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In vitro eye irritancy test of lauryl derivatives using the reconstructed rabbit corneal epithelium model
Toxicology in Vitro 23 (2009) 555–560
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Review
In vitro eye irritancy test of lauryl derivatives using the reconstructed rabbit corneal epithelium model Sanae Matsuda a,*, Masayoshi Hisama a, Hiroharu Shibayama a, Norihiko Itou b, Masahiro Iwaki c a
HBC Science Research Center Co., Ltd., 3-13-9, Higashinakamoto, Higashinari-ku, Osaka 537-0021, Japan Department of Ophthalmology, Yokohama City University School of Medicine, 3-9, Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan c Department of Pharmacy, School of Pharmacy, Kinki University, 3-4-1, Kowakae, Higashi-Osaka, Osaka 577-8502, Japan b
a r t i c l e
i n f o
Article history: Received 10 November 2008 Accepted 3 February 2009 Available online 11 February 2009 Keywords: Cosmetic integrants Eye irritation Rabbit corneal epithelial cell Rabbit corneal epithelium model Lauryl derivatives
a b s t r a c t The rabbit corneal epithelium model (RCE model) was developed as a three-dimensional in vitro model to replace animal testing for the assessment of eye tolerance. In the model, a stratified culture of rabbit corneal epithelial cells is grown at the air–liquid interface on an amniotic membrane acting as a parabasal membrane. The alkaline exposure was restored each day in the presence of no irritants, although with the addition of SLS, which is a major irritant, the restoration of deficit was inhibited on the RCE model in a dose-dependent manner. The results of this test were comparable with those of the Draize test, and thus, this method using the RCE model may prove to be a useful and sensitive in vitro eye irritation test. The lauryl fatty chain derivatives, such as polyoxyethylene (9) lauryl ether (PLE), sodium polyoxyethylene (2) lauryl ether sulfate (SPLE), mono glyceryl laurate (MGL), and sodium N-lauroyl-Lglutaminate (SLG), which are widely used as surfactants for toiletry products and cosmetics, were evaluated for in vitro eye irritation potential using the RCE model. SLS, PLE, SPLE, MGL, and SLG inhibited 88.7%, 59.2%, 69.0%, 47.5%, and 15.7% of the restoration of deletion 24 h after treatment at a concentration of 0.05%. The IC50 (50% inhibitory concentration) values of SLS, PLE, SPLE, MGL, and SLG were 0.002%, 0.021%, 0.005%, 0.056%, and 0.448%, respectively. These results indicated that a functional group at the end of lauryl chain is an important factor for inhibiting the restoration of deletion using the RCE model. Ó 2009 Elsevier Ltd. All rights reserved.
Contents 1. 2.
3.
4.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Test materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Cell and culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Light microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Eye irritation tests with the RCE model. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Imaging technique. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Preparation of the rabbit corneal epithelial model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Eye irritation test with the RCE model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Eye irritant effect of lauryl derivatives on the RCE model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discussion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
* Corresponding author. Tel.: +81 6 6974 7111; fax: +81 6 6974 7181. E-mail address: [email protected] (S. Matsuda). 0887-2333/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2009.02.003
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1. Introduction The in vivo Draize eye test (Draize, 1959), which has become the international standard assay for acute eye irritation (OECD TG 405) is often criticized for both ethical and scientific reasons. The 7th amendment of the cosmetic directive will lead to the ban of animal testing for cosmetic ingredients in 2009. Thus, alternative strategies are necessary which allow the testing of chemicals with wide physicochemical properties under conditions similar to in vivo exposure. Many efforts have been made in order to find reliable and relevant predictive models such as the chorioallantoic membrane (CAM) methods (Hagino et al., 1999), cell-based cytotoxicity methods (Uchiyama et al., 1999; Tani et al., 1999), reconstituted tissue models (Ohuchi et al., 1999; Jones et al., 2001), and isolated organ methods (Xu et al., 2000). Success in fully replacing the Draize eye irritation test with in vitro methods has not yet occurred, in part due to a lack of understanding of the underlying physiological mechanisms of eye irritation. The corneal epithelium is a nonkeratinized, mucosal epithelial multilayer that covers the front surface of the cornea and is essential for proper vision. In healthy corneas, the epithelium is in a constant state of turnover: superficial epithelial cells are shed, whereas cells derived from stem cells in the limbus migrate in and replenish the epithelial cell population (Thoft and Friend, 1983). However, in severe ocular surface injuries, such as chemical burns and Stevens-Johnson syndrome, the corneal epithelial cells are sometimes totally destroyed. The damage often extends to the limbal area of the cornea, where the corneal epithelial stem cells are located (Schermer et al., 1986; Cotsarelis et al., 1989). In a severely injured cornea, in which the limbal and central epithelia are both absent, the neighboring conjunctival epithelial cells invade the corneal surface, and visual acuity is severely obstructed (Shapiro et al., 1981; Tseng, 1996). The corneal epithelium has a capacity for rapid regeneration that depends on the self-renewal of corneal stem cells. Corneal epithelium consists of corneal stem cells, transient amplifying cells, postmitotic cells, and terminally differentiated cells. Both stem cells and transient amplifying cells exhibit an ability to proliferate, whereas postmitotic cells have lost this ability. Stem cells are imbued with the potential for self-renewal, and can proliferate extensively. An increasingly popular surgical procedure for ocular surface reconstruction in individuals with severe chemical or thermal burns or serious ocular surface disorders, such as Stevens-Johnson syndrome, ocular cicatricial pemphigoid, and recurrent pterygium (Tsubota et al., 1996; Lee and Tseng, 1997; Prabhasawat et al., 1997; Shimazaki et al., 1997, 1998; Tseng et al., 1998; Tsubota and Shimazaki, 1999), involves the use of preserved human amniotic membrane as a biologic drape to dress the bare stroma after the removal of abnormal conjunctival tissue. This approach is based on the report by Kim and Tseng (1995), in which the amniotic membrane is thought to inhibit conjunctival overgrowth and provide a good substrate for normal epithelial migration. The results of ocular surface reconstruction with amniotic membrane are generally good. It appears that there is no immune rejection when cells derived from the contralateral limbus or from the cornea of the patient’s relative are cultured and transplanted. It is currently not established whether, after healing, corneal epithelial cells reconstituting the new epithelium were derived from the engrafted cells or from host stem cells that remained on the ocular surface. We developed the Rabbit Corneal Epithelial (RCE) model using cultured rabbit corneal epithelial cells and an amniotic membrane as a parabasal membrane to evaluate in vitro the eye irritation potential of chemicals including pharmaceuticals, cosmetics and their raw ingredients. The amniotic membrane has superior physical
