A potential in vitro epidermal equivalent assay to determine sensitizer potency

Toxicology in Vitro 25 (2011) 347–357

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A potential in vitro epidermal equivalent assay to determine sensitizer potency Guilherme G. dos Santos a, Sander W. Spiekstra a, Shakun C. Sampat-Sardjoepersad a, Judith Reinders a, Rik J. Scheper b, Susan Gibbs a,⇑ a b

Department of Dermatology, VU University Medical Centre, Amsterdam, The Netherlands Department of Pathology, VU University Medical Centre, Amsterdam, The Netherlands

a r t i c l e

i n f o

Article history: Received 25 May 2010 Accepted 6 October 2010 Available online 19 October 2010 Keywords: Sensitizer Potency Epidermal-equivalent Cell-viability Cytotoxicity IL-1a

a b s t r a c t Most in vitro assays aim to distinguish sensitizers from non-sensitizers. Few aim to classify sensitizers according to potency. Here, we describe a potential method for classifying sensitizers according to their irritant potency with the aid of in house epidermal equivalents (EE). Sixteen sensitizers were applied topically in a dose response to EE for 24 h. The EE-EC50 value (effective chemical concentration required to reduce cell viability by 50%) and the EE-IL-1a10 value (chemical concentration which increases IL-1a secretion by 10-fold) were calculated. From 16 sensitizers, EE-EC50 and/or EE-IL-1a10 values were obtained from 12 skin sensitizers. EE-EC50 and IL-1a10 values decreased in proportion to increasing sensitizer potency. The in vitro assay correlated with existing in vivo mouse and human sensitization data (LLNA, HRIPT), and showed low intra- and inter-experimental variability. Additionally DNCB and resorcinol were correctly assessed as extreme and moderate sensitizers using commercial EE (EST1000™ and RHE™). In conclusion, our data supports the view that irritancy may in part be a factor determining sensitizer potency. Since this assay does not distinguish sensitizers from non-sensitizers, its potential application is in a tiered strategy, where Tier 1 identifies sensitizers which may then tested in Tier 2, this assay, which determines sensitizer potency. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Since all new ingredients of household products and cosmetics are potential allergens, the need to identify harmful substances is crucial. Until recently, the identification of potential sensitizers has been carried out using animal based test methods, such as the local lymph node assay (LLNA) (Kimber et al., 1989), the Guinea Pig Maximization Test (Magnusson and Kligman, 1969) and the Buehler Test (Buehler, 1965). In addition to the ethical issues

Abbreviations: ACD, allergic contact dermatitis; DC, dendritic cell; EE, epidermal equivalent; EC50, effective chemical concentration required to reduce cell viability by 50%; IL-1a10, chemical concentration which increases IL-1a secretion by 10fold; HRIPT, human repeat insult patch test; KC, keratinocyte; LLNA, local lymph node assay; RHE, reconstructed human epidermis; VUMC-EE, VU university medical centre in house epidermal equivalent; CA, cinnamic alcohol; CNM, cinnamaldehyde; DMSO, dimethyl sulfoxide; DNCB, dinitrochlorobenzene; EU, eugenol; GLY, glyoxal; HCPt, ammonium hexachloroplatinate; IE, isoeugenol; MBT, 2-mercaptobenzothiazole; MDBGN, 2-bromo-2-(bromomethyl) pentanedinitrile; MDI, phenylmethane diisocyanate; NBB, 4-nitrobenzylbromide; OXA, oxazolone; pPD, p-phenylenediamine; RES, resorcinol; TMA, trimellitic anhydride; TMTD, tetramethylthiuram disulfide. ⇑ Corresponding author. Address: Department of Dermatology, VU University Medical Centre, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands. Tel.: +31 20 4442815; fax: +31 20 4442816. E-mail address: [email protected] (S. Gibbs). 0887-2333/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2010.10.008

involved around animal testing, both the 7th Amendment to the Cosmetic Directive (Directive 76/768/EEC) and REACH (Registration, Evaluation and Authorization of Chemicals) (Williams et al., 2009) have demanded that novel in vitro methods should be developed for the future risk assessment of potential sensitizers for both the scientific and industrial communities (Goldberg and Hartung, 2006). In order to develop a battery of in vitro assays which will be capable of mimicking human sensitization, it is important first to identify the series of events which result in sensitization and then to break this series of events down into a series of in vitro assays. Allergic contact dermatitis (ACD) is an adaptive inflammatory response of the skin caused by contact of the skin with allergens (sensitizers). It is recognised as a delayed-type hypersensitivity immune response (type IV) (Kimber et al., 2002; Saint-Mezard et al., 2004). Allergens are small molecules which penetrate the outermost layer of the skin and bind to epidermal proteins in order to form a sensitising complex (Weltzien et al., 1996). The sensitising complex is then taken up by dendritic cells (DC) which in turn start to mature and migrate out of the skin, under the influence of skin derived cytokines and chemokines, towards the local lymph nodes where they present the allergen to T-cells (Ouwehand et al., 2008; Villablanca and Mora, 2008). Allergen specific priming of T cells can then result in sensitization of the individual to a particular

