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Short Time Exposure (STE) test in conjunction with Bovine Corneal Opacity and Permeability (BCOP) assay including histopathology to evaluate correspondence with the Globally Harmonized System (GHS) eye irritation classification of textile dyes
Toxicology in Vitro xxx (2015) xxx–xxx
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Short Time Exposure (STE) test in conjunction with Bovine Corneal Opacity and Permeability (BCOP) assay including histopathology to evaluate correspondence with the Globally Harmonized System (GHS) eye irritation classification of textile dyes Gisele Augusto Rodrigues Oliveira a,⇑, Rafael do Nascimento Ducas a, Gabriel Campos Teixeira a, Aline Carvalho Batista b, Danielle Palma Oliveira c, Marize Campos Valadares a a
Faculdade de Farmácia, Universidade Federal de Goiás – FF/UFG, Goiânia, Goiás, Brazil Faculdade de Odontologia, Universidade Federal de Goiás – FO/UFG, Goiânia, Goiás, Brazil c Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo – FCFRP/USP, Ribeirão Preto, São Paulo, Brazil b
a r t i c l e
i n f o
Article history: Received 8 June 2014 Revised 5 May 2015 Accepted 10 May 2015 Available online xxxx Keywords: Reactive Orange 16 Reactive Green 19 UN GHS Alternative method Eye irritation
a b s t r a c t Eye irritation evaluation is mandatory for predicting health risks in consumers exposed to textile dyes. The two dyes, Reactive Orange 16 (RO16) and Reactive Green 19 (RG19) are classified as Category 2A (irritating to eyes) based on the UN Globally Harmonized System for classification (UN GHS), according to the Draize test. On the other hand, animal welfare considerations and the enforcement of a new regulation in the EU are drawing much attention in reducing or replacing animal experiments with alternative methods. This study evaluated the eye irritation of the two dyes RO16 and RG19 by combining the Short Time Exposure (STE) and the Bovine Corneal Opacity and Permeability (BCOP) assays and then comparing them with in vivo data from the GHS classification. The STE test (first level screening) categorized both dyes as GHS Category 1 (severe irritant). In the BCOP, dye RG19 was also classified as GHS Category 1 while dye RO16 was classified as GHS no prediction can be made. Both dyes caused damage to the corneal tissue as confirmed by histopathological analysis. Our findings demonstrated that the STE test did not contribute to arriving at a better conclusion about the eye irritation potential of the dyes when used in conjunction with the BCOP test. Adding the histopathology to the BCOP test could be an appropriate tool for a more meaningful prediction of the eye irritation potential of dyes. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Various materials and chemicals can cause damage to the cornea, ranging from irritation and inflammation, which causes mild discomfort to tissue corrosion. They include household, industrial, agricultural, personal care products and cosmetics and may even include certain ocular drugs if incorrectly administered (Wilson et al., 2015). Synthetic dyes are used worldwide for coloring or intensifying the color of various products, such as textiles, paper, rubber, food, drink, leather products, enamels, and printing inks, and they are also used in the pharmaceutical and cosmetic industries (Spadaro et al., 1992; McMullan et al., 2001; Oliveira et al., 2010; Ferraz ⇑ Corresponding author at: Faculdade de Farmácia, Universidade Federal de Goiás, Rua 240, s/n, Setor Leste Universitário, CEP 74605-170, Goiânia, Goiás, Brazil. E-mail addresses: [email protected], [email protected] (G.A.R. Oliveira).
et al., 2011; Leme et al., 2014). Information on eye irritating properties is an essential part of the hazard identification of these compounds. This information is also used for risk assessment and risk management, e.g. to ensure occupational and consumer safety (Scheel et al., 2011). Two dyes, Reactive Orange 16 (RO16) and Reactive Green 19 (RG19), are used extensively in textile dyeing industries and are classified as Category 2A, irritating to eyes, based on the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS) in accordance with Occupational Safety and Health Administration – Hazard Communication Standard 29 CFR 1910 (OSHA HCS, 2015). The UN GHS is a system for standardizing and harmonizing the classification and labeling of a chemical in terms of its type, degree of hazard and labeling so that this information is easily understood when conveyed in a material safety data sheet for that chemical (Sakaguchi et al., 2011; UN, 2013). Although, in principle, GHS
http://dx.doi.org/10.1016/j.tiv.2015.05.007 0887-2333/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Oliveira, G.A.R., et al. Short Time Exposure (STE) test in conjunction with Bovine Corneal Opacity and Permeability (BCOP) assay including histopathology to evaluate correspondence with the Globally Harmonized System (GHS) eye irritation classification of textile dyes. Toxicol. in Vitro (2015), http://dx.doi.org/10.1016/j.tiv.2015.05.007
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encourages the use of information from non-animal methods for classification, the current definition of categories for eye irritation is based on the Draize rabbit eye test: irreversible eye effects (Category 1), reversible eye effects (Category 2A for irritating to eyes and Category 2B for mildly irritating to eyes) and not-classified (not an eye irritant; hereafter non-irritant) (Sakaguchi et al., 2011). In recent times, animal welfare considerations and enforcement of new regulations, such as the banning of cosmetics in animal eye irritation tests in the EU (EC Cosmetics Regulation No 1223/2009) are drawing much attention in reducing or replacing animal experiments with alternative methods (Sakaguchi et al., 2011; Hayashi et al., 2012; Kojima et al., 2013). Some in vitro methods for assessing the eye irritation potential of chemicals have been developed as alternatives to the in vivo Draize test. In 2009, the Organization for Economic Cooperation and Development (OECD) adopted the Bovine Corneal Opacity and Permeability assay (BCOP) for identifying ocular corrosives and severe irritants such as the OECD Test Guideline 437 (OECD, 2009). The BCOP test method is an organotypic model, which provides short-term maintenance of the normal physiological and biochemical functions of the bovine cornea in vitro. The damage is assessed through quantitative measurements of changes in corneal opacity and permeability with an opacitometer and a visible light spectrophotometer, respectively (OECD, 2013). A histopathological evaluation of corneas could be useful for identifying damage in tissue layers, which do not produce significant opacity or permeability (OECD, 2011; Cazelle et al., 2014). In 2013, the Short Time Exposure (STE) assay was submitted to OECD as an alternative method to animal research in the assessment of eye irritation potency and a draft for an OECD guideline is currently under review. The STE is a cytotoxicity test which measures the viability of SIRC (rabbit corneal cell line) cells to identify chemicals which induce serious eye damage or eye irritation under the UN GHS classification system after 5 min of exposure (OECD, 2014). Unfortunately, a single eye irritation test is inadequate for replacing the range of injuries measured by the Draize rabbit eye test (Hayashi et al., 2012; Verstraelen et al., 2013). However, Hayashi et al. (2012) demonstrated that the accuracy of predicting the GHS rankings was slightly improved by using a tiered approach combination of the Short Time Exposure (STE) test and the Bovine Corneal Opacity and Permeability (BCOP) assay. So with that background, this study evaluated the ocular toxicity of two dyes, RO16 and RG19, using the STE test in association with the BCOP assay (cytotoxicity, permeability, opacity and corneal histopathological parameters) and compared the categorization obtained from these assays to those obtained from the UN GHS eye irritation classification, which were obtained using in vivo methods (Draize test).
