1995.

INVITTOX Protocol number 64. The Neutral Red cytotoxicity assay. The cytotoxic effect of chemi-

cals upon cells, such as BALB/c 3T3 and HepG2, in culture is measured by highest tolerated dose

(HTD), cell viability (Neutral Red) and total cell protein (coomassie blue). 1992.

Isoherranen K, Punnonen K, Jansen C, Uotila P. Ultraviolet irradiation induces cyclooxygenase-2

expression in keratinocytes. Br J Dermatol 140 (6): 1017-1022, 1999.

ISO 6938: 1984. Textiles - Natural fi bres – Generic names and defi nitions.

ISO 2076: 1999. Textiles – Man-made fi bres – Generic names.

Jahkola A, Estlander T, Jolanki R, Kanerva L. Formaldehydi ammatti-ihotautien aiheuttajana. Työ ja

ihminen 3: 189-200, 1987.

James RC. Toxic effects of organic solvents. In the book: Williams PL and Burson JL. Industrial

toxicology. Safety and health applications in the workplace: 230-259, 502p. Van Nostrand Reinhold

1985 New York.

Jolanki R, Karvinen P, Paganus A, Pehu E, Penttilä P-L, Sainio E-L, Voutilainen H. Tuotetietous.

In: Haahtela T, Hannuksela M, Terho EO. Allergologia, Duodecim, Jyväskylä, Finland, 1999,

pp. 518-531.

Jäger I, Hafner C, Schneider K. Mutagenicity of different textile dye products in Salmonella

typhimurium and mouse lymphoma cells. Mutat Res 561(1-2): 35-44, 2004.

Järvholm B. Natural organic fi bers-health effects. Int Arch Occup Environ Health 73: S69-74, 2000.

Kakko I, Toimela T, Tähti H. The toxicity of pyrethroid compounds in neural cell cultures studied with

total ATP, mitochondrial enzyme activity and microscopic photographing. Environ Toxicol Pharmacol

15: 95-102, 2004.

Kalimo & Lahti. Kosketusihottumat. In: Haahtela T. Hannuksela M & Terho EO. Allergologia.

Duodecim. Jyväskylä,1999, pp.518-531.

Page 57: Textile Toxicity

57Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

References

Kalliala E. The Ecology of Textiles and textile Services – A Life Cycle Assessment Study on Best

Available Applications and Technologies for Hotel Textile Production and Services, Tampereen

teknillinen korkeakoulu, Julkaisu 214, 1997.

Kanerva L. Iho ja kemikaalit. In: Haahtela T and Björksten F. Allerginen kansa – Allergia kansan-

terveysongelmana. Duodecim, Suomen Akatemia, Vammala, Finland, 1998, pp. 81-94.

Karjalainen A, Aalto L, Jolanki R, Keskinen H, Mäkinen I, Salo A., 2002. Ammattitaudit 2001.

Työterveyslaitos, Helsinki.

Kauppinen T. ASA-relisterin vaikutukset työpaikoilla ja rekisteriin ilmoitettujen työntekijöiden

syöpävaara. Loppuraportti. Työterveyslaitos, Syöpärekisteri, Helsinki 1999.

Kaur A, Sandhu RS, Grover IS. Screening of azo dyes for mutagenicity with Ames/Salmonella

assay. Environ Mol Mutagen 22: 188–190, 1993.

Keneklis T. Fiber reactive dye toxicological profi les. U.S. Consumer Product Safety Commission,

Washington D.C., Contact No.CPSC-C-81-1110,p.271, 1981.

Kennedy SW, Lorenzen A, James CA, Collins BT. Ethoxyresorufi n-O-deethylase and porphyrin

analysis in chicken embryo hepatocyte cultures with a fl uorescence multiwall plate reader. Anal

Biochem 211: 102-112, 1993.

Kennedy SW, Jones SP, Bastien LJ. Effi cient analysis of cytochrome P4501A catalytic activity,

porphyrins and total proteins in chicken embryo hepatocyte cultures with a fl uorescence plate

reader. Anal Biochem 226: 362-370, 1995.

Khan AA, Husain Q. Decolorization and removal of textile and non-textile dyes from polluted waste-

water and dyeing effl uent by using potato (Solanum tuberosum) soluble and immobilized polyphenol

oxidase. Bioresour Technol 98(5): 1012-1020, 2007.