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allergen (Edele et al., 2007). Repeated exposure to the same allergen after sensitization can result in elicitation and symptoms of ACD (Kimber et al., 2002). The battery of assays being developed within the FP6 European financed project, Sens-it-iv is based on mimicking sensitization of humans in vitro. Current assays under development are based on (i) increased IL-18 production by epidermal keratinocytes (Corsini et al., 2009); (ii) changes in the maturation status of dendritic cells by assessing changes in biomarker expression, e.g.: IL-8 secretion, p38MAP kinase activity, CD86 surface marker expression (Dos Santos et al., 2009; Mitjans et al., 2008; Ouwehand et al., 2010; Toebak et al., 2006); (iii) Langerhans cell migration towards dermis derived CXCL12 (Ouwehand et al., 2008); and (iv) in vitro T cell priming (Messi et al., 2003). In addition to the Sens-it-iv project, a number of other assays are under development by others. These include a non-cell based peptide reactivity assay, the ARE-regulated luciferase activity assay using the cell line AREc32 (Natsch and Emter, 2008) and DC assays based on changes in biomarker expression (reviewed by Dos Santos et al., 2009). Until now, the main aim of these assays is to distinguish a sensitizer from a non-sensitizer. Currently very few assays address the problem of determining sensitizer potency, i.e. whether the sensitizer is a weak, intermediate or strong sensitizer. The assay described in this manuscript addresses sensitizer potency. It describes the very first phase in sensitization where the sensitizer penetrates the stratum corneum and reaches the viable keratinocytes within the epidermis. The proposed sensitizer potency assay is based on the clinical observation that there is a clear role for irritancy in allergic contact dermatitis due to the irritant property of many allergens (Agner et al., 2002; McLelland et al., 1991; Pedersen et al., 2004). We hypothesized that the method which we recently proposed to determine irritant potency may also be used to determine sensitizer potency in vitro (Spiekstra et al., 2009). This assay makes use of in vitro reconstructed human epidermal equivalents (EE). Preliminary results showed that topical exposure to irritants in a dose dependant manner followed by assessment of changes in epidermal metabolism/viability (MTT assay) and IL-1a release may not only distinguish an irritant from a non-irritant, but may also be able to determine irritant potency (in contrast to the recently validated ECVAM irritation assay (Spielmann et al., 2007)). The stronger the irritant, the greater the decrease in metabolic activity/viability of the EE and the greater the release of IL-1a observed. In this study we expand on our previous report and determine whether the assay may also be suitable to rank the sensitizer potency of a test panel of chemicals supplied by ECVAM. The effective chemical concentration required to reduce cell metabolism/viability to 50% of the maximum value (EE-EC50) was used as the primary parameter to rank sensitizer potency: the lower the EC50 value, the stronger the sensitising potency of the chemical. The second parameter was the effective chemical concentration required to result in a 10-fold increase in secretion of IL-1a (EEIL-1a10) compared to vehicle exposed cultures. For the chemicals which did show irritant properties and where an EE-EC50 and/or EE-IL-1a10 value could be calculated, values were compared with the LLNA and the human repeated insult patch test (HRIPT) data in order to correlate our in vitro results with available in vivo data on sensitizer potency. In addition, similar experiments were performed with submerged primary keratinocyte (KC) cultures.

2. Materials and methods 2.1. Culture of keratinocytes Epidermal KC were isolated from neonatal foreskins obtained from routine surgical procedures essentially as described earlier

(Spiekstra et al., 2005). Primary KC were seeded and cultured in DMEM/Hams F12 (ICN biomedicals, Irvine, CA) (3:1), supplemented with 1% ultroserG (BioSepra S.A, Cergy-Saint-Christophe, France), 1 lM hydrocortisone, 1 lM isoproteronol, 0.1 M insulin and 1 ng/mL KGF. Unless otherwise stated, all additives were purchased from Sigma (St. Louis, MO). Cultures were incubated at 37 °C and 7.5% CO2. Healthy human foreskins were obtained and handled according to the Declaration of Helsinki. 2.2. Culture of epidermal equivalents Epidermal equivalents were cultured as previously described (Spiekstra et al., 2009). In short, secondary KC were seeded into a 12 mm diameter transwell (pore size of 0.4 lm; Corning, NY, USA) and grown submerged in medium containing DMEM/Hams F12 (3:1), 1% ultroserG, 1 lM hydrocortisone, 1 lM isoproteronol, 0.1 lM insulin and 1 ng/mL KGF for 1 week. Cultures were then lifted to the air–liquid interface and cultured for further 4 days in KC culture medium (DMEM/Hams F12 (3:1), 0.2% ultroser G, 1 lM hydrocortisone, 1 lM isoproterenol, 0.1 lM insulin, 1.0  10 5 M L-carnitine, 1.0  10 2 M L-serine, and 2 ng/mL KGF). After 4 days, 50 lg/mL ascorbic acid was added and EE were cultured further for 10 days. Unless otherwise stated, all additives were purchased from Sigma (St. Louis, MO). EE were incubated at 37 °C, 7.5% CO2 and medium was refreshed twice a week. 2.3. EST1000™ and RHE™ EST1000™ (Epidermal Skin Test-1000) (CellSystemsÒ Biotechnology GmbH, St. Katharinen, Germany) and RHE™ (Reconstructed Human Epidermis) (SkinEthicÒ Laboratories, Nice, France) are commercially available reconstructed epidermal models derived from normal human KC. Reconstructed epidermal cultures were maintained according to the supplier in EST1000™ or RHE™ Maintenance Medium for 24 h at 37 °C, 5% CO2. After 24 h of incubation, EST1000™ and RHE™ cultures were exposed to chemicals as described below. 2.4. Chemical exposure All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA), unless indicated otherwise. Cultures were exposed to chemicals in a dose dependent manner: CA, CNM, DNCB, EU, IE, MDI, NBB, pPD and RES were dissolved in DMSO (0.1% v/v for submerged KC and 1% v/v for EE). GLY (Acros Organics, Geel, Belgium), HCPt, TMA were dissolved in water. MBT (Acros Organics, Geel, Belgium), MDBGN, OXA and TMTD were dissolved in acetone/olive oil (4:1) for EE and 0.1% DMSO for submerged KC. At least three independent experiments were performed for each tested chemical, each with internal duplicates. Submerged keratinocytes: KC were seeded into a 12-well plate (Corning, NY, USA) at a density of 3  105 cells per well. The next day, cells were refreshed with new medium (primary KC medium) containing freshly prepared chemicals or vehicle and exposed for 24 h. Epidermal equivalents: Finn Chamber filter paper discs 7.5 or 11 mm (Epitest LTD Oy, Finland) were impregnated with chemicals or vehicle controls and applied topically to the VUMC-EE/ EST1000™/RHE™ stratum corneum. Cultures were further incubated for 24 h. After chemical exposure, metabolic activity was determined by MTT assay (see below). All tested chemicals in this study were supplied by ECVAM as part of the FP6 financed project Sens-it-iv (www.sens-it-iv.eu) and were selected due to data being available from LLNA for comparison.