2. Material and methods 2.1. Material Benzalkonium chloride (CAS 8001-54-5); imidazole (CAS 28832-4); trichloroacetic acid 30% (CAS 76-03-9); 2,6-dichlorobenzoyl chloride (CAS 4659-45-4); ethyl-2-methylacetoacetate (CAS 609-14-3); glycerol (CAS 56-81-5); and n-hexane (CAS 110-54-3) were selected as standard chemicals to confirm intra-laboratory reproducibility in our research group (data not shown). The dyes Reactive Orange 16 (RO16; Remazol Brilliant Orange 3R; dye content 70%; CAS No.: 12225-83-1) and Reactive Green 19 (RG19; dye content 65%; CAS No. 61931-49-5) were purchased from Sigma–Aldrich (St Louis, MO, USA). Both dyes are classified as
Category 2A, irritating to eyes, based on the UN GHS classification system in accordance with OSHA HCS 29 CFR 1910. Phosphate buffered saline (PBS) was used as the vehicle to prepare the dye solutions. The chemical structures of each dye are presented in Fig. 1. 2.2. Tiered approach for assessing eye irritation The two dyes RO16 and RG19 were evaluated by arranging the STE test (DRAFT TG, 2014) as a first level screening, followed by the BCOP assay with histopathological analysis (OECD, 2011, 2013), as a tiered approach to assess the range of UN GHS eye irritation classifications. 2.2.1. STE test SIRC (rabbit corneal cell line) cells were cultivated in Eagle’s MEM (Sigma–Aldrich) containing an antibiotic mixture (50 UI/mL penicillin and 50 lg/mL streptomycin), 2 mM L-glutamine and supplemented with 10% v/v fetal bovine serum (invitrogen) at 37 °C, 5% CO2 and 96% relative humidity. When the cells reached confluence as determined by microscopic analysis, they were collected by trypsinization (trypsin-EDTA solution; Sigma–Aldrich). The STE test was carried out according to the draft for the OECD guideline (OECD, 2014). 3 103 cells per well were cultured in a 96-well plate at 37 °C, 5% CO2 and 96% relative humidity for 5 days and exposed to 200 lL of each dye, RO16 and RG19, at 5% and 0.05% prepared in PBS for 5 min. Six wells were used for each concentration and three independent tests were conducted for each dye. After exposure, the cells were washed twice with 200 lL of PBS and 200 lL of MTT solution (0.5 mg/mL of culture medium) were added. After a 2 h reaction period in an incubator (37 °C, 5% CO2), the MTT formazan was extracted with DMSO for 20 min and absorbance was measured at 570 nm with a plate reader (ELISA). The same steps were performed with the control group (PBS) and the positive control (benzalkonium chloride 5%; UN GHS Category 1). Cell viability was compared to the solvent control (relative viability) to estimate potential serious eye damage or eye irritation caused by the two dyes RO16 and RG19. Eye irritation or serious eye damage are classified by the STE test as follow: a test substance is classified as UN GHS Category 1 (serious eye damage) when the 5% and 0.05% concentrations both produce a cell viability of 670%; it is classified as UN GHS No Category (not classified for eye irritation or serious eye damage) when the 5% and 0.05% concentrations both produce a cell viability of >70%; it is unable to be classified when the 5% concentration produces a cell viability of 670% and the 0.05% produces a cell viability of >70%. 2.2.2. BCOP assay The BCOP assay was performed according to the OECD TG 437 (OECD, 2013). Briefly, eyes were collected at a slaughterhouse as soon as possible after death and completely immersed in Hanks’ Balanced Salt Solution (HBSS). To minimize contamination during transport, the eyes were kept on wet ice during collection and transportation and in medium HBSS containing penicillin at 100 IU/mL and streptomycin at 100 lg/mL. Isolated corneas were mounted in holders and filled to excess with pre-warmed Eagle’s Minimum Essential Medium (EMEM) (posterior chamber). The device was then equilibrated at 32 ± 1 °C for at least 1 h and holders were calibrated at 74, 150 and 224 opacity units. The corneas were exposed to dyes RO16 and RG19 at 20% w/v prepared in PBS. After 4 h of exposure, test dilutions and controls (PBS or benzalkonium chloride 5%) were removed from the anterior chamber and the epithelium was washed at least three times to eliminate excess dyes, since both are hydrosoluble, so as to avoid interference in the spectrophotometer measurement. Corneal effects were
Please cite this article in press as: Oliveira, G.A.R., et al. Short Time Exposure (STE) test in conjunction with Bovine Corneal Opacity and Permeability (BCOP) assay including histopathology to evaluate correspondence with the Globally Harmonized System (GHS) eye irritation classification of textile dyes. Toxicol. in Vitro (2015), http://dx.doi.org/10.1016/j.tiv.2015.05.007
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Fig. 1. Chemical structures of the two dyes (A) Reactive Orange 16 and (B) Reactive Green 19.
measured by decreased light transmission (corneal opacity) using an opacitometer resulting in opacity values measured on a continuous scale and increased passage of sodium fluorescein dye (permeability). One mL of sodium fluorescein solution (4 mg/mL) was added to the anterior chamber of the corneal holder, which interfaces with the epithelial side of the cornea, while the posterior chamber, which interfaces with the endothelial side of the cornea, was filled with fresh EMEM. The holder was then incubated in a horizontal position for 90 min at 32 ± 1 °C. The amount of sodium fluorescein that crossed into the posterior chamber was quantitatively measured with a spectrophotometer at 490 nm. The opacity and permeability assessments of the cornea after exposure to the dyes were combined to derive an In Vitro Irritancy Score (IVIS), used to classify the irritancy level of each exposed group as follows:
IVIS ¼ mean opacity value þ ð15 mean permeability valueÞ Eye irritation was classified by the BCOP assay as follow: a test substance with an IVIS 6 3 was categorized as UN GHS No Category (not classified for eye irritation or serious eye damage); a test substance with a 3 < IVIS 6 55 was categorized as UN GHS No prediction can be made; and, a test substance with an IVIS > 55 was categorized as UN GHS Category 1 (corrosive or severe irritant). A histopathological analysis was also used to further characterize the degree and depth of corneal damage (OECD, 2011). The corneas were preserved in 10% phosphate buffered formalin (pH 7.4) at room temperature. Two sagittal macroscopic cross sections of the organ were obtained. These specimens were dehydrated in graded ethanol (70–100%), cleared in xylene, embedded in paraffin, sectioned at about 5 lm using a microtome (Leica RM 2155, Heidelberg, BW, Germany) and stained with hematoxylin and eosin. Histopathological slices were analyzed using a light microscope (Axio Scope A1 Carl Zeiss, Jena, TH, Germany) at 40 magnification. Photographic representations (AxioCam MRc Carl Zeiss camera) of the corneas for each exposed textile dye and control group were made and analyzed in AxioVs40 V 4.7.2.0 Carl Zeis software. A histomorphometric study measured the thickness of the epithelium and stroma at nine locations in each region (Fig. 2). Differences in mean values between the groups were analyzed with the Mann-Whitney U test to compare the 2 groups (control x RO16 and control x RG19) and the Kruskal-Wallis test followed by the Dunn’s multiple comparisons test (p < 0.05) was used for comparison between the 3 groups.