properties of strength and elasticity as well as desirable biological potencies such as low antigenicity, histocompatibility and good adhesion ability to the cell, and so is a useful scaffold for the RCE model. Generally, cytotoxicity tests using cultured cells have the advantage of being simple and quick with, a low evaluation cost. However, when using cultured cells alone in media, the testing of water insoluble materials is sometimes difficult as the test substances may precipitate out in the media. In contrast to the conventional monolayer culture system suspended in media, the RCE model has a dry surface. Therefore, it is useful for both soluble and insoluble substances including various forms of cosmetic products. The aim of the development of RCE model was to evaluate a new three-dimensional epithelial model cultivated from rabbit corneal cells to replace animal testing in the assessment of eye tolerance. Lauryl fatty chain derivatives are one group of surfactants which includes anionic, cationic and nonionic surfactants with various functional groups. Surfactants containing lauryl fatty chain are widely used for toiletry products and cosmetics. Of those, the lauryl derivatives such as polyoxyethylene (9) lauryl ether (PLE), sodium polyoxyethylene (2) lauryl ether sulfate (SPLE), mono glyceryl laurate (MGL), and sodium N-lauroyl-L-glutaminate (SLG) are frequently used in shampoo as a foaming agent and in skin-care cosmetics as an emulsifier. Therefore, it is important to evaluate the eye safety of lauryl fatty chain derivatives and to elucidate the mechanism of action of eye irritation. Thus we focused on the mechanisms of eye injury, and examined new in vitro endpoints which would be more predictive of the biogenic response to chemical injury. This should help to develop new or improved in vitro methods that could proceed to formal validation. In this study, we have developed the RCE model using an amniotic membrane as a parabasal membrane, and used this model to evaluate the in vitro eye irritation potential of lauryl derivatives, PLE, SPLE, MGL, and SLG. 2. Materials and methods 2.1. Test materials Sodium lauryl sulfate (SLS) was obtained from Wako Pure Chemical Industries, Co., Ltd. (Tokyo, Japan). Polyoxyethylene (9) lauryl ether (PLE), sodium polyoxyethylene (2) lauryl ether sulfate (SPLE), mono glyceryl laurate (MGL), and sodium N-lauroyl-L-glutaminate (SLG), which were selected from the Japanese Standard of Cosmetic Ingredients or Japanese Pharmacopoeia, were obtained from a supplier of cosmetic ingredients. The culture medium M-stars A, supplemented hormonal epithelial medium (SHEM), and amniotic membrane were obtained from ArBlast Co., Ltd. (Kobe, Japan). 2.2. Cell and culture Rabbit corneal epithelial cells were provided by ArBlast Co., Ltd. (Kobe, Japan) and were co-cultured along with inactivated 3T3 fibroblasts, as described previously (Rheinwald and Green, 1975). For the three-dimensional cell culture, intact and denuded pig amniotic membranes were spread, epithelial basement membrane side up, on the bottom of polyester membrane culture inserts. After that, the culture inserts were placed in a 6-well plate containing the treated 3T3 fibroblasts, and the cultured rabbit corneal epithelial cells were inoculated with a density of 5.0 105 cells/well on 6-well culture inserts. The culture was submerged into the previously described M-stars A medium for 7 days, and then exposed to air by lowering the medium level (airlifting). After airlifting, the suitability of the rabbit corneal epithelial (RCE) model was con-
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firmed by examining the multilayer structure formation and the flatness of the most outer layer with light microscopy.
tion, the restoration rate for the deletion is calculated for each time.
2.3. Light microscopy
2.5. Imaging technique
The multilayer structure formation of RCE model was by evaluated by light microscopy of histological vertical section. The RCE models were fixed with 10% buffered neutral formaldehyde. After that, the samples were routinely processed by cryostat, and were stained with hematoxylin and eosin.
The RCE model was observed and images were recorded with a Stereomicroscope SZX 10 (Olympus, Tokyo, Japan). The images of RCE model were analyzed using image analysis, and the area of deletion was measured. Image analysis was performed using the public domain Java image processing program, Image J v3.91 software (http://rsb.info.nih.gov/ij), which was developed at the National Institute of Health (NIH) to assist in clinical and scientific image analyzes.
2.4. Eye irritation tests with the RCE model The eye irritation potency of test samples was estimated by the measurement of inhibition of restoration against alkaline exposure. A medical sponge, with a diameter of 8 mm and impregnated with 1 N sodium hydroxide solution, was laid on the RCE model to produce a deletion. The RCE model containing a deletion was cultured in a SHEM medium containing the test samples at 37 °C under 5% CO2 for 5 min. After incubation, the test substance was removed from the RCE model by washing with the assay medium. The RCE model was further cultured in a SHEM medium at 37 °C in 5% CO2 for 96 h after treatment. The area of deletion after alkaline exposure was measured as an index of inhibition of restoration against deletion induced by alkaline exposure using the imaging technique every 24 h for 96 h after treatment. By comparison of the area of deletion after incubation with that before sample irrita-
2.6. Statistical analysis The inhibitory effect on restoration against alkaline exposure was expressed as the mean ± standard error (SE) of three independent experiments, and subsequent inspection of means was evaluated using the Student’s t-test between two groups at a significance level of p < 0.05 and p < 0.01. 3. Results 3.1. Preparation of the rabbit corneal epithelial model Rabbit corneal epithelial cells have been used to develop a three-dimensional in vitro model of the rabbit corneal epithelium (RCE model). Rabbit corneal epithelial cells form a stratified culture when grown at the air–liquid interface on an amniotic membrane acting as a parabasal membrane in a cell culture insert with Mstars A medium. The RCE model was confirmed to be a multilayer of well-stratified corneal cells on the amniotic membrane using a histological vertical section (Fig. 1). 3.2. Eye irritation test with the RCE model
Fig. 1. Histological vertical section of the RCE Model.
