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Prediction of ocular irritancy of prototype shampoo formulations by the isolated rabbit eye (IRE) test and bovine corneal opacity and permeability (BCOP) assay
Toxicology in Vitro 15 (2001) 95±103
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Prediction of ocular irritancy of prototype shampoo formulations by the isolated rabbit eye (IRE) test and bovine corneal opacity and permeability (BCOP) assay K.J. Cooper a,*, L.K. Earl a, J. Harbell b, H. Raabe b a
Safety and Environment Assurance Centre, Toxicology Unit, Unilever Research, Colworth House, Sharnbrook, Bedfordshire MK44 1LQ, UK b Institute for In Vitro Sciences, Inc., Gaithersburg, Maryland, USA Accepted 11 October 2000
Abstract The isolated rabbit eye (IRE) test and bovine corneal opacity and permeability (BCOP) assay were evaluated for their ability to predict the eye irritation potential of a range of hair shampoo formulations, some containing a novel non-surfactant ingredient known to be an ocular irritant. The additional endpoints of corneal swelling and histological examination were incorporated into the standard BCOP protocol. Historic Draize data were available for several of the formulations and served as a reference. The standard BCOP assay (without histology) failed to distinguish between shampoos of low and high irritant potential, when exposure times of 10 and 60 min were employed (for undiluted and 10% dilution of the shampoos, respectively) and the in vitro score classi®ed the majority of formulations as mild. The incorporation of the histological endpoint to the BCOP protocol allowed discrimination between formulations of diering irritancy, and should be included to augment the standard BCOP protocol. Corneal swelling values did not, however, correlate with the irritant potential of the shampoos tested. The IRE which includes the endpoints of corneal swelling and histopathological scoring produced classi®cations of irritancy that were fairly consistent with in vivo data and distinguished between the high and low irritant potential shampoos. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Ocular irritation; Bovine corneal opacity and permeability assay; Isolated rabbit eye test; Shampoo formulations
1. Introduction In an eort to identify a replacement for the in vivo eye test (Draize et al., 1944), a number of alternative in vitro methods have been devised (Frazier et al., 1987). Many of these methods are based on cytotoxic endpoints; however, a model comprising a multicellular penetration barrier is more likely to have a closer anity with both in vivo results and ultimately with accidental human exposure. The isolated rabbit eye (IRE) test was ®rst proposed by Burton et al. (1981), as a means of screening for severe irritants without using live animals. A good correlation was obtained with in vivo results for a range of Abbreviations: IRE, isolated rabbit eye; BCOP, bovine corneal opacity and permeability; HBSS, Hanks' balanced salt solution; MEM, minimum essential medium; * Corresponding author. Tel.: +44-1234-222018; fax: +44-1234222122. E-mail address: [email protected] (K.J. Cooper).
chemicals with dierent in vivo irritancies. In addition, the method is capable of distinguishing between mild and moderate eye irritants, such as `baby' and normal `adult' shampoos, and is used in-house as part of a tiered safety evaluation strategy for materials and formulations of interest to Unilever, in order to obviate the need for in vivo testing. A collaborative study on the evaluation of alternative methods to the eye irritation test, sponsored by the Commission of the European Communities (1991) found the IRE to be an accurate predictor of ocular irritancy, based on in vivo classi®cations for 21 chemicals tested. The IRE confers the advantage that animals are not bred exclusively for the purpose, and have previously been used in dermal irritation studies; however, the use of laboratory animals in any guise remains an emotive issue. The BCOP assay was introduced by Gautheron et al. (1992) as a method for screening process intermediates for worker safety. In a multinational interlaboratory study (Gautheron et al., 1994) using Draize data as the reference, BCOP data correctly predicted whether a
0887-2333/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0887-2333(00)00060-6
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compound would be irritating or non-irritating for 44 of the 52 compounds. The method was also found to be easily transferable between participating laboratories, and confers the ethical advantage of using abattoir waste, as well as the practical advantage of the high throughput of slaughterhouse animals. In the European Community/Home Oce (EC/HO) study (Balls et al., 1995), for a group of 59 chemicals, concordance with in vivo data was, however, relatively poor for both the IRE and the BCOP. Several of the chemicals for which the ocular irritation was signi®cantly under-predicted by the opacity and permeability endpoints in the original BCOP studies, were recently re-evaluated using histology as an additional endpoint. Appreciable pathologic changes were observed in both the epithelium and stroma, showing that the tissue was damaged by the exposure to the