Fig. 2. Photomicrographic representation (10 magnification) of histomorphometric measurements of bovine corneal epithelium and stroma from different locations of each region of control group.
Table 1 STE testing results of the two dyes RO16 and RG19. Exposure
NC RO16 RG19 PC
UN GHS classification systema
Cell viability At 5 (%)
At 0.05 (%)
100 47 41.50 22
100 51 44 24
No category Category 1 Category 1 Category 1
NC: negative control; PBS. PC: positive control; benzalkonium chloride 5%. Prediction model based on OECD Draft (2014). a Correspondence between STE test classification and UN GHS classification system.
3. Results 3.1. STE test The viability of SIRC cells after exposure to 5% and 0.05% of the two dyes RO16 and RG19 for 5 min and the STE test categorization defined by the UN GHS classification system are shown in Table 1. Both dyes produced a cell viability of less than 70% with 5% and 0.05% concentration and were classified as UN GHS Category 1,
Please cite this article in press as: Oliveira, G.A.R., et al. Short Time Exposure (STE) test in conjunction with Bovine Corneal Opacity and Permeability (BCOP) assay including histopathology to evaluate correspondence with the Globally Harmonized System (GHS) eye irritation classification of textile dyes. Toxicol. in Vitro (2015), http://dx.doi.org/10.1016/j.tiv.2015.05.007
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which means that they induce serious eye damage. Therefore, the two dyes, RO16 and RG19 UN GHS Category 2A, were predicted as UN GHS Category 1 according to the STE test classification, which could mean an overestimation. 3.2. BCOP assay The results for the eye-hazard potential of the two textile dyes measured by their ability to induce opacity and increased permeability in isolated bovine corneas are shown in Table 2. As expected, the corneas exposed to PBS produced no changes in corneal opacity. On the other hand, both dyes were capable of inducing opacity in bovine corneas, in particular dye RG19. With regard to permeability, the amount of sodium fluorescein dye which permeated through all corneal cell layers after exposure to both dyes was insignificant, generating absorbance values of 0.002 for dye RO16 and of 0.001 for RG19. Based on these two parameters, dye RG19 was categorized as UN GHS Category 1 (corrosive or severe irritant) since its IVIS was greater than 55, while dye RO16 which presented an IVIS of 12.027 was categorized as UN GHS no prediction can be made (Table 2). As a complementary parameter for the BCOP assay, a histopathology analysis was performed to determine the depth and degree of the lesions within the exposed corneas. Although no permeability was observed after exposure to both dyes, a microscopic evaluation showed a loss of superficial epithelial lining (upper wing layer) and mild cytoplasmic vacuolation (black arrows) in the squamous layers of the corneas, induced by exposure to dye RO16 (Fig. 3B). There was no evidence of damage to the Bowman’s Layer. Dye RG19 caused damage to the epithelial structure of the corneas that resulted in areas with significant epithelial cell loss (Fig. 3C) and intense cytoplasmic and nuclear vacuolation (Fig. 3D). Moderate collagen matrix vacuolation (Fig. 3C and D) was also observed after exposure to dye RG19. In addition to histopathology, a histomorphometry study was performed to investigate the depth of the lesions after exposure to the two dyes. According to Table 3, histomorphometry revealed that the RG19 group showed a lesser thickness of the epithelium with 131.08 lm when compared to the RO16 (146.51 lm) and control (166.22 lm) groups; however this effect was not significant (p > 0.05). Significant pathologic changes in corneal stroma confirmed that the tissue was damaged by exposure to the two dyes RG19 (p < 0.001) and RO16 (p < 0.019) (Table 3). These results taken together showed that dye RG19 induced more severe damage than dye RO16 in bovine cornea (Fig. 3A–D). 4. Discussion To reduce the risk of exposure to dangerous substances all manufactured consumer products and their ingredients must be tested and their eye irritation potential assessed so that the public can be
Table 2 BCOP testing results of the two dyes RO16 and RG19. Exposure
Opacity
Permeability
IVIS
UN GHS classification systema
NC RO16 RG19 PC
3.000 12.000 113.340 86.330
0.000 0.002 0.001 2.297
3.000 12.027 112.760 120.791
No category No prediction can be made Category 1 Category 1
NC: negative control; PBS. PC: positive control; benzalkonium chloride 5%. IVIS: In Vitro Irritancy Score. Prediction model based on OECD TG 437 (2013). a Correspondence between BCOP irritation category and UN GHS classification system.
assured of their safety, or warned of the associated dangers (Wilson et al., 2015). The two dyes Reactive Orange 16 and Reactive Green 19 are used extensively in textile dyeing industries and are classified as Category 2A, irritating to eyes, based on the UN GHS classification system in accordance with OSHA HCS 29 CFR 1910 (OSHA HCS, 2015). Our results showed that both dyes are cytotoxic for SIRC cells (cell viability 6 70%) and therefore, they were classified as UN GHS Category 1 (serious eye damage) with irreversible effects on the eye (Table 1). The cytotoxic effect of chemicals on corneal epithelial cells is an important mode of action, which leads to corneal epithelium damage and eye irritation (OECD Draft TG, 2014). Hayashi et al. (2012) assessed the correspondence between the three STE rankings (rank 1: minimally irritant; rank 2: moderately irritant; rank 3: severely irritant) according to Takahashi et al. (2008) and GHS rank classifications (not classified for eye irritation, category 1 and category 2) for 51 chemicals. The authors observed that there was 68.6% accuracy between the STE and GHS rankings. Our results showed that the two dyes RO16 and RG19 UN GHS Category 2A were identified as UN GHS Category 1 by the STE assay (Table 1), confirming 100% overprediction of this in vitro cytotoxicity assay in relation to ocular irritation potential (serious eye damage). Takahashi et al. (2011) also evaluated the correspondence between the STE and GHS irritation categories for 109 chemicals and obtained an accuracy of 87%. The authors considered that the STE test is a promising alternative method for assessing eye irritation due to its high intra-laboratory reproducibility as well as its excellent predictability of eye irritation. Moreover, considering that an eye droplet of 50 lL is excreted from the human eye in 1–2 min by natural excretion and exposure period to a chemical is short, the STE test developed in 5 min of exposure time has advantage over conventional cytotoxicity tests, which use exposure periods of 24–48 h for evaluation (Takahashi et al., 2011). According to the BCOP assay, dye RG19 was classified as corrosive or severe irritant (UN GHS Category 1) and dye RO16 as UN GHS no prediction can be made. Both dyes induced opacity in isolated bovine corneas, but neither caused changes in corneal permeability (Table 2). A similar fact was observed by Cooper et al. (2001) who re-evaluated several chemicals for which eye irritation was significantly under-predicted by the opacity and permeability parameter, using histology as an additional endpoint. Their analysis showed appreciable pathological changes in both corneal epithelium and stroma, which confirmed that the tissue was damaged by exposure to the chemicals. In our study, although dye RO16 was classified by the BCOP assay (Table 2), as UN GHS no prediction can be made, it induced a loss of superficial epithelial lining and mild cytoplasmic vacuolation in the squamous layers of bovine corneas (Fig. 3B). Dye RG19 also caused severe damage to the epithelial structure of the corneas resulting in areas with cell loss and cytoplasmic and nuclear vacuolations (Fig. 3C and D). Epithelial vacuolation can be caused by membrane lysis suggesting the likelihood of serious eye damage. Besides, the presence of very slight epithelial vacuolation and moderate epithelial erosion obtained in an in vivo test could identify a chemical as EU CLP/UN GHS Category 1 (Cazelle et al., 2014). Corroborating these data, the histomorphometric alteration of corneal stroma confirmed that the tissue was damaged by exposure to the two dyes RO16 and RG19 (Table 3). In this case, the BCOP assay seems to identify dye RO16 as Category 2 (IVIS of 12 close to 3 and pathologic damage) and its categorization remains similar to that of the UN GHS while dye RG19 is overestimated when compared to the UN GHS classification system. This fact could be related to the various differences between humans,
Please cite this article in press as: Oliveira, G.A.R., et al. Short Time Exposure (STE) test in conjunction with Bovine Corneal Opacity and Permeability (BCOP) assay including histopathology to evaluate correspondence with the Globally Harmonized System (GHS) eye irritation classification of textile dyes. Toxicol. in Vitro (2015), http://dx.doi.org/10.1016/j.tiv.2015.05.007
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Fig. 3. Photomicrographic representations of corneal histopathology evaluation. Representative full thickness (epithelium, stroma, and endothelium) cross section of corneas exposed to (A) negative control (PBS), (B) Reactive Orange 16, (C) and (D) Reactive Green 19. The arrows show the cytoplasmic vacuolization. Each cornea was processed for hematoxylin and eosin staining.