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2.5. MTT assay and IL-1a ELISA

VUMC-EE, EST1000™ and RHE™ and submerged KC were washed with PBS. Thiazolyl blue tetrazolium bromide (MTT) (Sigma) solution in PBS (0.5 mL of a 5 mg/mL solution) was added and cultures were incubated for 2 h under standard culture conditions. The formazan crystals were then dissolved in 0.5 mL isopropanol (Merck) overnight at room temperature. Absorbance of the formazan dissolved in isopropanol was measured at 570 nm and expressed as a percentage relative to the absorbance value of VUMC-EE/ EST1000™/RHE™/KC_exposed to vehicle controls (n P 3).

The MTT assay is based on the ability of a mitochondrial dehydrogenase enzyme from viable cells to cleave the tetrazolium rings of the yellow MTT reagent solution and form dark blue formazan crystals (Mosmann, 1983). Therefore, the MTT assay measures mitochondrial activity, which is representative of cell viability, by quantifying dehydrogenase activity. After 24 h chemical exposure, filter paper discs impregnated with chemicals were removed from

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Fig. 1. Decrease in EE cell viability and increase in IL-1a secretion after chemical exposure. EE were topically exposed to a panel of extreme, strong, moderate and weak contact sensitizers for 24 h in a dose response manner. (A) Changes in cell viability/metabolic activity were determined using MTT assay. (B) Changes in IL-1a secretion were determined by ELISA. Relative MTT and IL-1a values are shown compared to vehicle (V) (DMSO, water or AOO – see Section 2). Statistical analysis was performed with Student’s t-test, with *p < 0.05, **p < 0.01, ***p < 0.001 vs. vehicle treated EE, n P 3 ± SEM.

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The amount of IL-1a present in culture supernatants were determined by ELISA as previously described (Spiekstra et al., 2007). 2.6. Statistical analysis and determination of EE- and KC-EC50 and IL-1a10 values The EC50 value is the effective chemical concentration required to reduce metabolic activity (corresponding to cell viability) to 50% of the maximum value. The 100% value for cell viability corresponds to the vehicle control (0.1% or 1% DMSO, water or ace-

tone/olive oil 4:1). EC50 values from all epidermal equivalents (EE-EC50) were obtained by non-linear regression analysis (r2 > 0.9) based on changes in metabolic activity (MTT) using GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego, CA, USA). The EE-IL-1a10 value is the effective chemical concentration required to result in a 10-fold increase in secretion of IL-1a (EE-IL-1a10) compared to vehicle exposed cultures. For all chemical exposures, at least three independent experiments were performed each having an internal duplicate. The data presented are expressed as mean ± SD. Statistical significance was determined by Student’s t-test and differences were considered

G.G. dos Santos et al. / Toxicology in Vitro 25 (2011) 347–357 Table 1 Panel of test chemicals listed according to their skin sensitising capacity in the local lymph node assay (LLNA), human repeated insult patch test (HRIPT), EE-EC50 and EE-IL-1a10 values. Contact sensitizer

LLNA-EC3 (%)

Extreme OXA 0.003a NBB 0.05b DNCB 0.08c Strong pPD 0.16b Moderate IE 1.2b MDBGN 1.3d GLY 1.4b MBT 1.7b CNM 3.0b TMTD 5.2b RES 5.5e Weak EU 13b CA 21b Respiratory sensitizers MDI na HcPT na TMA 0.22c

HRIPT

EE-EC50 (%)

EE-IL-1a10 (%)

na na 5.5f

1.31 0.07 0.13

– 0.08 0.12

–

3.08

250 320h 345* na 590g na na

0.55 0.76 12.07 – 1.99 – 2.64

0.33 1.06 19.15 3.89 2.17 – 2.33

5905g 4724g

4.46 0.86

1.56 1.08

na na na

– – –

-

6.9f g

Mouse LLNA-EC3(%) values and human HRIPT maximal no observed effect level (NOEL) (lg/cm2) of chemicals (Basketter et al., 2005) or chemical dose per skin area leading to a sensitization incidence of 5% (DSA05) (Schneider and Akkan, 2004) or chemical dose-induced perceived deterioration of the dermatitis (Bruze et al., 2006) are shown. Information on LLNA and HRIPT is derived from the references as indicated in superscript letters. The EE-EC50 value is the effective chemical concentration required to reduce EE metabolic activity (corresponding to cell viability) to 50% of the maximum value. The EE-IL-1a10 value is the effective chemical concentration required to result in a 10-fold increase in secretion of IL-1a (EE-IL1a10) compared to vehicle exposed cultures (see Section 2). (na) no data available; (–) no EE-EC50 or EE-IL1a5 value obtained from dose response. a Roberts et al. (2007). b Gerberick et al. (2005). c Kimber et al. (2003). d Basketter et al. (2007a). e Kern et al. (2010). f Schneider and Akkan (2004). g Basketter et al. (2005). h Bruze et al. (2006). * Basketter et al. (unpublished data).