chemicals. However, these changes were not re¯ected in proportionally increased opacity or permeability. These data suggest that histological examination can provide important supplemental data to the opacity and permeability endpoints in assessing the degree and depth of injury (Curren et al., 1999). Both methods use the metric of corneal opacity, which in relation to accidental human exposure to an irritant is arguably the most important parameter, since it provides information on how visual acuity may be impaired following accidental exposure. In this investigation, seven shampoos were tested in the IRE and the BCOP. For the BCOP assay the additional endpoints of corneal swelling (assessed by water uptake of treated corneas) and histological evaluation were included. The shampoo formulations diered in irritant potential, ranging from a fairly mild, leading brand baby shampoo to a relatively aggressive adult shampoo formulation. 2. Materials and methods 2.1. Test materials The seven shampoo formulations were assigned the arbitrary codes A through G (Table 1); based on historic data the major irritant categories were represented by these materials. In vivo (modi®ed Draize) data were available for shampoos A through D, shampoos E and F were standard IRE reference controls which, based on original in vivo (modi®ed Draize) data and market history, were considered to be moderate ocular irritants; shampoo G was a branded baby shampoo known from market history to be mild. Shampoo A consisted of a base only, common also to shampoos B±D, but with increasing levels of an oxidising agent, ingredient `X', which is non-surfactant and known to be an ocular irritant, with B containing the lowest concentration and D the highest. It should be noted that the same surfactant system was common to shampoos A through D.
Table 1 Test shampoo formulations and in vivo scores (MAS) Code
Formulation
In vivo score (MAS)
Classi®cationa
A B C D E F G
Base Base+1.5% ingredient X Base+3.0% ingredient X Base+6.0% ingredient X Reference control Reference control Baby shampoo
14.3 30.0 59.0 77.0 No data No data No data
Mildb Moderateb Extremeb Extremeb Moderate (predicted)b,c Moderate (predicted)b,c Mild (predicted)c
a b c
Classi®cations derived from: In vivo (modi®ed Draize) data Market history.
Shampoos E±G did not contain ingredient `X' and were based on standard shampoo surfactant systems. 2.2. BCOP assay 2.2.1. Preparation of corneas and treatment The BCOP assay was performed according to modi®cations of the procedures described by Gautheron et al. (1992), the additional endpoints of corneal swelling and histological evaluation were also included. Bovine eyes were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised and then placed in Hanks' balanced salt solution (HBSS), supplemented with penicillin/streptomycin, and transported to the laboratory on ice packs. The corneas were grossly examined for damage and those exhibiting defects were discarded. The connective tissue surrounding the eyeball was carefully removed and the cornea excised such that a 2±3 mm rim of sclera was present around the cornea. The isolated corneas were then stored in a petri dish containing HBSS until they were mounted in a corneal holder. Each cornea was mounted in a holder with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was then positioned on top of the cornea and the screws were tightened. Starting with the posterior compartment, the two compartments of the corneal holder were then ®lled with Eagle's minimum essential medium (MEM) without phenol red, with 1% fetal bovine serum (complete MEM). The corneal holders were incubated at 321 C for a minimum of 1 h. Following incubation, the corneal holders were removed from the incubator and the medium from both compartments was replaced with fresh medium. The opacity was determined for each cornea using a Spectro Designs OP-KIT opacitometer. Three corneas whose opacity readings were approximately the median of all the corneas were selected as the negative control corneas. The medium was then removed from the anterior part of the holder and replaced with an aliquot of 750 ml
K.J. Cooper et al. / Toxicology in Vitro 15 (2001) 95±103
of test material, dilution of test material or positive control. Five corneas were used per treatment. The concentrations and exposure times employed were derived from the work of Gautheron et al. (1992), in which surfactants were tested at up to 10% (w/v) for 30 min exposure. When selecting the test regimen, consideration was also given to the fact that the degree of corneal injury elicited by ingredient `X' could not be predicted. Consequently, the test materials were tested undiluted, and at a dilution of 10% (w/v) prepared in complete MEM, for incubation times of 10 and 60 min, respectively. In a separate, time course study using three corneas per treatment, the baby shampoo, G and shampoo D (highest level of active), were tested neat for 10, 30 and 60 min and also at 10% solutions for 10, 30, 60 and 120 min incubation. All treated corneas were incubated at 321 C. The concurrent positive control, ethanol, was tested for 10 min in two corneas. When the test material was removed, the epithelial side of the cornea was washed at least three times with complete MEM containing phenol red to ensure total removal of the test material, the corneas were then given a ®nal rinse with complete MEM (without phenol red). The anterior compartment was re®lled with complete MEM and the opacity determined. The corneas were then incubated for a total of approximately 2 h at 321 C. The 2 h included the actual exposure time and postexposure incubation. Thus, the 120-min exposure group did not receive a post-rinsing incubation and only one post-exposure opacity reading was taken. At the completion of the incubation period, a second measure of opacity (except the 120-min exposure group) was performed. The values obtained at this second opacity measurement were used in calculating the corneal opacity. 