Table 3 Histomorphometry analysis results of the two dyes RO16 and RG19. Exposure
Epithelium (mean ± SD)
Stroma (mean ± SD)
NC RO16 RG19
166.22 ± 28.7 lm 146.51 ± 32.62 lm 131.08 ± 56.49 lm
730.95 ± 23.17 lm 829.62 ± 19.66 lm* 1011.52 ± 81.75 lm*
NC: negative control; PBS. * p < 0.05.
rabbits and bovines, which include anatomical differences such as the presence of a nictitating membrane, or third eyelid, in the
rabbit. It has been suggested that this membrane removes and/or excludes irritating substances from the corneal surface of the eyes (ICCVAM, 2006). Furthermore, corneal thickness can vary from species to species. The corneal thickness of the bovine eye is 0.8 mm, while that of the human eye is approximately 0.5 mm, and that of the rabbit is about 0.37 mm. In addition, the number of epithelial cell layers in the cornea ranges from five to seven in rabbits, compared to an average of five in humans and 10–14 in bovines (Cooper et al., 2001; ICCVAM, 2006). Thicknesses of the structural components of the cornea also differ between species. For example, Descemet’s
Please cite this article in press as: Oliveira, G.A.R., et al. Short Time Exposure (STE) test in conjunction with Bovine Corneal Opacity and Permeability (BCOP) assay including histopathology to evaluate correspondence with the Globally Harmonized System (GHS) eye irritation classification of textile dyes. Toxicol. in Vitro (2015), http://dx.doi.org/10.1016/j.tiv.2015.05.007
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membrane in humans is of about 5–10 lm while in rabbits it ranges from 7 to 8 lm (ICCVAM, 2006). It can be concluded that histopathology could be useful in determining the likelihood of delayed effects and of specific modes of action, discriminating borderline cases, and evaluating the depth of injury, proposed as a measure of the reversibility or irreversibility of effects (OECD, 2011; Cazelle et al., 2014). When considering the tiered approach of combining STE test as first level screening followed by the BCOP assay including histopa thological/histomorphometric analysis for assessing eye irritation, the two dyes were capable of inducing serious eye damage (Category 1) or eye irritation (Category 2). The RG19 was classified as UN GHS Category 1, which means it has irreversible effects on the eye while the RO16 could be identified as Category 2. In such cases, the histological examination could be used to confirm the level of corneal irritation non-predicted by the opacity and permeability endpoints and provide important supplementary information about the types of lesions and the depth of damage that a substance could induce in the cornea and not confirmed by increased opacity or permeability (Cooper et al., 2001). Cazelle et al. (2014) also concluded that the use of histopathological analysis as an additional endpoint to the ICE test was suitable for identifying EU CLP/UN GHS Category 1 and making a better discrimination between Categories 1 and 2. 5. Conclusions In summary, our findings demonstrated that the STE test did not contribute to arriving at a better conclusion about the eye irritation potential of the dyes when used in conjunction with the BCOP test. Adding the histopathology to the BCOP assay could be an appropriate means towards making a more meaningful prediction of the eye irritation potential of dyes. However, it is recommended that more dyes be investigated in order to confirm the BCOP assay with histopathological analysis as a promising alternative for in vivo eye irritation testing. Conflict of interest The authors declare there were no conflicting interests. Acknowledgements This research was supported by the Faculdade de Farmácia, Universidade Federal de Goiás, CNPq, FINEP, FAPEG and CAPES. References Cazelle, E., Eskes, C., Hermann, M., Jones, P., McNamee, P., Prinsen, M., Taylor, H., Wijnands, M.V., 2014. Suitability of histopathology as an additional endpoint to the isolated chicken eye test for classification of non-extreme pH detergent and cleaning products. Toxicol. in Vitro 28 (4), 657–666. Cooper, K.J., Earl, L.K., Harbell, J., Raabe, H., 2001. Prediction of ocular irritancy of prototype shampoo formulations by the isolated rabbit eye (IRE) test and bovine corneal opacity and permeability (BCOP) assay. Toxicol. in Vitro 15 (2), 95–103. European Parliament and Council, 2009. Regulation (EC) No 1223/2009 of the European Parliament and of the Council of 30 November 2009 on cosmetic. Official J. Eur. Union (2009). Ferraz, E.R., Umbuzeiro, G.A., de-Almeida, G., Caloto-Oliveira, A., Chequer, F.M., Zanoni, M.V., Dorta, D.J., Oliveira, D.P., 2011. Differential toxicity of Disperse Red
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Oliveira, G.A.R., Ferraz, E.R.A., Chequer, F.M.D., Grando, M.D., Angeli, J.P.F., Tsuboy, M.S., Marcarini, J.C., Mantovani, M.S., Osugi, M.E., Lizier, T.M., Zanoni, M.V.B., Oliveira, D.P., 2010. Chlorination treatment of aqueous samples reduces, but does not eliminate, the mutagenic effect of the azo dyes Disperse Red 1, Disperse Red 13 and Disperse Orange 1. Mutat. Res. 703, 200–208. OSHA HCS, Occupational Safety & Health Administration, 2015 . Sakaguchi, H., Ota, N., Omori, T., Kuwahara, H., Sozu, T., Takagi, Y., Takahashi, Y., Tanigawa, K., Nakanishi, M., Nakamura, T., Morimoto, T., Wakuri, S., Okamoto, Y., Sakaguchi, M., Hayashi, T., Hanji, T., Watanabe, S., 2011. Validation study of the short time exposure (STE) test to assess the eye irritation potential of chemicals. Toxicol. in Vitro 25 (4), 796–809. Scheel, J., Kleber, M., Kreutz, J., Lehringer, E., Mehling, A., Reisinger, K., Steiling, W., 2011. Eye irritation potential: usefulness of the HET-CAM under the globally harmonized system of classification and labeling of chemicals (GHS). Regul. Toxicol. Pharmacol. 59 (3), 471–492. Spadaro, J.T., Gold, M.H., Renganathan, V., 1992. Degradation of azo dyes by the lignin-degrading fungus Phanerochaete chrysosporium. Appl. Environ. Microbiol. 58 (8), 2397–2401. Takahashi, Y., Koike, M., Honda, H., Ito, Y., Sakaguchi, H., Suzuki, H., Nishiyama, N., 2008. Development of the short time exposure (STE) test: an in vitro eye irritation test using SIRC cells. Toxicol. in Vitro 22 (3), 760–770. Takahashi, Y., Hayashi, K., Abo, T., Koike, M., Sakaguchi, H., Nishiyama, N., 2011. The short time exposure (STE) test for predicting eye irritation potential: intralaboratory reproducibility and correspondence to globally harmonized system (GHS) and EU eye irritation classification for 109 chemicals. Toxicol. in Vitro 25 (7), 1425–1434. UN, 2013. 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Short Time Exposure (STE) test in conjunction with Bovine Corneal Opacity and Permeability (BCOP) assay including histopathology to evaluate correspondence with the Globally Harmonized System (GHS) eye irritation classification of textile dyes Gisele Augusto Rodrigues Oliveira a,⇑, Rafael do Nascimento Ducas a, Gabriel Campos Teixeira a, Aline Carvalho Batista b, Danielle Palma Oliveira c, Marize Campos Valadares a a
Faculdade de Farmácia, Universidade Federal de Goiás – FF/UFG, Goiânia, Goiás, Brazil Faculdade de Odontologia, Universidade Federal de Goiás – FO/UFG, Goiânia, Goiás, Brazil c Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo – FCFRP/USP, Ribeirão Preto, São Paulo, Brazil b