significant for p < 0.05. EC50 as well as IL-1a10 were compared with LLNA-EC3 and HRIPT by non-parametric (Spearman) and parametric correlation (Pearson) with two-tailed p values and confidence interval of 99% using GraphPad software. Correlations were considered significant for p < 0.05. 3. Results 3.1. Determination of epidermal equivalent EC50 (EE-EC50) values after chemical exposure The EE-EC50 value indicates the effective chemical concentration required to reduce cell viability to 50% of the maximum value. Therefore, in order to determine an EE-EC50 value, a decrease in at least 50% in metabolic activity must be obtained after chemical exposure. In this study, EE were topically exposed in a dose dependent manner for 24 h to 16 sensitizers in order to determine whether or not there was a relationship between EE-EC50 values and sensitizer potency. The 16 chemicals consisted of 13 skin sensitizers which according to the LLNA were classified as extreme (DNCB, NBB and OXA), strong (pPD), moderate (CNM, GLY, IE, MBT, MDBGN, RES and TMTD), and weak (CA, EU) skin sensitizers and three respiratory sensitizers (HCPt, MDI and TMA). From the 13 skin sensitizers tested, EE-EC50 values could be obtained for 10 chemicals (CA, CNM, DNCB, EU, GLY, IE, MDBGN, NBB, OXA

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and RES) (Fig. 1, Table 1). In general, an increasing EE-EC50 value was observed with decreasing sensitizer potency indicating a correlation between EE-EC50 value and sensitizer potency. Three of the 13 skin sensitizers (pPD, MBT and TMTD) were not able to reduce EE metabolic activity at the highest tested soluble concentration of the chemical and therefore EE-EC50 values could not be determined for these chemicals. For one of these skin sensitizers (pPD), this was due to interference in the MTT colorimetric assay. For MBT and TMTD this was due to maximum solubility being reached in the vehicles. Of the 10 contact sensitizers which showed dose dependent decrease in metabolic activity, the intra- and interexperiment variation was determined. For all 10 contact sensitizers both intra- and inter-experiment variation was very low, indicating a high reproducibility of the EE model to assess chemical cytotoxicity. Fig. 2 shows the intra- and inter-experiment variation for 1 extreme (NBB), 1 moderate (RES) and 1 weak (EU) skin sensitizer. Notably, none of the three respiratory sensitizers tested (HCPt, MDI and TMA) were able to result in a decrease in metabolic activity after topical exposure to EE and therefore no EE-EC50 could be determined. 3.2. Determination of EE-IL-1a10 values after chemical exposure The EE-IL-1a10 value indicates the chemical concentration which results in 10-fold increase in IL-1a secretion compared to vehicle exposed cultures. EE-IL-1a10 values could be obtained for 11 of the 13 skin sensitizers (CA, CNM, DNCB, EU, GLY, IE, MBT, MDBGN, NBB, pPD and RES) (see Fig. 1B, Table 1). In general, a higher EE-IL-1a10 value was observed with decreasing sensitizer potency. Interestingly an EE-IL-1a10 value could be obtained for pPD and MBT whereas no EE-EC50 value could be obtained for these chemicals. In contrast no EE-IL-1a10 value could be obtained for OXA whereas an EE-EC50 value could be obtained. As with EE-EC50, none of the respiratory sensitizers nor TMTD were able to produce a EE-IL1a10 value. 3.3. In vitro vs. in vivo correlation: Comparison of EE-EC50 and EE-IL1a10 with LLNA-EC3 and HRIPT data For the chemicals from which an EE-EC50 and/or EE-IL1a10 value was obtained, it was next determined whether these values correlated to the sensitizer potency. The calculated EE-EC50 (r2 P 0.9) and EE-IL1a10 were compared to existing LLNA-EC3 and HRIPT data from literature using the Spearman correlation (Tables 1 and 2). From the 10 contact sensitizers of which EEEC50 could be calculated (CA, CNM, DNCB, EU, GLY, IE, MDBGN, NBB, OXA and RES), seven sensitizers showed a good correlation with LLNA (Fig. 3, Table 2). Three of the 10 sensitizers (CA, GLY and OXA) showed a poor correlation with the LLNA-EC3 data. CA exhibited more cytotoxicity than was expected, and GLY and OXA exhibited less cytotoxicity than expected. This is emphasized when these three chemicals were removed from the analyses: Spearman correlation changed from r = 0.5758; p = 0.0883 to r = 1.0000; p = 0.0004. When only CA and GLY were removed from the analysis a significant correlation with LLNA-EC3 was observed (r = 0.762; p = 0.037) (Table 2; Fig. 3). From the 10 skin sensitizers of which EE-EC50 could be calculated, HRIPT data was available for only seven of these chemicals (CA, CNM, DNCB, EU, GLY, IE and MDBGN). A correlation between EE-EC50 values and HRIPT data was noted, which became statistically significant after removal of data for CA and GLY (no human data is available for OXA); Spearman correlation changed from r = 0.7500; p = 0.0663 to r = 1.0000; p = 0.0167 (Fig. 3; Table 2). Next we determined whether EE-IL1a10 values also correlated to LLNA-EC3 and HRIPT (NOEL/DSA05) data. LLNA-EC3 vs. EE-IL1a10 showed a clearly poorer correlation to that observed

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A Intra-experiment variation

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Fig. 2. Intra- and inter-experiment variation between three independent experiments is low in EE exposed to contact sensitizers. (A) Intra-experiment variation between internal duplicates for each chemical concentration tested is negligible. Data from a single representative experiment for NBB, RES and EU is shown. (B) Low inter-experiment variation is shown by data from three independent experiments; internal duplicates are averaged for each experiment.