The RCE model was exposed in the central part using a medical sponge impregnated with a 1 N sodium hydroxide solution to produce the deletion. Fig. 2 shows the time-course of macroscopic restoration of the deletion on the RCE model grown in the presence of sodium lauryl sulfate (SLS), which is a known irritant. Previously, SLS was evaluated with the Draize test, and showed the Draize score on a dose-dependent manner (Bagley et al., 1999), and so
Fig. 2. Macroscopic views of the RCE Model treated with 0.005% SLS.
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used with an eye irritation test of RCE model as a standard irritant. When there was no irritant added, the restoration process had started, and it was almost complete after 96 h on the RCE model. Restoration of the deletion was clearly inhibited by the treatment of 0.005% SLS. When the deletion was observed under an inverted microscope, there appeared to be a lower degree of restoration of the deletion in the RCE model irritated by SLS than in the non-irritated model. Although, using SLS as the irritant, the restoration of deletion was generally inhibited in a dose- and time-dependent manner (Fig. 3). However, at the high concentration of 0.05% tested, SLS did not inhibit the deletion in a time-dependent manner, and instead still inhibited 86.4% of the restoration of the deletion 96 h later.
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Time (hours) Fig. 3. Time-course of the restoration of alkaline deletion treatment of SLS on the RCE model. RCE model containing an alkaline deletion was treated with SLS at concentrations of 0.001% (j), 0.005% (d), 0.01% (), and 0.05% (N), and cultured in a SHEM medium at 37 °C in 5% CO2 for 96 h. Control (e) was not treated with SLS. The area of deletion was measured as an index of inhibition of restoration against deletion induced by alkaline exposure using the imaging technique every 24 h. Data are expressed as a percentage of control. Bars represent mean ± SE of three independent experiments. Significantly different from the non-treated control group at p < 0.05 () and p < 0.01 ().
3.3. Eye irritant effect of lauryl derivatives on the RCE model The inhibitory effects of the lauryl derivatives, polyoxyethylene (9) lauryl ether (PLE), sodium polyoxyethylene (2) lauryl ether sulfate (SPLE), mono glyceryl laurate (MGL), and sodium N-lauroyl-Lglutaminate (SLG) were determined by the RCE model with an alkaline deletion. Fig. 4 shows macroscopic restoration of the deletion on the RCE model grown in the presence of the lauryl derivatives PLE, SPLE, MGL, and SLG at concentrations of 0.01% and 0.05%. When the RCE model was used without added irritant, the deletion was restored on the RCE model 24 h after treatment. Restoration of the deletion was clearly inhibited by the treatment of PLE, SPLE, MGL, and SLG at a concentration of 0.05%. When the deletion was observed under an inverted microscope, there appeared to be some differences in the restoration of the deletion in the RCE model depending on the various irritants used. In case of treatment with SLG, the deletion was restored more than in the presence of the other lauryl derivatives. All lauryl derivatives inhibited the restoration of the deletion in a dose- and time-dependent manner from 24 h to 96 h after treatment. The inhibition of the restoration of deletion decreased slightly from 24 h to 96 h after the treatment (Fig. 5). SLS, PLE, SPLE, MGL, and SLG inhibited 88.7%, 59.2%, 69.0%, 47.5%, and 15.7% of the restoration of deletion 24 h after treatment at a concentration of 0.050%, and the IC50 (50% inhibitory concentration) values of SLS, PLE, SPLE, MGL, and SLG were 0.002%, 0.021%, 0.005%, 0.056%, and 0.448%, respectively (Fig. 6A). SLS, PLE, SPLE, MGL, and SLG inhibited 86.4%, 42.0%, 49.0%, 34.5%, and 19.5% of the restoration of deletion 96 h after treatment at a concentration of 0.05%, and the IC50 values of SLS, PLE, SPLE, MGL, and SLG were 0.007%, 0.063%, 0.052%, 0.069%, and 0.340%, respectively (Fig. 6B). This result indicated that SLS has the highest level of eye irritation of the lauryl derivatives in the RCE model at both 24 h and 96 h after treatment. According to the IC50 value, the inhibition by SLS was 10 times more potent that the other lauryl derivatives. 4. Discussion The rabbit corneal epithelium (RCE) model was developed as a three-dimensional in vitro model by using a stratified culture of rabbit corneal epithelial cells grown at the air–liquid interface on an amniotic membrane as a parabasal membrane. This allows an
Fig. 4. Microscopic views of the RCE model treated with lauryl derivatives (PLE, SPLE, MGL, and SLG).
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Time (hours) Fig. 5. Time-course of the restoration of alkaline deletion with 0.05% lauryl derivatives treatment on the RCE model. RCE model containing an alkaline deletion was treated with SLS (e), PLE (h), SPLE (s), MGL (), and SLG (4) at a concentration of 0.05%, and cultured in a SHEM medium at 37 °C in 5% CO2 for 96 h. The area of deletion was measured as an index of inhibition of restoration against deletion induced by alkaline exposure using the imaging technique every 24 h. Data are expressed as a percentage of control. Bars represent mean ± SE of three independent experiments. Significantly different from the non-treated control group at p < 0.05 () and p < 0.01 ().
Inhibitory effect of restoration rate (% of control)
artificial corneal epithelium (reconstituted rabbit corneal epithelium) to be prepared which exhibits barrier characteristics and paracellular permeability similar to those of native rabbit cornea (Fig. 1). The deletion with alkaline exposure was restored each day in the presence of no irritants, although with the addition of sodium lauryl sulfate (SLS) as a positive irritant, the restoration of deletion was inhibited on the RCE model in a dose-dependent manner (Figs. 2 and 3). SLS, which is a major irritant, also increased the Draize score in a dose-dependent manner (Bagley et al., 1999).