a r t i c l e
i n f o
Article history: Received 8 June 2014 Revised 5 May 2015 Accepted 10 May 2015 Available online xxxx Keywords: Reactive Orange 16 Reactive Green 19 UN GHS Alternative method Eye irritation
a b s t r a c t Eye irritation evaluation is mandatory for predicting health risks in consumers exposed to textile dyes. The two dyes, Reactive Orange 16 (RO16) and Reactive Green 19 (RG19) are classified as Category 2A (irritating to eyes) based on the UN Globally Harmonized System for classification (UN GHS), according to the Draize test. On the other hand, animal welfare considerations and the enforcement of a new regulation in the EU are drawing much attention in reducing or replacing animal experiments with alternative methods. This study evaluated the eye irritation of the two dyes RO16 and RG19 by combining the Short Time Exposure (STE) and the Bovine Corneal Opacity and Permeability (BCOP) assays and then comparing them with in vivo data from the GHS classification. The STE test (first level screening) categorized both dyes as GHS Category 1 (severe irritant). In the BCOP, dye RG19 was also classified as GHS Category 1 while dye RO16 was classified as GHS no prediction can be made. Both dyes caused damage to the corneal tissue as confirmed by histopathological analysis. Our findings demonstrated that the STE test did not contribute to arriving at a better conclusion about the eye irritation potential of the dyes when used in conjunction with the BCOP test. Adding the histopathology to the BCOP test could be an appropriate tool for a more meaningful prediction of the eye irritation potential of dyes. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Various materials and chemicals can cause damage to the cornea, ranging from irritation and inflammation, which causes mild discomfort to tissue corrosion. They include household, industrial, agricultural, personal care products and cosmetics and may even include certain ocular drugs if incorrectly administered (Wilson et al., 2015). Synthetic dyes are used worldwide for coloring or intensifying the color of various products, such as textiles, paper, rubber, food, drink, leather products, enamels, and printing inks, and they are also used in the pharmaceutical and cosmetic industries (Spadaro et al., 1992; McMullan et al., 2001; Oliveira et al., 2010; Ferraz ⇑ Corresponding author at: Faculdade de Farmácia, Universidade Federal de Goiás, Rua 240, s/n, Setor Leste Universitário, CEP 74605-170, Goiânia, Goiás, Brazil. E-mail addresses: [email protected], [email protected] (G.A.R. Oliveira).
et al., 2011; Leme et al., 2014). Information on eye irritating properties is an essential part of the hazard identification of these compounds. This information is also used for risk assessment and risk management, e.g. to ensure occupational and consumer safety (Scheel et al., 2011). Two dyes, Reactive Orange 16 (RO16) and Reactive Green 19 (RG19), are used extensively in textile dyeing industries and are classified as Category 2A, irritating to eyes, based on the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS) in accordance with Occupational Safety and Health Administration – Hazard Communication Standard 29 CFR 1910 (OSHA HCS, 2015). The UN GHS is a system for standardizing and harmonizing the classification and labeling of a chemical in terms of its type, degree of hazard and labeling so that this information is easily understood when conveyed in a material safety data sheet for that chemical (Sakaguchi et al., 2011; UN, 2013). Although, in principle, GHS
http://dx.doi.org/10.1016/j.tiv.2015.05.007 0887-2333/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Oliveira, G.A.R., et al. Short Time Exposure (STE) test in conjunction with Bovine Corneal Opacity and Permeability (BCOP) assay including histopathology to evaluate correspondence with the Globally Harmonized System (GHS) eye irritation classification of textile dyes. Toxicol. in Vitro (2015), http://dx.doi.org/10.1016/j.tiv.2015.05.007
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encourages the use of information from non-animal methods for classification, the current definition of categories for eye irritation is based on the Draize rabbit eye test: irreversible eye effects (Category 1), reversible eye effects (Category 2A for irritating to eyes and Category 2B for mildly irritating to eyes) and not-classified (not an eye irritant; hereafter non-irritant) (Sakaguchi et al., 2011). In recent times, animal welfare considerations and enforcement of new regulations, such as the banning of cosmetics in animal eye irritation tests in the EU (EC Cosmetics Regulation No 1223/2009) are drawing much attention in reducing or replacing animal experiments with alternative methods (Sakaguchi et al., 2011; Hayashi et al., 2012; Kojima et al., 2013). Some in vitro methods for assessing the eye irritation potential of chemicals have been developed as alternatives to the in vivo Draize test. In 2009, the Organization for Economic Cooperation and Development (OECD) adopted the Bovine Corneal Opacity and Permeability assay (BCOP) for identifying ocular corrosives and severe irritants such as the OECD Test Guideline 437 (OECD, 2009). The BCOP test method is an organotypic model, which provides short-term maintenance of the normal physiological and biochemical functions of the bovine cornea in vitro. The damage is assessed through quantitative measurements of changes in corneal opacity and permeability with an opacitometer and a visible light spectrophotometer, respectively (OECD, 2013). A histopathological evaluation of corneas could be useful for identifying damage in tissue layers, which do not produce significant opacity or permeability (OECD, 2011; Cazelle et al., 2014). In 2013, the Short Time Exposure (STE) assay was submitted to OECD as an alternative method to animal research in the assessment of eye irritation potency and a draft for an OECD guideline is currently under review. The STE is a cytotoxicity test which measures the viability of SIRC (rabbit corneal cell line) cells to identify chemicals which induce serious eye damage or eye irritation under the UN GHS classification system after 5 min of exposure (OECD, 2014). Unfortunately, a single eye irritation test is inadequate for replacing the range of injuries measured by the Draize rabbit eye test (Hayashi et al., 2012; Verstraelen et al., 2013). However, Hayashi et al. (2012) demonstrated that the accuracy of predicting the GHS rankings was slightly improved by using a tiered approach combination of the Short Time Exposure (STE) test and the Bovine Corneal Opacity and Permeability (BCOP) assay. So with that background, this study evaluated the ocular toxicity of two dyes, RO16 and RG19, using the STE test in association with the BCOP assay (cytotoxicity, permeability, opacity and corneal histopathological parameters) and compared the categorization obtained from these assays to those obtained from the UN GHS eye irritation classification, which were obtained using in vivo methods (Draize test).