Table 2 Correlation of local lymph node assay (LLNA) and human repeated insult patch test (HRIPT) with epidermal equivalent (EE) data. Spearman r

p

LLNA vs. EE-EC50 10 sensitizers Without CA/GLY Without CA/GLY/OXA

0.5758 0.7619 1.0000

0.0883 0.0368 0.0004

LLNA vs. EE-IL1a10 11 sensitizers Without CA/GLY Without CA/GLY/pPD

0.4273 0.5500 0.7857

0.1928 0.1328 0.0279

HRIPT vs. EE-EC50 Seven sensitizers Without CA/GLY

0.7500 1.000

0.0663 0.0167

HRIPT vs. EE-IL1a10 Eight sensitizers Without CA/GLY Without CA/GLY/pPD

0.3538 0.3414 0.9000

0.3894 0.2972 0.0883

Spearman correlation r and p values are shown. r value indicates degree of correlation between different parameters (1.0 = absolute correlation) and p value determines how significant the correlation is. Correlations were considered significant when p < 0.5. Chemicals involved in each correlation are shown in Fig. 3.

for LLNA-EC3 vs. EE-EC50 (Fig. 3; Table 2). When selecting the same seven sensitizers (NBB, DNCB, IE, MDBGN, CNM, RES and EU) from

which data was available for both EE-IL1a10 and EE-EC50, then the correlation was as follows: for LLNA-EC3 vs. EE-EC50 Spearman: r = 1.000; p = 0.0004 and for EE-IL1a10 vs. LLNA Spearman: r = 0.8929; p = 0.0123. Therefore, in general EE-EC50 values correlated to both LLNA and HRIPT data. However three chemicals showed a clear deviation. EE-IL1a10 values correlated to a lesser extent with both LLNA and HRIPT data. IL-1a did permit values for MBT and pPD, but not OXA, to be obtained. 3.4. Technology transfer to a commercially available reconstructed epidermis model Next we determined whether the assay was transferable to two different commercially available reconstructed epidermis models (RHE™ and EST1000™) or whether it was restricted only to our in house EE (VUMC-EE). Technology transfer is important when considering potential implementation of an in vitro assay on a broad scale. First, all three EE were exposed to the vehicle 1% DMSO in order to determine whether or not the vehicle itself had an effect on the different EE. Notably, whereas 1% DMSO had a negligible effect on cell viability of the VUMC-EE, it resulted in approximately 20% decrease in cell viability in both commercial models. Regarding IL-1a secretion, basal secretion from VUMC-EE was similar to RHE™ and lower than that for EST1000™. Exposure to the 1% DMSO resulted in a slight increase in IL-1a secretion from

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EE-EC50 (%)

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C

0.1

1

10

100

0.01 1

10

D

100

100

1000

10000

8 skin sensitizers

100

6* 10

11

12

6*

3

10

78 9

5

1

0.01 0.001 0.01

10*

0.1

11 skin sensitizers

0.1

7

4

3

2 0.01 0.001 0.01

5

1

4

3

9

10

0.1

EE-IL1α10x (%)

7 skin sensitizers

12 5

7

1

10*

10* 3

4

9

1. OXA 2. NBB 3. DNCB 4. IE 5. MDBGN 6. GLY 7. CNM 8. RES 9. EU 10. CA 11. MBT 12. pPD

4

0.1

2 0.01

0.1

1

10

100

LLNA-EC3 (%)

1

10

100

1000

10000

HRIPT (μg/cm2)

Fig. 3. Correlation of EE potency assay EE-EC50 and EE-IL-1a10 with local lymph node assay (LLNA) and human repeated insult patch test (HRIPT) data. Correlation of EE-EC50 data obtained from skin sensitizers with (A) LLNA-EC3 and (B) HRIPT data. Correlation of EE-IL1a10 data obtained with (C) LLNA-EC3 and (D) HRIPT. Only CA*, GLY* and OXA* showed a poor correlation with LLNA. HRIPT data was not available for MBT, NBB, OXA, RES and TMTD. For the comparison EE-EC50 vs. LLNA and HRIPT: MBT, pPD and TMTD were excluded since no EE-EC50 value was not obtained. OXA and pPD were excluded from the comparison of EE-IL1a10 vs. LLNA and HRIPT since likewise no EE-IL1a10 value was obtained. Corresponding non-parametric Spearman correlation r values and two-tailed p values are shown (99% confidence interval) in Table 2.