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This result indicated that the eye irritation potency of test samples may be estimated by the measurement of inhibition of restoration against alkaline exposure on the RCE model. When the time–activity relationships were examined, it became evident that the different inhibitory potencies between the lauryl derivatives at a concentration of 0.05% 24 h after irritation are greater than those at subsequent hours (Fig. 5). These results indicated that the inhibitory effect is most sensitive 24 h after treatment and so research into the eye irritation of test samples should be analyzed at this time point on the RCE model. As for the structure-activity relationships, the lauryl derivatives had distinctly different inhibitory potencies against the restoration of deletion, according to their substitution patterns. The inhibitory effects of the restoration of deletion decreased in the order SLS > SPLE > PLE > MGL > SLG (Figs. 4 and 6). The difference in structure between the lauryl derivatives, SLS, SPLE, PLE, MGL, and SLG is a functional group at the end of lauryl fatty chain. These results indicated that the functional group at the end of lauryl fatty chain is an important factor for inhibiting the restoration of deletion in the order sodium sulfate > sodium polyoxyethylene ether sulfate > polyoxyethylene ether > glycerate > sodium glutaminate. SLS inhibited the restoration of deletion greater than all the other lauryl derivatives tested on the eye irritancy test with this RCE model. These results were comparable with those of the Draize test (Ohno et al., 1999), and therefore, the RCE model may be a useful in vitro eye irritation test. In addition, the small difference in inhibitory effects between the lauryl derivatives, which cannot be confirmed with the Draize test, was remarkably confirmed using the RCE model. Thus, the RCE model may be a sensitive one. In conclusion, the rabbit corneal epithelial (RCE) model was prepared by culture of rabbit corneal cells on amniotic membrane in a cell culture insert. The irritancy effects of lauryl derivatives were determined by measurement of the inhibitory effect of the restoration of an alkaline deletion on the RCE model as a guideline. SLS inhibited the restoration of deletion greater than other lauryl derivatives, and was suggested to be the strongest eye irritant of the lauryl derivatives tested. These results indicated that the functional group at the end of lauryl fatty chain is an important factor
Inhibitory effect of restoration rate (% of control)
Inhibitory effect of restoration rate (% of control)
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Fig. 6. Inhibitory effects of the restoration of alkaline deletion with lauryl derivatives treatment 24 h and 96 h on the RCE model. RCE model containing an alkaline deletion was treated with SLS, PLE, SPLE, MGL, and SLG at a concentration of 0.005%, 0.001%, 0.05%, 0.1%, and 0.5%, and cultured in a SHEM medium at 37 °C in 5% CO2 for 24 h and 96 h. The area of deletion was measured as an index of inhibition of restoration against deletion induced by alkaline exposure using the imaging technique 24 h (A) and 96 h (B) after treatment. Data are expressed as a percentage of control. Bars represent mean ± SE of three independent experiments. Significantly different from the non-treated control group at p < 0.05 () and p < 0.01 ().
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for the degree of eye irritation. The results of this test were comparable with those of the Draize test, and thus, this modification of the RCE model may prove to be a useful and sensitive in vitro eye irritation test. However, the eye irritation test with RCE model was preliminary evaluated by with only a few compounds in a very limited chemical space, and so may not exhibit their expected effects in vivo if they are adversely affected by factor such as absorption, bio-disposition and metabolism after they are incorporated into the human eye. Further studies by many sorts of chemicals with wide physicochemical properties the eye irritation test with RCE model and Comparison between this test, and in vivo and other in vitro eye irritation tests, are needed to determine the efficacy of this test for in vitro model supposed to replace the Draize eye irritation test.
References Bagley, D.M., Gardner, J.R., Holland, G., Lewis, R.W., Vrijhof, H., Walker, A.P., 1999. Eye irritation: updated reference chemicals data bank. Toxicol. In Vitro 13 (3), 505–510. Cotsarelis, G., Cheng, S.Z., Dong, G., Sun, T.T., Lavker, R.M., 1989. Existence of slowcycling limbal epithelial basal cells that can be preferentially stimulated to proliferate: implications on epithelial stem cells. Cell 57, 201–209. Draize, J.H., 1959. Appraisal of the Safety of Chemicals in Food, Drugs and Cosmetics, ed. by the Association of Food and Drug Officials of the United States, Austin TX. Hagino, S., Kinoshita, S., Tani, N., Nakamura, T., Ono, N., Konishi, K., Iimura, H., Kojima, H., Ohno, Y., 1999. Interlaboratory validation of the in vitro eye irritation tests for cosmetic ingredients (2) chorioallantonic membrane (CAM) test. Toxicol. In Vitro 13, 99–113. Jones, P.A., Budynsky, E., Cooper, K.J., Decker, D., Griffiths, H.A., Fentem, J.H., 2001. Comparative evaluation of five in vitro tests for assessing the eye irritation potential of hair-care products. Aitern. Lab. Anim. 29, 669–692. Kim, J.C., Tseng, S.C.G., 1995. Transplantation of preserved human amniotic membrane for surface reconstruction in severely damaged rabbit corneas. Cornea 14, 473–484. Lee, S.H., Tseng, S.C.G., 1997. Amniotic membrane transplantation for persistent epithelial defects with ulceration. Am. J. Ophthalmol. 123, 303–312. Ohno, Y., Kaneko, T., Kobayashi, T., Inoue, I., Kuroiwa, Y., Yoshida, T., Fujii, T., Matsuda, T., Ohno, T., Hayashi, M., Momma, J., Akiyama, J., Chiba, K., Ikeda, N., Imanishi, Y., Itagaki, H., Kakishima, H., Kasai, Y., Kurishita, H., Kojima, H., Matsukawa, S., Nakamura, T., Ohkoshi, K., Okumura, H., Saijo, K., Sakamoto, K., Suzuki, T., Takano, K., Tatsumi, H., Tani, N., Usami, M., Watanabe, R., 1999. Interlaboratory validation of the in vitro eye irritation tests for cosmetic ingredients. (1) Overviews of the validation study and Draize scores for the evaluation of the tests. Toxicol. In vitro 13, 73–98.