2. Material and methods 2.1. Material Benzalkonium chloride (CAS 8001-54-5); imidazole (CAS 28832-4); trichloroacetic acid 30% (CAS 76-03-9); 2,6-dichlorobenzoyl chloride (CAS 4659-45-4); ethyl-2-methylacetoacetate (CAS 609-14-3); glycerol (CAS 56-81-5); and n-hexane (CAS 110-54-3) were selected as standard chemicals to confirm intra-laboratory reproducibility in our research group (data not shown). The dyes Reactive Orange 16 (RO16; Remazol Brilliant Orange 3R; dye content 70%; CAS No.: 12225-83-1) and Reactive Green 19 (RG19; dye content 65%; CAS No. 61931-49-5) were purchased from Sigma–Aldrich (St Louis, MO, USA). Both dyes are classified as
Category 2A, irritating to eyes, based on the UN GHS classification system in accordance with OSHA HCS 29 CFR 1910. Phosphate buffered saline (PBS) was used as the vehicle to prepare the dye solutions. The chemical structures of each dye are presented in Fig. 1. 2.2. Tiered approach for assessing eye irritation The two dyes RO16 and RG19 were evaluated by arranging the STE test (DRAFT TG, 2014) as a first level screening, followed by the BCOP assay with histopathological analysis (OECD, 2011, 2013), as a tiered approach to assess the range of UN GHS eye irritation classifications. 2.2.1. STE test SIRC (rabbit corneal cell line) cells were cultivated in Eagle’s MEM (Sigma–Aldrich) containing an antibiotic mixture (50 UI/mL penicillin and 50 lg/mL streptomycin), 2 mM L-glutamine and supplemented with 10% v/v fetal bovine serum (invitrogen) at 37 °C, 5% CO2 and 96% relative humidity. When the cells reached confluence as determined by microscopic analysis, they were collected by trypsinization (trypsin-EDTA solution; Sigma–Aldrich). The STE test was carried out according to the draft for the OECD guideline (OECD, 2014). 3 103 cells per well were cultured in a 96-well plate at 37 °C, 5% CO2 and 96% relative humidity for 5 days and exposed to 200 lL of each dye, RO16 and RG19, at 5% and 0.05% prepared in PBS for 5 min. Six wells were used for each concentration and three independent tests were conducted for each dye. After exposure, the cells were washed twice with 200 lL of PBS and 200 lL of MTT solution (0.5 mg/mL of culture medium) were added. After a 2 h reaction period in an incubator (37 °C, 5% CO2), the MTT formazan was extracted with DMSO for 20 min and absorbance was measured at 570 nm with a plate reader (ELISA). The same steps were performed with the control group (PBS) and the positive control (benzalkonium chloride 5%; UN GHS Category 1). Cell viability was compared to the solvent control (relative viability) to estimate potential serious eye damage or eye irritation caused by the two dyes RO16 and RG19. Eye irritation or serious eye damage are classified by the STE test as follow: a test substance is classified as UN GHS Category 1 (serious eye damage) when the 5% and 0.05% concentrations both produce a cell viability of 670%; it is classified as UN GHS No Category (not classified for eye irritation or serious eye damage) when the 5% and 0.05% concentrations both produce a cell viability of >70%; it is unable to be classified when the 5% concentration produces a cell viability of 670% and the 0.05% produces a cell viability of >70%. 2.2.2. BCOP assay The BCOP assay was performed according to the OECD TG 437 (OECD, 2013). Briefly, eyes were collected at a slaughterhouse as soon as possible after death and completely immersed in Hanks’ Balanced Salt Solution (HBSS). To minimize contamination during transport, the eyes were kept on wet ice during collection and transportation and in medium HBSS containing penicillin at 100 IU/mL and streptomycin at 100 lg/mL. Isolated corneas were mounted in holders and filled to excess with pre-warmed Eagle’s Minimum Essential Medium (EMEM) (posterior chamber). The device was then equilibrated at 32 ± 1 °C for at least 1 h and holders were calibrated at 74, 150 and 224 opacity units. The corneas were exposed to dyes RO16 and RG19 at 20% w/v prepared in PBS. After 4 h of exposure, test dilutions and controls (PBS or benzalkonium chloride 5%) were removed from the anterior chamber and the epithelium was washed at least three times to eliminate excess dyes, since both are hydrosoluble, so as to avoid interference in the spectrophotometer measurement. Corneal effects were
Please cite this article in press as: Oliveira, G.A.R., et al. Short Time Exposure (STE) test in conjunction with Bovine Corneal Opacity and Permeability (BCOP) assay including histopathology to evaluate correspondence with the Globally Harmonized System (GHS) eye irritation classification of textile dyes. Toxicol. in Vitro (2015), http://dx.doi.org/10.1016/j.tiv.2015.05.007
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Fig. 1. Chemical structures of the two dyes (A) Reactive Orange 16 and (B) Reactive Green 19.
measured by decreased light transmission (corneal opacity) using an opacitometer resulting in opacity values measured on a continuous scale and increased passage of sodium fluorescein dye (permeability). One mL of sodium fluorescein solution (4 mg/mL) was added to the anterior chamber of the corneal holder, which interfaces with the epithelial side of the cornea, while the posterior chamber, which interfaces with the endothelial side of the cornea, was filled with fresh EMEM. The holder was then incubated in a horizontal position for 90 min at 32 ± 1 °C. The amount of sodium fluorescein that crossed into the posterior chamber was quantitatively measured with a spectrophotometer at 490 nm. The opacity and permeability assessments of the cornea after exposure to the dyes were combined to derive an In Vitro Irritancy Score (IVIS), used to classify the irritancy level of each exposed group as follows:
IVIS ¼ mean opacity value þ ð15 mean permeability valueÞ Eye irritation was classified by the BCOP assay as follow: a test substance with an IVIS 6 3 was categorized as UN GHS No Category (not classified for eye irritation or serious eye damage); a test substance with a 3 < IVIS 6 55 was categorized as UN GHS No prediction can be made; and, a test substance with an IVIS > 55 was categorized as UN GHS Category 1 (corrosive or severe irritant). A histopathological analysis was also used to further characterize the degree and depth of corneal damage (OECD, 2011). The corneas were preserved in 10% phosphate buffered formalin (pH 7.4) at room temperature. Two sagittal macroscopic cross sections of the organ were obtained. These specimens were dehydrated in graded ethanol (70–100%), cleared in xylene, embedded in paraffin, sectioned at about 5 lm using a microtome (Leica RM 2155, Heidelberg, BW, Germany) and stained with hematoxylin and eosin. Histopathological slices were analyzed using a light microscope (Axio Scope A1 Carl Zeiss, Jena, TH, Germany) at 40 magnification. Photographic representations (AxioCam MRc Carl Zeiss camera) of the corneas for each exposed textile dye and control group were made and analyzed in AxioVs40 V 4.7.2.0 Carl Zeis software. A histomorphometric study measured the thickness of the epithelium and stroma at nine locations in each region (Fig. 2). Differences in mean values between the groups were analyzed with the Mann-Whitney U test to compare the 2 groups (control x RO16 and control x RG19) and the Kruskal-Wallis test followed by the Dunn’s multiple comparisons test (p < 0.05) was used for comparison between the 3 groups.