VUMC-EE and a greater secretion from both commercial EE. Therefore in conclusion, the vehicle 1% DMSO had a negligible effect on VUMC-EE but resulted in slight decrease in cell viability and increase in IL-1a secretion from both commercial EE. Next, RHE™ and EST1000™ were exposed to DNCB and RES in an identical manner to our in house model. After chemical exposure, EE-EC50 and EE-IL1a10 values were calculated (Fig. 4; Table 3). Despite the difference in the effect of 1% DMSO vehicle, similar EE-EC50 values were obtained for all three EE models since EE-EC50 values are calculated using relative cell viability compared to vehicle rather than absolute cell viability. With regards to EE-IL1a10, a 10-fold increase in IL-1a secretion relative to the vehicle was not reached for either RHE™ and EST1000™ after exposure to DNCB or RES. Therefore IL-1a2 values were calculated (Table 3). Very interestingly, the EE-IL1a10 values obtained for the VUMC-EE exposed to both DNCB and RES were similar to EE-IL1a2 values obtained for both commercial models with both chemicals (Table 3). This shows that a 2-fold increase in IL-1a secretion relative to the vehicle by both commercial models correlates to the same chemical concentration which results in 10-fold increase in IL-1a secretion by the VUMC-EE. Taken together, these results indicate that the assay protocol may be suitable for a wide range of EE, including commercially available EE. However it should be noted that only two chemicals have been tested in the different EE models until now. 3.5. EC50 and IL-a10 values from submerged keratinocytes do not correlate to sensitizer potency Next we determined whether or not conventional, submerged keratinocyte cultures could be used as an alternative to 3D organo-

typic EE cultures. Submerged KC were exposed to the same panel of 16 sensitizers (CA, CNM, DNCB, EU, GLY, HCPt, IE, MBT, MDBGN, MDI, NBB, OXA, pPD, RC, TMA and TMTD) as EE. Neither a KC-EC50 or a KC-IL1a10 value could be obtained for CA, HcPT or MDI, and only a KC-EC50 could be obtained for OXA and GLY. As shown in Fig. 5, a very poor correlation was observed for both KC-EC50 and KC-IL1a10 when comparing with LLNA and HRIPT data. Removal of GLY and OXA from the EC50 correlation did not improve these findings (KC-EC50 vs. LLNA: r = 0.1727, p = 0.6147; KC-IL1a10 vs. HRIPT: r = 0.0857, p = 0.9194). As with EE, KC-EC50 and KC-IL1a10 values could not be obtained for two respiratory sensitizers (HcPT or MDI) whereas EC50 values were now obtained for TMA (respiratory sensitizer) and TMTD. 4. Discussion Our data supports the view that irritancy, may in part be a factor in determining sensitizer potency. Seven of the tested sensitizers showed a good correlation between irritancy/cytotoxicity and sensitizer potency. However, this correlation cannot be taken as proof for a causal relationship between irritancy and sensitization since it is possible that chemical reactivity is the common denominator. This was indeed shown to be the case for genotoxicity and sensitization where strongly reactive molecules were shown to be both genotoxic and sensitising without any suggestion of there being a causal relationship between the two (Mekenyan et al., 2010). Whether our observed sensitizer cytotoxicity comes from (i) a simple narcotic/membrane toxicity; (ii) reactive toxicity modifying proteins and depleting glutathione or (iii) specific pharmacological actions is currently unknown. However, if the main driver for a molecule is (i) membrane toxicity, then the extent of

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EE models + 1%DMSO

B

EE models + 1%DMSO

IL-1α secretion (pg/mL)

Cell viability (MTT % of unexposed)

A 100 75 50 25

RHETM

VUMC-EE

unexposed

50

25

0

EST-1000TM

0

75

0 veh

0 veh

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RHE

0 veh

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EST-1000TM

C DNCB

Cell viability (MTT % of vehicle)

VUMC-EE

RHETM 125

125 *

100

***

75

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***

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100

75

0

2

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*** V

2

IL-1α secretion (pg/mL)

***

4

6

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8

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Fig. 4. Technology transfer to commercially available reconstructed epidermis models (EST1000™ and RHE™). (A) Effect of vehicle 1% DMSO on cell viability (MTT assay). (B) Effect of vehicle 1% DMSO on IL-1a secretion (ELISA). (C) DNCB and RES show dose dependent decrease in metabolic activity and increase in IL-1a secretion as observed with in house EE (VUMC EE), indicating assay technology is transferable to commercially available reconstructed epidermis models. All data represent three independent batches of EE each performed in duplicate unless indicated otherwise. Statistical analysis was performed with Student’s t-test, with *p < 0.05, **p < 0.01, ***p < 0.001 versus vehicle treated EE, n = 3 ± SEM.

the general alarm signal formation resulting from membrane damage (e.g.: extent of cytokine and chemokine release) may be related

to sensitization potency (Spiekstra et al., 2005). If for a molecule option (ii) is the driver, then toxicity would directly correlate to

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Notably, all three respiratory sensitizers and two of the skin sensitizers (MBT and TMTD) did not demonstrate any degree of cytotoxicity in our assay. Furthermore, CA, GLY and OXA were clear outliers. This result shows that not all chemicals show a correlation between irritancy and sensitizer potency and that additional factors must also be involved. Our results support the view that bioavailability and peptide reactivity, independent of irritancy, may also be involved in determining sensitizer potency of some chemicals since these chemicals with the exception of CA are extreme to moderate sensitizers. Interestingly, the same three respiratory sensitizers investigated in our study also behaved differently to the skin sensitizers in the keratinocyte IL-18 study of Corsini et al. (2009) where in contrast to skin sensitizers, the respiratory sensitizers did not result in an increase intracellular IL-18 production. Whether or not this is a property of respiratory sensitizers in general or a property of these three particular chemicals is currently unknown. Similar findings to ours were reported by Basketter et al. (2007b) who compared the sensitising potency of 55 chemicals tested in the LLNA with the ability of the chemicals to produce pro-inflammatory (danger) signal release, measured as a function of relative skin irritancy in guinea pigs. A trend toward the more sensitising chemicals being more irritant was also observed. However, several chemicals departed from the trend. This finding was consistent with their hypothesis that chemicals that were stronger sensitizers might appear so, in part, as a consequence not only due to their greater (pro)electrophilic reactivity, but also due to their ability to stimulate the production of inflammatory signals. However, they suggested that the limited trend observed in their study indicated that skin irritation is either a poor measure of danger signals, or that such signals may not be related to sensitizer potency

Table 3 Technology is transferable to commercially available reconstructed epidermis models (RHE™ and EST1000™).