Ohuchi, J., Kasai, Y., Sakamoto, K., Ohnuma, M., Kitamura, M., Kawasaki, Y., Kakishima, H., Suzuki, K., Kuwahara, H., Imanishi, Y., Tatsumi, H., Kotani, M., Inoue, K., Okumura, H., Arashima, M., Kurishita, A., Kinoshita, S., Tani, N., Kojima, H., Nakamura, T., Suzuki, K., Ishibashi, T., Hori, H., Takahashi, H., Nishikawa, T., Kitano, Y., Ohno, Y., 1999. Interlaboratory validation of the in vitro eye irritation tests for cosmetic ingredients (6) evaluation of MATREX. Toxicol. In Vitro 13, 153–162. Prabhasawat, P., Barton, K., Burkett, G., Tseng, S.C.G., 1997. Comparison of conjunctival autografts, amniotic membrane grafts, and primary closure for pteryguim excision. Ophthalmologe 104, 974–985. Rheinwald, J.G., Green, H., 1975. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6, 331–343. Schermer, A., Galvin, S., Sun, T.T., 1986. Differentiation-related expression of a major 64 K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells. J. Cell Biol. 103, 49–62. Shapiro, M.S., Friend, J., Thoft, R.A., 1981. Corneal re-epithelialization from the conjunctiva. Invest. Ophthalmol. Vis. Sci. 21, 135–142. Shimazaki, J., Yang, H.Y., Tsubota, K., 1997. Amniotic membrane transplantation for ocular surface reconstruction in patients with chemical and thermal burns. Ophthalmologe 104, 2068–2076. Shimazaki, J., Shinozaki, N., Tsubota, K., 1998. Transplantation of amniotic membrane and limbal autograft for patients with recurrent pterygium associated with symblepharon. Br. J. Ophthalmol. 82, 235–240. Tani, N., Kinoshita, S., Okamoto, Y., Kotani, M., Itagaki, H., Murakami, N., Sugiura, S., Usami, M., Kato, K., Kojima, H., Ohno, T., Saijo, K., Kato, M., Hayashi, M., Ohno, Y., 1999. Interlaboratory validation of the in vitro eye irritation tests for cosmetic ingredients (8) evaluation of cytotoxicity tests on SIRC cells. Toxicol. In Vitro 13, 175–187. Thoft, R.A., Friend, J., 1983. The X, Y, Z hypothesis of corneal epithelial maintenance. Invest. Ophthalmol. Vis. Sci. 24, 1442–1443. Tseng, S.C.G., 1996. Regulation and clinical implications of corneal epithelium stem cells. Mol. Biol. Rep. 23, 47–58. Tseng, S.C., Prabhasawat, P., Barton, K., Gray, T., Meller, D., 1998. Amniotic membrane transplantation with or without limbal allografts for corneal surface reconstruction in patients with limbal stem cell deficiency. Arch. Ophthalmol. 116, 431–441. Tsubota, K., Satake, Y., Ohyama, M., Toda, I., Takano, Y., Ono, M., Shinozaki, N., Shimazaki, J., 1996. Surgical reconstruction of the ocular surface in advanced ocular cicatricial pemphigoid and Stevens-Johnson syndrome. Am. J. Ophthalmol. 122, 38–52. Tsubota, K., Shimazaki, J., 1999. Surgical treatment of children blinded by StevensJohnson syndrome. Am. J. Ophthalmol. 128, 573–581. Uchiyama, T., Akiyama, J., Miyai, E., Sakamoto, K., Takino, Y., Ohnuma, M., Ohkosi, K., Okamoto, Y., Morito, Y., Kojima, H., Okumura, H., Sawamura, J., Ikeda, N., Sumida, Y., Chiba, K., Makino, I., Kawakami, K., Yamamoto, R., Torishima, H., Yanase, H., Miyajima, A., Sunouchi, M., Hayashi, M., Ohno, Y., 1999. Interlaboratory validation of the in vitro eye irritation tests for cosmetic ingredients (7) evaluation of cytotoxicity test by CornePack. Toxicol. In Vitro 13, 163–173. Xu, K.P., Li, X.F., Yu, F.S., 2000. Corneal organ culture model for assessing epithelial responses to surfactants. Toxicol. Sci. 58, 306–314.
Contents lists available at ScienceDirect
Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit
Review
In vitro eye irritancy test of lauryl derivatives using the reconstructed rabbit corneal epithelium model Sanae Matsuda a,*, Masayoshi Hisama a, Hiroharu Shibayama a, Norihiko Itou b, Masahiro Iwaki c a
HBC Science Research Center Co., Ltd., 3-13-9, Higashinakamoto, Higashinari-ku, Osaka 537-0021, Japan Department of Ophthalmology, Yokohama City University School of Medicine, 3-9, Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan c Department of Pharmacy, School of Pharmacy, Kinki University, 3-4-1, Kowakae, Higashi-Osaka, Osaka 577-8502, Japan b
a r t i c l e
i n f o
Article history: Received 10 November 2008 Accepted 3 February 2009 Available online 11 February 2009 Keywords: Cosmetic integrants Eye irritation Rabbit corneal epithelial cell Rabbit corneal epithelium model Lauryl derivatives
a b s t r a c t The rabbit corneal epithelium model (RCE model) was developed as a three-dimensional in vitro model to replace animal testing for the assessment of eye tolerance. In the model, a stratified culture of rabbit corneal epithelial cells is grown at the air–liquid interface on an amniotic membrane acting as a parabasal membrane. The alkaline exposure was restored each day in the presence of no irritants, although with the addition of SLS, which is a major irritant, the restoration of deficit was inhibited on the RCE model in a dose-dependent manner. The results of this test were comparable with those of the Draize test, and thus, this method using the RCE model may prove to be a useful and sensitive in vitro eye irritation test. The lauryl fatty chain derivatives, such as polyoxyethylene (9) lauryl ether (PLE), sodium polyoxyethylene (2) lauryl ether sulfate (SPLE), mono glyceryl laurate (MGL), and sodium N-lauroyl-Lglutaminate (SLG), which are widely used as surfactants for toiletry products and cosmetics, were evaluated for in vitro eye irritation potential using the RCE model. SLS, PLE, SPLE, MGL, and SLG inhibited 88.7%, 59.2%, 69.0%, 47.5%, and 15.7% of the restoration of deletion 24 h after treatment at a concentration of 0.05%. The IC50 (50% inhibitory concentration) values of SLS, PLE, SPLE, MGL, and SLG were 0.002%, 0.021%, 0.005%, 0.056%, and 0.448%, respectively. These results indicated that a functional group at the end of lauryl chain is an important factor for inhibiting the restoration of deletion using the RCE model. Ó 2009 Elsevier Ltd. All rights reserved.
Contents 1. 2.
3.