Fig. 2. Photomicrographic representation (10 magnification) of histomorphometric measurements of bovine corneal epithelium and stroma from different locations of each region of control group.
Table 1 STE testing results of the two dyes RO16 and RG19. Exposure
NC RO16 RG19 PC
UN GHS classification systema
Cell viability At 5 (%)
At 0.05 (%)
100 47 41.50 22
100 51 44 24
No category Category 1 Category 1 Category 1
NC: negative control; PBS. PC: positive control; benzalkonium chloride 5%. Prediction model based on OECD Draft (2014). a Correspondence between STE test classification and UN GHS classification system.
3. Results 3.1. STE test The viability of SIRC cells after exposure to 5% and 0.05% of the two dyes RO16 and RG19 for 5 min and the STE test categorization defined by the UN GHS classification system are shown in Table 1. Both dyes produced a cell viability of less than 70% with 5% and 0.05% concentration and were classified as UN GHS Category 1,
Please cite this article in press as: Oliveira, G.A.R., et al. Short Time Exposure (STE) test in conjunction with Bovine Corneal Opacity and Permeability (BCOP) assay including histopathology to evaluate correspondence with the Globally Harmonized System (GHS) eye irritation classification of textile dyes. Toxicol. in Vitro (2015), http://dx.doi.org/10.1016/j.tiv.2015.05.007
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which means that they induce serious eye damage. Therefore, the two dyes, RO16 and RG19 UN GHS Category 2A, were predicted as UN GHS Category 1 according to the STE test classification, which could mean an overestimation. 3.2. BCOP assay The results for the eye-hazard potential of the two textile dyes measured by their ability to induce opacity and increased permeability in isolated bovine corneas are shown in Table 2. As expected, the corneas exposed to PBS produced no changes in corneal opacity. On the other hand, both dyes were capable of inducing opacity in bovine corneas, in particular dye RG19. With regard to permeability, the amount of sodium fluorescein dye which permeated through all corneal cell layers after exposure to both dyes was insignificant, generating absorbance values of 0.002 for dye RO16 and of 0.001 for RG19. Based on these two parameters, dye RG19 was categorized as UN GHS Category 1 (corrosive or severe irritant) since its IVIS was greater than 55, while dye RO16 which presented an IVIS of 12.027 was categorized as UN GHS no prediction can be made (Table 2). As a complementary parameter for the BCOP assay, a histopathology analysis was performed to determine the depth and degree of the lesions within the exposed corneas. Although no permeability was observed after exposure to both dyes, a microscopic evaluation showed a loss of superficial epithelial lining (upper wing layer) and mild cytoplasmic vacuolation (black arrows) in the squamous layers of the corneas, induced by exposure to dye RO16 (Fig. 3B). There was no evidence of damage to the Bowman’s Layer. Dye RG19 caused damage to the epithelial structure of the corneas that resulted in areas with significant epithelial cell loss (Fig. 3C) and intense cytoplasmic and nuclear vacuolation (Fig. 3D). Moderate collagen matrix vacuolation (Fig. 3C and D) was also observed after exposure to dye RG19. In addition to histopathology, a histomorphometry study was performed to investigate the depth of the lesions after exposure to the two dyes. According to Table 3, histomorphometry revealed that the RG19 group showed a lesser thickness of the epithelium with 131.08 lm when compared to the RO16 (146.51 lm) and control (166.22 lm) groups; however this effect was not significant (p > 0.05). Significant pathologic changes in corneal stroma confirmed that the tissue was damaged by exposure to the two dyes RG19 (p < 0.001) and RO16 (p < 0.019) (Table 3). These results taken together showed that dye RG19 induced more severe damage than dye RO16 in bovine cornea (Fig. 3A–D). 4. Discussion To reduce the risk of exposure to dangerous substances all manufactured consumer products and their ingredients must be tested and their eye irritation potential assessed so that the public can be
Table 2 BCOP testing results of the two dyes RO16 and RG19. Exposure
Opacity
Permeability
IVIS
UN GHS classification systema
NC RO16 RG19 PC
3.000 12.000 113.340 86.330
0.000 0.002 0.001 2.297
3.000 12.027 112.760 120.791
No category No prediction can be made Category 1 Category 1
NC: negative control; PBS. PC: positive control; benzalkonium chloride 5%. IVIS: In Vitro Irritancy Score. Prediction model based on OECD TG 437 (2013). a Correspondence between BCOP irritation category and UN GHS classification system.
assured of their safety, or warned of the associated dangers (Wilson et al., 2015). The two dyes Reactive Orange 16 and Reactive Green 19 are used extensively in textile dyeing industries and are classified as Category 2A, irritating to eyes, based on the UN GHS classification system in accordance with OSHA HCS 29 CFR 1910 (OSHA HCS, 2015). Our results showed that both dyes are cytotoxic for SIRC cells (cell viability 6 70%) and therefore, they were classified as UN GHS Category 1 (serious eye damage) with irreversible effects on the eye (Table 1). The cytotoxic effect of chemicals on corneal epithelial cells is an important mode of action, which leads to corneal epithelium damage and eye irritation (OECD Draft TG, 2014). Hayashi et al. (2012) assessed the correspondence between the three STE rankings (rank 1: minimally irritant; rank 2: moderately irritant; rank 3: severely irritant) according to Takahashi et al. (2008) and GHS rank classifications (not classified for eye irritation, category 1 and category 2) for 51 chemicals. The authors observed that there was 68.6% accuracy between the STE and GHS rankings. Our results showed that the two dyes RO16 and RG19 UN GHS Category 2A were identified as UN GHS Category 1 by the STE assay (Table 1), confirming 100% overprediction of this in vitro cytotoxicity assay in relation to ocular irritation potential (serious eye damage). Takahashi et al. (2011) also evaluated the correspondence between the STE and GHS irritation categories for 109 chemicals and obtained an accuracy of 87%. The authors considered that the STE test is a promising alternative method for assessing eye irritation due to its high intra-laboratory reproducibility as well as its excellent predictability of eye irritation. Moreover, considering that an eye droplet of 50 lL is excreted from the human eye in 1–2 min by natural excretion and exposure period to a chemical is short, the STE test developed in 5 min of exposure time has advantage over conventional cytotoxicity tests, which use exposure periods of 24–48 h for evaluation (Takahashi et al., 2011). According to the BCOP assay, dye RG19 was classified as corrosive or severe irritant (UN GHS Category 1) and dye RO16 as UN GHS no prediction can be made. Both dyes induced opacity in isolated bovine corneas, but neither caused changes in corneal permeability (Table 2). A similar fact was observed by Cooper et al. (2001) who re-evaluated several chemicals for which eye irritation was significantly under-predicted by the opacity and permeability parameter, using histology as an additional endpoint. Their analysis showed appreciable pathological changes in both corneal epithelium and stroma, which confirmed that the tissue was damaged by exposure to the chemicals. In our study, although dye RO16 was classified by the BCOP assay (Table 2), as UN GHS no prediction can be made, it induced a loss of superficial epithelial lining and mild cytoplasmic vacuolation in the squamous layers of bovine corneas (Fig. 3B). Dye RG19 also caused severe damage to the epithelial structure of the corneas resulting in areas with cell loss and cytoplasmic and nuclear vacuolations (Fig. 3C and D). Epithelial vacuolation can be caused by membrane lysis suggesting the likelihood of serious eye damage. Besides, the presence of very slight epithelial vacuolation and moderate epithelial erosion obtained in an in vivo test could identify a chemical as EU CLP/UN GHS Category 1 (Cazelle et al., 2014). Corroborating these data, the histomorphometric alteration of corneal stroma confirmed that the tissue was damaged by exposure to the two dyes RO16 and RG19 (Table 3). In this case, the BCOP assay seems to identify dye RO16 as Category 2 (IVIS of 12 close to 3 and pathologic damage) and its categorization remains similar to that of the UN GHS while dye RG19 is overestimated when compared to the UN GHS classification system. This fact could be related to the various differences between humans,
Please cite this article in press as: Oliveira, G.A.R., et al. Short Time Exposure (STE) test in conjunction with Bovine Corneal Opacity and Permeability (BCOP) assay including histopathology to evaluate correspondence with the Globally Harmonized System (GHS) eye irritation classification of textile dyes. Toxicol. in Vitro (2015), http://dx.doi.org/10.1016/j.tiv.2015.05.007
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Fig. 3. Photomicrographic representations of corneal histopathology evaluation. Representative full thickness (epithelium, stroma, and endothelium) cross section of corneas exposed to (A) negative control (PBS), (B) Reactive Orange 16, (C) and (D) Reactive Green 19. The arrows show the cytoplasmic vacuolization. Each cornea was processed for hematoxylin and eosin staining.