Khan WA, Das M, Stick S. Induction of epidermal NAD(P): quinone reductase by chemical carcino-

gens: A possible mechanism for the detoxifi cation. Biochem Biophys Res Commun 146: 126-133,

1987.

Kidd DA, Johnson M, Clements J. Development of an in vitro corrosion/irritation prediction assay

using the EpiDerm skin model. Toxicol In Vitro 21(7): 1292-7, 2007.

Kissa E. Repellent fi nishes. In: Lewin M. and Sello S.B., 1983. Handbook of fi ber science and

technology: chemical processing of fi bers and fabrics: volume II: Functional fi nishes, Part B. Marcel

Dekker, Inc., New York, USA, 1984, pp.144-204.

Page 58: Textile Toxicity

58 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

Kaisa Klemola: Textile Toxicity

Page 59: Textile Toxicity

59Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

References

Page 60: Textile Toxicity

60 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

Kaisa Klemola: Textile Toxicity

Page 61: Textile Toxicity

61Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

References

Page 62: Textile Toxicity

62 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

Kaisa Klemola: Textile Toxicity

Page 63: Textile Toxicity

63Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

References

Page 64: Textile Toxicity

64 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

Kaisa Klemola: Textile Toxicity

Page 65: Textile Toxicity

65Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

References

Page 66: Textile Toxicity

66 Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

Kaisa Klemola: Textile Toxicity

Page 67: Textile Toxicity

67Kuopio Univ. Publ. C. Nat. and Environ. Sci. 241:1-67, 2008

References

Page 68: Textile Toxicity

Kuopio University Publications C. Natural and Environmental Sciences C 218. Madetoja, Elina. Novel process line approach for model-based optimization in papermaking. 2007. 125 p. Acad. Diss. C 219. Hyttinen, Marko. Formation of organic compounds and subsequent emissions from ventilation filters. 2007. 80 p. Acad. Diss. C 220. Plumed-Ferrer, Carmen. Lactobacillus plantarum: from application to protein expression. 2007. 60 p. Acad. Diss. C 221. Saavalainen, Katri. Evaluation of the mechanisms of gene regulation on the chromatin level at the example of human hyaluronan synthase 2 and cyclin C genes. 2007. 102 p. Acad. Diss. C 222. Koponen, Hannu T. Production of nitrous oxide (N2O) and nitric oxide (NO) in boreal agricultural soils at low temperature. 2007. 102 p. Acad. Diss. C 223. Korkea-aho, Tiina. Epidermal papillomatosis in roach (Rutilus rutilus) as an indicator of environmental stressors. 2007. 53 p. Acad. Diss. C 224. Räisänen, Jouni. Fourier transform infrared (FTIR) spectroscopy for monitoring of solvent emission rates from industrial processes. 2007. 75 p. Acad. Diss. C 225. Nissinen, Anne. Towards ecological control of carrot psyllid (Trioza apicalis). 2008. 128 p. Acad. Diss. C 226. Huttunen, Janne. Approximation and modellingerrors in nonstationary inverse problems. 2008. 56 p. Acad. Diss. C 227. Freiwald, Vera. Does elevated ozone predispose northern deciduous tree species to abiotic and biotic stresses? 2008. 109 p. Acad. Diss. C 228. Semenov, Dmitry. Distance sensing with dynamic speckles. 2008. 63 p. Acad. Diss. C 229. Höytö, Anne. Cellular responses to mobile phone radiation: proliferation, cell death and related effects. 2008. 102 p. Acad. Diss. C 230. Hukkanen, Anne. Chemically induced resistance in strawberry (Fragaria × ananassa) and arctic bramble (Rubus arcticus): biochemical responses and efficacy against powdery mildew and downy mildew diseases. 2008. 98 p. Acad. Diss. C 231. Hanhineva, Kati. Metabolic engineering of phenolic biosynthesis pathway and metabolite profiling of strawberry (Fragaria × ananassa). 2008. 80 p. Acad. Diss. C 232. Nissi, Mikko. Magnetic resonance parameters in quantitative evaluation of articular cartilage: studies on T₁ and T₂ relaxation time. 2008. 83 p. Acad. Diss.