4.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Test materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Cell and culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Light microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Eye irritation tests with the RCE model. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Imaging technique. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Preparation of the rabbit corneal epithelial model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Eye irritation test with the RCE model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Eye irritant effect of lauryl derivatives on the RCE model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discussion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
* Corresponding author. Tel.: +81 6 6974 7111; fax: +81 6 6974 7181. E-mail address: [email protected] (S. Matsuda). 0887-2333/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2009.02.003
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1. Introduction The in vivo Draize eye test (Draize, 1959), which has become the international standard assay for acute eye irritation (OECD TG 405) is often criticized for both ethical and scientific reasons. The 7th amendment of the cosmetic directive will lead to the ban of animal testing for cosmetic ingredients in 2009. Thus, alternative strategies are necessary which allow the testing of chemicals with wide physicochemical properties under conditions similar to in vivo exposure. Many efforts have been made in order to find reliable and relevant predictive models such as the chorioallantoic membrane (CAM) methods (Hagino et al., 1999), cell-based cytotoxicity methods (Uchiyama et al., 1999; Tani et al., 1999), reconstituted tissue models (Ohuchi et al., 1999; Jones et al., 2001), and isolated organ methods (Xu et al., 2000). Success in fully replacing the Draize eye irritation test with in vitro methods has not yet occurred, in part due to a lack of understanding of the underlying physiological mechanisms of eye irritation. The corneal epithelium is a nonkeratinized, mucosal epithelial multilayer that covers the front surface of the cornea and is essential for proper vision. In healthy corneas, the epithelium is in a constant state of turnover: superficial epithelial cells are shed, whereas cells derived from stem cells in the limbus migrate in and replenish the epithelial cell population (Thoft and Friend, 1983). However, in severe ocular surface injuries, such as chemical burns and Stevens-Johnson syndrome, the corneal epithelial cells are sometimes totally destroyed. The damage often extends to the limbal area of the cornea, where the corneal epithelial stem cells are located (Schermer et al., 1986; Cotsarelis et al., 1989). In a severely injured cornea, in which the limbal and central epithelia are both absent, the neighboring conjunctival epithelial cells invade the corneal surface, and visual acuity is severely obstructed (Shapiro et al., 1981; Tseng, 1996). The corneal epithelium has a capacity for rapid regeneration that depends on the self-renewal of corneal stem cells. Corneal epithelium consists of corneal stem cells, transient amplifying cells, postmitotic cells, and terminally differentiated cells. Both stem cells and transient amplifying cells exhibit an ability to proliferate, whereas postmitotic cells have lost this ability. Stem cells are imbued with the potential for self-renewal, and can proliferate extensively. An increasingly popular surgical procedure for ocular surface reconstruction in individuals with severe chemical or thermal burns or serious ocular surface disorders, such as Stevens-Johnson syndrome, ocular cicatricial pemphigoid, and recurrent pterygium (Tsubota et al., 1996; Lee and Tseng, 1997; Prabhasawat et al., 1997; Shimazaki et al., 1997, 1998; Tseng et al., 1998; Tsubota and Shimazaki, 1999), involves the use of preserved human amniotic membrane as a biologic drape to dress the bare stroma after the removal of abnormal conjunctival tissue. This approach is based on the report by Kim and Tseng (1995), in which the amniotic membrane is thought to inhibit conjunctival overgrowth and provide a good substrate for normal epithelial migration. The results of ocular surface reconstruction with amniotic membrane are generally good. It appears that there is no immune rejection when cells derived from the contralateral limbus or from the cornea of the patient’s relative are cultured and transplanted. It is currently not established whether, after healing, corneal epithelial cells reconstituting the new epithelium were derived from the engrafted cells or from host stem cells that remained on the ocular surface. We developed the Rabbit Corneal Epithelial (RCE) model using cultured rabbit corneal epithelial cells and an amniotic membrane as a parabasal membrane to evaluate in vitro the eye irritation potential of chemicals including pharmaceuticals, cosmetics and their raw ingredients. The amniotic membrane has superior physical
properties of strength and elasticity as well as desirable biological potencies such as low antigenicity, histocompatibility and good adhesion ability to the cell, and so is a useful scaffold for the RCE model. Generally, cytotoxicity tests using cultured cells have the advantage of being simple and quick with, a low evaluation cost. However, when using cultured cells alone in media, the testing of water insoluble materials is sometimes difficult as the test substances may precipitate out in the media. In contrast to the conventional monolayer culture system suspended in media, the RCE model has a dry surface. Therefore, it is useful for both soluble and insoluble substances including various forms of cosmetic products. The aim of the development of RCE model was to evaluate a new three-dimensional epithelial model cultivated from rabbit corneal cells to replace animal testing in the assessment of eye tolerance. Lauryl fatty chain derivatives are one group of surfactants which includes anionic, cationic and nonionic surfactants with various functional groups. Surfactants containing lauryl fatty chain are widely used for toiletry products and cosmetics. Of those, the lauryl derivatives such as polyoxyethylene (9) lauryl ether (PLE), sodium polyoxyethylene (2) lauryl ether sulfate (SPLE), mono glyceryl laurate (MGL), and sodium N-lauroyl-L-glutaminate (SLG) are frequently used in shampoo as a foaming agent and in skin-care cosmetics as an emulsifier. Therefore, it is important to evaluate the eye safety of lauryl fatty chain derivatives and to elucidate the mechanism of action of eye irritation. Thus we focused on the mechanisms of eye injury, and examined new in vitro endpoints which would be more predictive of the biogenic response to chemical injury. This should help to develop new or improved in vitro methods that could proceed to formal validation. In this study, we have developed the RCE model using an amniotic membrane as a parabasal membrane, and used this model to evaluate the in vitro eye irritation potential of lauryl derivatives, PLE, SPLE, MGL, and SLG. 2. Materials and methods 2.1. Test materials Sodium lauryl sulfate (SLS) was obtained from Wako Pure Chemical Industries, Co., Ltd. (Tokyo, Japan). Polyoxyethylene (9) lauryl ether (PLE), sodium polyoxyethylene (2) lauryl ether sulfate (SPLE), mono glyceryl laurate (MGL), and sodium N-lauroyl-L-glutaminate (SLG), which were selected from the Japanese Standard of Cosmetic Ingredients or Japanese Pharmacopoeia, were obtained from a supplier of cosmetic ingredients. The culture medium M-stars A, supplemented hormonal epithelial medium (SHEM), and amniotic membrane were obtained from ArBlast Co., Ltd. (Kobe, Japan). 2.2. Cell and culture Rabbit corneal epithelial cells were provided by ArBlast Co., Ltd. (Kobe, Japan) and were co-cultured along with inactivated 3T3 fibroblasts, as described previously (Rheinwald and Green, 1975). For the three-dimensional cell culture, intact and denuded pig amniotic membranes were spread, epithelial basement membrane side up, on the bottom of polyester membrane culture inserts. After that, the culture inserts were placed in a 6-well plate containing the treated 3T3 fibroblasts, and the cultured rabbit corneal epithelial cells were inoculated with a density of 5.0 105 cells/well on 6-well culture inserts. The culture was submerged into the previously described M-stars A medium for 7 days, and then exposed to air by lowering the medium level (airlifting). After airlifting, the suitability of the rabbit corneal epithelial (RCE) model was con-
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firmed by examining the multilayer structure formation and the flatness of the most outer layer with light microscopy.
tion, the restoration rate for the deletion is calculated for each time.