DNCB EE-EC50 EE-IL-1a2 EE-IL-1a10 RES EE-EC50 EE-IL-1a2 EE-IL-1a10

VUMC-EE

RHE™

EST1000™

6.49 ± 0.11 (r2 = 0.86) 0.03% (1.5 mM) 0.12% (6 mM)

4.58 ± 0.36 (r2 = 0.83) 0.13% (6.2 mM) not reached

6.75 ± 0.28 (r2 = 0.94) 0.13% (6.4 mM) not reached

218 ± 12.8 (r2 = 0.96) 0.83% (75 mM) 2.33% (212 mM)

313 ± 41.4 (r2 = 0.87) 2,75% (250 mM) Not reached

474 ± 6.73* 2.31% (210 mM) Not reached

Technology transfer to commercially available reconstructed epidermis models (RHETM and EST1000™). Exposure of RHE™ and EST1000™ to DNCB and RES results in similar EE-EC50 values to those obtained using in house EE (VUMC-EE). VUMC-EE-IL-1a10 value correlated to EE-IL-1a2 value obtained from both commercial models. Values are calculated from dose response data obtained from three independent EE batches each performed in duplicate. Non-linear regression (curvefit) analysis using dose response formulas (r2 > 0.8) based on changes in metabolic activity (MTT) or IL-1a release were performed using GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego, CA, USA). * For RES, EST1000™ EE-EC50 value was estimated manually according to Spiekstra et al. (2007) due to non-convergence of dose response data with software program.

reactivity and in turn sensitization potential. This may be the case for the highly reactive molecules DNCB and NBB. If for a molecule option (iii) is the driver, then specific properties of the molecule may result in, e.g.; greater activation of antigen presenting cells and/or increased specific T cell frequency compared to other molecules. This may possibly be the case for OXA since this extreme sensitizer only has marginal irritant properties in our study.

KC-EC50 (%)

A 1 0.1

13 12

1

0.01 0.001

C 1

0.00001 0.01

6 5

1

10

100

9

1000

10000

0.00001 1

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10 sensitizers ( r = 0.5030; p = 0.1440)

7

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0.0001

14

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7 sensitizers ( r = 0.1071; p = 0.8397)

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KC-IL1α10x (%)

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13 sensitizers ( r = 0.0109; p = 0.9716)

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7

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0.0001

1. OXA 2. NBB 3. DNCB 4. IE 5. MDBGN 6. GLY 7. CNM 8. RES 9. EU 10. CA 11. MBT 12. pPD 13. TMA 14. TMTD

0.00001 0.1

1

10

LLNA-EC3 (%)

100

1

10

100

HRIPT

1000

10000

(μg/cm2)

Fig. 5. Correlation of KC-EC50 and KC-IL-1a10 with local lymph node assay (LLNA) and human repeated insult patch test (HRIPT) data. Correlation of KC-EC50 data obtained from skin sensitizers with (A) LLNA-EC3 and (B) HRIPT data. Correlation of KC-IL1a10 data obtained with (C) LLNA-EC3 and (D) HRIPT. HRIPT data was not available for MBT, NBB, OXA, RES, TMTD, HcPT, MDI and TMA. For the comparison KC-EC50 vs. LLNA and HRIPT: CA, MDI and HcPT were excluded since no EE-EC50 value was not obtained. OXA, GLY, CA, pPD, TMTD, HcPT and MDI were excluded from the comparison of EE-IL1a10 vs. LLNA and HRIPT since likewise no EE-IL1a10 value was obtained. Corresponding non-parametric Spearman correlation r values and two-tailed p values are shown (99% confidence interval).