Table 3 Histomorphometry analysis results of the two dyes RO16 and RG19. Exposure
Epithelium (mean ± SD)
Stroma (mean ± SD)
NC RO16 RG19
166.22 ± 28.7 lm 146.51 ± 32.62 lm 131.08 ± 56.49 lm
730.95 ± 23.17 lm 829.62 ± 19.66 lm* 1011.52 ± 81.75 lm*
NC: negative control; PBS. * p < 0.05.
rabbits and bovines, which include anatomical differences such as the presence of a nictitating membrane, or third eyelid, in the
rabbit. It has been suggested that this membrane removes and/or excludes irritating substances from the corneal surface of the eyes (ICCVAM, 2006). Furthermore, corneal thickness can vary from species to species. The corneal thickness of the bovine eye is 0.8 mm, while that of the human eye is approximately 0.5 mm, and that of the rabbit is about 0.37 mm. In addition, the number of epithelial cell layers in the cornea ranges from five to seven in rabbits, compared to an average of five in humans and 10–14 in bovines (Cooper et al., 2001; ICCVAM, 2006). Thicknesses of the structural components of the cornea also differ between species. For example, Descemet’s
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membrane in humans is of about 5–10 lm while in rabbits it ranges from 7 to 8 lm (ICCVAM, 2006). It can be concluded that histopathology could be useful in determining the likelihood of delayed effects and of specific modes of action, discriminating borderline cases, and evaluating the depth of injury, proposed as a measure of the reversibility or irreversibility of effects (OECD, 2011; Cazelle et al., 2014). When considering the tiered approach of combining STE test as first level screening followed by the BCOP assay including histopa thological/histomorphometric analysis for assessing eye irritation, the two dyes were capable of inducing serious eye damage (Category 1) or eye irritation (Category 2). The RG19 was classified as UN GHS Category 1, which means it has irreversible effects on the eye while the RO16 could be identified as Category 2. In such cases, the histological examination could be used to confirm the level of corneal irritation non-predicted by the opacity and permeability endpoints and provide important supplementary information about the types of lesions and the depth of damage that a substance could induce in the cornea and not confirmed by increased opacity or permeability (Cooper et al., 2001). Cazelle et al. (2014) also concluded that the use of histopathological analysis as an additional endpoint to the ICE test was suitable for identifying EU CLP/UN GHS Category 1 and making a better discrimination between Categories 1 and 2. 5. Conclusions In summary, our findings demonstrated that the STE test did not contribute to arriving at a better conclusion about the eye irritation potential of the dyes when used in conjunction with the BCOP test. Adding the histopathology to the BCOP assay could be an appropriate means towards making a more meaningful prediction of the eye irritation potential of dyes. However, it is recommended that more dyes be investigated in order to confirm the BCOP assay with histopathological analysis as a promising alternative for in vivo eye irritation testing. Conflict of interest The authors declare there were no conflicting interests. Acknowledgements This research was supported by the Faculdade de Farmácia, Universidade Federal de Goiás, CNPq, FINEP, FAPEG and CAPES. References Cazelle, E., Eskes, C., Hermann, M., Jones, P., McNamee, P., Prinsen, M., Taylor, H., Wijnands, M.V., 2014. Suitability of histopathology as an additional endpoint to the isolated chicken eye test for classification of non-extreme pH detergent and cleaning products. Toxicol. in Vitro 28 (4), 657–666. Cooper, K.J., Earl, L.K., Harbell, J., Raabe, H., 2001. Prediction of ocular irritancy of prototype shampoo formulations by the isolated rabbit eye (IRE) test and bovine corneal opacity and permeability (BCOP) assay. Toxicol. in Vitro 15 (2), 95–103. European Parliament and Council, 2009. Regulation (EC) No 1223/2009 of the European Parliament and of the Council of 30 November 2009 on cosmetic. Official J. Eur. Union (2009). Ferraz, E.R., Umbuzeiro, G.A., de-Almeida, G., Caloto-Oliveira, A., Chequer, F.M., Zanoni, M.V., Dorta, D.J., Oliveira, D.P., 2011. Differential toxicity of Disperse Red
1 and Disperse Red 13 in the Ames test, HepG2 cytotoxicity assay, and Daphnia acute toxicity test. Environ. Toxicol. 26 (5), 489–497. Hayashi, K., Mori, T., Abo, T., Koike, M., Takahashi, Y., Sakaguchi, H., Nishiyama, N., 2012. A tiered approach combining the short time exposure (STE) test and the bovine corneal opacity and permeability (BCOP) assay for predicting eye irritation potential of chemicals. J. Toxicol. Sci. 37 (2), 269–280. ICCVAM, Interagency Coordinating Committee on the Validation of Alternative Methods, 2006. Introduction and rationale for the proposed use of in vitro test methods to identify ocular corrosives and severe irritants. Disponível em:
Please cite this article in press as: Oliveira, G.A.R., et al. Short Time Exposure (STE) test in conjunction with Bovine Corneal Opacity and Permeability (BCOP) assay including histopathology to evaluate correspondence with the Globally Harmonized System (GHS) eye irritation classification of textile dyes. Toxicol. in Vitro (2015), http://dx.doi.org/10.1016/j.tiv.2015.05.007