2.3. Light microscopy
2.5. Imaging technique
The multilayer structure formation of RCE model was by evaluated by light microscopy of histological vertical section. The RCE models were fixed with 10% buffered neutral formaldehyde. After that, the samples were routinely processed by cryostat, and were stained with hematoxylin and eosin.
The RCE model was observed and images were recorded with a Stereomicroscope SZX 10 (Olympus, Tokyo, Japan). The images of RCE model were analyzed using image analysis, and the area of deletion was measured. Image analysis was performed using the public domain Java image processing program, Image J v3.91 software (http://rsb.info.nih.gov/ij), which was developed at the National Institute of Health (NIH) to assist in clinical and scientific image analyzes.
2.4. Eye irritation tests with the RCE model The eye irritation potency of test samples was estimated by the measurement of inhibition of restoration against alkaline exposure. A medical sponge, with a diameter of 8 mm and impregnated with 1 N sodium hydroxide solution, was laid on the RCE model to produce a deletion. The RCE model containing a deletion was cultured in a SHEM medium containing the test samples at 37 °C under 5% CO2 for 5 min. After incubation, the test substance was removed from the RCE model by washing with the assay medium. The RCE model was further cultured in a SHEM medium at 37 °C in 5% CO2 for 96 h after treatment. The area of deletion after alkaline exposure was measured as an index of inhibition of restoration against deletion induced by alkaline exposure using the imaging technique every 24 h for 96 h after treatment. By comparison of the area of deletion after incubation with that before sample irrita-
2.6. Statistical analysis The inhibitory effect on restoration against alkaline exposure was expressed as the mean ± standard error (SE) of three independent experiments, and subsequent inspection of means was evaluated using the Student’s t-test between two groups at a significance level of p < 0.05 and p < 0.01. 3. Results 3.1. Preparation of the rabbit corneal epithelial model Rabbit corneal epithelial cells have been used to develop a three-dimensional in vitro model of the rabbit corneal epithelium (RCE model). Rabbit corneal epithelial cells form a stratified culture when grown at the air–liquid interface on an amniotic membrane acting as a parabasal membrane in a cell culture insert with Mstars A medium. The RCE model was confirmed to be a multilayer of well-stratified corneal cells on the amniotic membrane using a histological vertical section (Fig. 1). 3.2. Eye irritation test with the RCE model
Fig. 1. Histological vertical section of the RCE Model.
The RCE model was exposed in the central part using a medical sponge impregnated with a 1 N sodium hydroxide solution to produce the deletion. Fig. 2 shows the time-course of macroscopic restoration of the deletion on the RCE model grown in the presence of sodium lauryl sulfate (SLS), which is a known irritant. Previously, SLS was evaluated with the Draize test, and showed the Draize score on a dose-dependent manner (Bagley et al., 1999), and so
Fig. 2. Macroscopic views of the RCE Model treated with 0.005% SLS.
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used with an eye irritation test of RCE model as a standard irritant. When there was no irritant added, the restoration process had started, and it was almost complete after 96 h on the RCE model. Restoration of the deletion was clearly inhibited by the treatment of 0.005% SLS. When the deletion was observed under an inverted microscope, there appeared to be a lower degree of restoration of the deletion in the RCE model irritated by SLS than in the non-irritated model. Although, using SLS as the irritant, the restoration of deletion was generally inhibited in a dose- and time-dependent manner (Fig. 3). However, at the high concentration of 0.05% tested, SLS did not inhibit the deletion in a time-dependent manner, and instead still inhibited 86.4% of the restoration of the deletion 96 h later.
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Time (hours) Fig. 3. Time-course of the restoration of alkaline deletion treatment of SLS on the RCE model. RCE model containing an alkaline deletion was treated with SLS at concentrations of 0.001% (j), 0.005% (d), 0.01% (), and 0.05% (N), and cultured in a SHEM medium at 37 °C in 5% CO2 for 96 h. Control (e) was not treated with SLS. The area of deletion was measured as an index of inhibition of restoration against deletion induced by alkaline exposure using the imaging technique every 24 h. Data are expressed as a percentage of control. Bars represent mean ± SE of three independent experiments. Significantly different from the non-treated control group at p < 0.05 () and p < 0.01 ().
3.3. Eye irritant effect of lauryl derivatives on the RCE model The inhibitory effects of the lauryl derivatives, polyoxyethylene (9) lauryl ether (PLE), sodium polyoxyethylene (2) lauryl ether sulfate (SPLE), mono glyceryl laurate (MGL), and sodium N-lauroyl-Lglutaminate (SLG) were determined by the RCE model with an alkaline deletion. Fig. 4 shows macroscopic restoration of the deletion on the RCE model grown in the presence of the lauryl derivatives PLE, SPLE, MGL, and SLG at concentrations of 0.01% and 0.05%. When the RCE model was used without added irritant, the deletion was restored on the RCE model 24 h after treatment. Restoration of the deletion was clearly inhibited by the treatment of PLE, SPLE, MGL, and SLG at a concentration of 0.05%. When the deletion was observed under an inverted microscope, there appeared to be some differences in the restoration of the deletion in the RCE model depending on the various irritants used. In case of treatment with SLG, the deletion was restored more than in the presence of the other lauryl derivatives. All lauryl derivatives inhibited the restoration of the deletion in a dose- and time-dependent manner from 24 h to 96 h after treatment. The inhibition of the restoration of deletion decreased slightly from 24 h to 96 h after the treatment (Fig. 5). SLS, PLE, SPLE, MGL, and SLG inhibited 88.7%, 59.2%, 69.0%, 47.5%, and 15.7% of the restoration of deletion 24 h after treatment at a concentration of 0.050%, and the IC50 (50% inhibitory concentration) values of SLS, PLE, SPLE, MGL, and SLG were 0.002%, 0.021%, 0.005%, 0.056%, and 0.448%, respectively (Fig. 6A). SLS, PLE, SPLE, MGL, and SLG inhibited 86.4%, 42.0%, 49.0%, 34.5%, and 19.5% of the restoration of deletion 96 h after treatment at a concentration of 0.05%, and the IC50 values of SLS, PLE, SPLE, MGL, and SLG were 0.007%, 0.063%, 0.052%, 0.069%, and 0.340%, respectively (Fig. 6B). This result indicated that SLS has the highest level of eye irritation of the lauryl derivatives in the RCE model at both 24 h and 96 h after treatment. According to the IC50 value, the inhibition by SLS was 10 times more potent that the other lauryl derivatives. 4. Discussion The rabbit corneal epithelium (RCE) model was developed as a three-dimensional in vitro model by using a stratified culture of rabbit corneal epithelial cells grown at the air–liquid interface on an amniotic membrane as a parabasal membrane. This allows an
Fig. 4. Microscopic views of the RCE model treated with lauryl derivatives (PLE, SPLE, MGL, and SLG).