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but rather to the acquisition of skin sensitization. The authors did note that different vehicles were used in the assays and that this may have complicated their analysis. As already stated above, the three respiratory sensitizers, TMTD and MDI did not exhibit any cytotoxic properties in our study. Also pPD gave no EE-EC50 value and was a major outlier in the EE-IL1a10 analysis. The reason for these findings may be related to chemical solubility, and for pPD interference with the MTT assay. When the solubility of a chemical is limiting, a wider range of vehicles could be tested in addition to the water, DMSO (1%) and acetone olive oil (4:1) used in this study in order to determine the best vehicle for the assay. Care should be taken though that the vehicle itself does not influence the readout of the assay. For example, non-irritant vehicle concentrations should always be used. One should be aware of evaporation of some vehicles which may influence the final chemical concentration (e.g.: acetone). Also the hydrophilic (e.g.: water, DMSO) and hydrophobic (e.g.: olive oil) nature of the vehicle may effect the release of a chemical and its subsequent penetration into the epidermis. Hydrophobic chemicals penetrate faster from hydrophilic vehicles. Therefore for a hydrophobic molecule, bioavailability from water is much greater compared to olive oil. In this study we used three different vehicles. We first tested whether the chemical was soluble in the vehicle and then whether cytotoxicity resulting in an EE-EC50 value could be obtained. The order of selecting the vehicle was as follows; water then DMSO 1% and the acetone olive oil 4:1 in order to limit these vehicle issues as much as possible. Of the 12 skin sensitizers which could be assessed using EEEC50 and/or EE-IL1a10 in the EE potency assay, eight showed a good correlation with mouse LLNA and/or (human) HRIPT data (NBB, DNCB, IE, MDBGN, CNM, RES, EU and MBT). The EE-EC50 parameter correlated well with both mouse and human data. EE-IL1a10 correlated to a better extent with human than it did with mouse data. The reason for this is currently unknown. Importantly, as seen when selecting the seven chemicals (NBB, DNCB, IE, MDBGN, CNM, RES and EU) from which both an EEEC50 and an EE-IL1a10 values could be determined, EE-EC50 showed better correlation with LLNA data than EE-IL1a10. For these same chemicals, EE-EC50 vs LLNA Spearman: r = 1.000; p = 0.0004 and for EE-IL1a10 vs. LLNA Spearman: r = 0.8929; p = 0.0123 values were obtained. Therefore, when further developing the assay in the future EE-EC50 values should be taken as the primary readout in the assay and EE-IL1a10 could provide supportive information. Moreover, it is possible that combined assessment of both markers will prove beneficial when a more extended chemical panel is tested. Only a limited number of in vitro studies have been reported that address sensitizer potency (Welss et al., 2007). A complicating factor in comparing these studies and our study is that correlation of the data to LLNA data is performed using different statistical means in the different studies, and few as well as different chemicals have been investigated. For example, Sakaguchi et al. (2009), exposed THP-1 cells to a panel of 21 chemical sensitizers and correlated the increase in CD86 (EC150) and CD54 (EC200) surface marker expression to LLNA data using Pearson in their correlation. Of the 21 sensitizers tested, only 12 increased CD86 or CD54 expression and were included in their correlation. Notably CA, GLY and OXA were not included. In contrast to Sakaguchi et al., Corsini et al. (2009) used the Spearman correlation. They correlated intracellular production of IL-18 produced by the keratinocyte cell line NCTC 2544 after exposure to 11 sensitising chemicals from the Sens-it-iv test panel (excluding OXA) with LLNA data. A very different type of in vitro assay is the non-cell based peptide reactivity assay based on the ability of a chemical to react with two synthetic peptides containing either a single

cysteine or lysine (Gerberick et al., 2007, 2009). Interestingly, the peptide reactivity result for OXA did correlate well with the LLNA data, however only minimal reactivity was found for the moderate sensitizer RES. In our EE potency study the opposite was observed, OXA gave a poor correlation whereas RES gave a good correlation with LLNA data. Taken together, these results indicate that if limitations are taken into account such as chemical solubility/instability/metabolism, then in vitro assays may have the potential to assess sensitizer potency. At the moment it is too early to say whether one assay performs better than another since all assays are in the research phase or are just entering pre-validation. However it would be interesting and important to determine whether the different assays are able to compliment each other with regards to chemicals which perform poorly in one particular assay and well in another assay. In contrast to EE, no significant correlation was found between KC-EC50 or KC-IL-1a10 and LLNA or HRIPT data, even when removing the 2 major EE outliers (OXA, GLY – no result for CA) from the analysis. The major difference between KC and EE is the presence of the stratum corneum and therefore barrier function in EE compared to KC. Our experiments on EE potency show a very low intra- and inter- experiment variability. Furthermore, the protocol was transferable from our in house EE to two different commercially available EEs (RHE™ and EST-1000™). This indicates that our protocol to assess sensitizer potency is robust. Similar results were obtained between the three models for the strong sensitizer DNCB and the moderate sensitizer RES despite variations in barrier competency between the EE (as shown by cytotoxic effect of 1% DMSO). Very interestingly, the EE-IL1a10 values obtained for the VUMC-EE were similar to EE-IL1a2 values obtained for both commercial models. Even though the VUMC-EE showed lower IL-1a basal vehicle levels and a greater inducible capacity, our results suggest that with regards to the commercial models it may be possible to use EE-IL1a2 values to supplement EE-EC50 data. However, only two chemicals have been tested until date and this needs confirming with an extended chemical panel. The EE potency assay may be able to rank sensitizer potency in vitro. The assay is limited however to the group of sensitizers which also have irritant properties. Since this assay is not able to discriminate sensitizers from non-sensitizers, we propose a tiered strategy. Tier 1 would aim to distinguish sensitizers from non-sensitizers, e.g.: peptide reactivity assay (Gerberick et al., 2007, 2009), dendritic cell-based assay (Dos Santos et al., 2009; Toebak et al., 2006) or keratinocyte IL-18 assay (Corsini et al., 2009), and Tier 2 would determine or confirm sensitizer potency of the selected sensitizers. All of these assays are currently unproven since they are still in the research phase or are just entering pre-validation. Therefore, multiple assays are being developed so that the most optimal testing strategy can be selected in the future. Since the EE potency assay protocol can easily be applied to commercially available EE, it will be possible in the future to test the large panels of chemicals required for further (pre-) validation and risk assessment in laboratories with limited cell and tissue culture know-how. This is of importance when considering wide spread implementation of an in vitro assay in the future. 5. Conflict of interest statement None declared. Acknowledgments This study was financed by the EU project Sens-it-iv (Grant #018681) ‘‘Novel strategies for in vitro assessment of allergens”.

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