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Time (hours) Fig. 5. Time-course of the restoration of alkaline deletion with 0.05% lauryl derivatives treatment on the RCE model. RCE model containing an alkaline deletion was treated with SLS (e), PLE (h), SPLE (s), MGL (), and SLG (4) at a concentration of 0.05%, and cultured in a SHEM medium at 37 °C in 5% CO2 for 96 h. The area of deletion was measured as an index of inhibition of restoration against deletion induced by alkaline exposure using the imaging technique every 24 h. Data are expressed as a percentage of control. Bars represent mean ± SE of three independent experiments. Significantly different from the non-treated control group at p < 0.05 () and p < 0.01 ().
Inhibitory effect of restoration rate (% of control)
artificial corneal epithelium (reconstituted rabbit corneal epithelium) to be prepared which exhibits barrier characteristics and paracellular permeability similar to those of native rabbit cornea (Fig. 1). The deletion with alkaline exposure was restored each day in the presence of no irritants, although with the addition of sodium lauryl sulfate (SLS) as a positive irritant, the restoration of deletion was inhibited on the RCE model in a dose-dependent manner (Figs. 2 and 3). SLS, which is a major irritant, also increased the Draize score in a dose-dependent manner (Bagley et al., 1999).
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This result indicated that the eye irritation potency of test samples may be estimated by the measurement of inhibition of restoration against alkaline exposure on the RCE model. When the time–activity relationships were examined, it became evident that the different inhibitory potencies between the lauryl derivatives at a concentration of 0.05% 24 h after irritation are greater than those at subsequent hours (Fig. 5). These results indicated that the inhibitory effect is most sensitive 24 h after treatment and so research into the eye irritation of test samples should be analyzed at this time point on the RCE model. As for the structure-activity relationships, the lauryl derivatives had distinctly different inhibitory potencies against the restoration of deletion, according to their substitution patterns. The inhibitory effects of the restoration of deletion decreased in the order SLS > SPLE > PLE > MGL > SLG (Figs. 4 and 6). The difference in structure between the lauryl derivatives, SLS, SPLE, PLE, MGL, and SLG is a functional group at the end of lauryl fatty chain. These results indicated that the functional group at the end of lauryl fatty chain is an important factor for inhibiting the restoration of deletion in the order sodium sulfate > sodium polyoxyethylene ether sulfate > polyoxyethylene ether > glycerate > sodium glutaminate. SLS inhibited the restoration of deletion greater than all the other lauryl derivatives tested on the eye irritancy test with this RCE model. These results were comparable with those of the Draize test (Ohno et al., 1999), and therefore, the RCE model may be a useful in vitro eye irritation test. In addition, the small difference in inhibitory effects between the lauryl derivatives, which cannot be confirmed with the Draize test, was remarkably confirmed using the RCE model. Thus, the RCE model may be a sensitive one. In conclusion, the rabbit corneal epithelial (RCE) model was prepared by culture of rabbit corneal cells on amniotic membrane in a cell culture insert. The irritancy effects of lauryl derivatives were determined by measurement of the inhibitory effect of the restoration of an alkaline deletion on the RCE model as a guideline. SLS inhibited the restoration of deletion greater than other lauryl derivatives, and was suggested to be the strongest eye irritant of the lauryl derivatives tested. These results indicated that the functional group at the end of lauryl fatty chain is an important factor
Inhibitory effect of restoration rate (% of control)
Inhibitory effect of restoration rate (% of control)
S. Matsuda et al. / Toxicology in Vitro 23 (2009) 555–560
100
B
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0.005
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0.05
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Dose (%) SLS
PLE
SPLE
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Fig. 6. Inhibitory effects of the restoration of alkaline deletion with lauryl derivatives treatment 24 h and 96 h on the RCE model. RCE model containing an alkaline deletion was treated with SLS, PLE, SPLE, MGL, and SLG at a concentration of 0.005%, 0.001%, 0.05%, 0.1%, and 0.5%, and cultured in a SHEM medium at 37 °C in 5% CO2 for 24 h and 96 h. The area of deletion was measured as an index of inhibition of restoration against deletion induced by alkaline exposure using the imaging technique 24 h (A) and 96 h (B) after treatment. Data are expressed as a percentage of control. Bars represent mean ± SE of three independent experiments. Significantly different from the non-treated control group at p < 0.05 () and p < 0.01 ().
560
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for the degree of eye irritation. The results of this test were comparable with those of the Draize test, and thus, this modification of the RCE model may prove to be a useful and sensitive in vitro eye irritation test. However, the eye irritation test with RCE model was preliminary evaluated by with only a few compounds in a very limited chemical space, and so may not exhibit their expected effects in vivo if they are adversely affected by factor such as absorption, bio-disposition and metabolism after they are incorporated into the human eye. Further studies by many sorts of chemicals with wide physicochemical properties the eye irritation test with RCE model and Comparison between this test, and in vivo and other in vitro eye irritation tests, are needed to determine the efficacy of this test for in vitro model supposed to replace the Draize eye irritation test.
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