2.2.2. Permeability measurements After the ®nal opacity measurement was performed, the medium was removed from both chambers of the holder. The posterior compartment was re®lled with fresh complete MEM. 1 ml of a 4 mg/ml ¯uorescein solution was added to the anterior compartment. The corneas were then incubated in a horizontal position, anterior side up for approximately 90 min at 321 C. An aliquot of medium was removed from the posterior chamber and placed into tubes. A volume of 360 ml were placed into designated wells of a 96-well plate. The optical density at 490 nm (OD490) was determined using a Molecular Devices Vmax kinetic microplater reader. If the OD490 of a test or control sample was greater than 2000, a 1 in 5 dilution of the sample in MEM was made. Samples of the 1 in 5 dilution were transferred to speci®ed wells on the 96-well plate. The plate was reread and the ®nal reading recorded. 2.2.3. Corneal swelling measurements Once the opacity measurements (or the permeability measurements for the time course study) were completed
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the last cornea from each treatment group was removed from the chamber and three 8-mm punches were taken from around the edge of the cornea. The punches were taken towards the corneal centre and transferred onto a mesh screen in a petri dish above a moistened paper towel (to prevent corneal dehydration). The punches were then weighed. Treated tissue weights were compared to control tissue weights to assess the uptake of water (swelling) by the corneas. 2.2.4. Histology After determination of ¯uorescein permeability, the corneas were ®xed for 12 h in Davidson's Fixative. Sections were made from the paran blocks, from which haematoxylin and eosin stained slides were made. Corneal sections were examined for the presence of changes in the epithelial, stromal and endothelial components of the tissue. 2.2.5. Presentation of data 2.2.5.1. Opacity measurement. The change in opacity for each cornea was calculated by subtracting the pretreatment opacity readings from the ®nal opacity readings. The corrected opacity value of each cornea was calculated by subtracting the average change in opacity of the negative control corneas from that of each treated cornea. The mean opacity value of each treatment group was calculated by averaging the mean corrected opacity values of the treated corneas for each treatment condition. 2.2.5.2. Permeability measurement. The corrected OD490 was calculated by subtracting the mean OD490 of the negative control corneas from the OD490 value of each treated cornea. The mean OD490 value of each treatment group was calculated by averaging the corrected OD490 values of the treated corneas for that treatment condition. The following formula was used to determine the in vitro score: In vitro Score Mean Opacity Value 15 Mean OD490 Value 2.2.5.3. Relative corneal swelling. Calculated by comparing the combined weights of the three punches from the test article treated cornea at each exposure time with the combined weights of the three punches from a negative (solvent) control cornea. Data were expressed as percent increase in weight of the treated corneas to the negative control. 2.2.5.4. Irritancy classification from opacity and permeability data. The following system (Table 2) was established by Sina et al. (1995), based on 10 min exposure data from a wide range of liquid pharmaceutical intermediates.
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Table 2 BCOP standard protocol irritancy classi®cation scheme
Table 3 IRE classi®cation scheme
In vitro score
Irritancy classi®cation
Classi®cationa
Opacity (score)
0±25 25.1±55 55.1 and above
Mild Moderate Severe
Corneal swelling (%)
Epithelial cell layers eroded
Very slight Slight Moderate Severe
None (0) Slight opacity (1±2) Slight±moderate (2±3) Moderate±severe (3±4)
11% 12±25 26±35 >35
0±2 3±4 5±6 7±8
2.3. Isolated rabbit eye (IRE) test The isolated rabbit eye test was ®rst proposed by Burton et al. (1981). Rabbits are not bred exclusively for this purpose, eyes are enucleated from animals which have already been used for other purposes at a nearby laboratory and then transported to the testing facility with minimum delay. On arrival, eyes were placed in clamps and mounted in a maintenance chamber, which provides optimum conditions; water sleeving maintains the temperature at 31 C and a saline drip irrigates the anterior corneal surface. The eyes were left in the maintenance chamber for a short period to stabilise. Observations of the eyes during this period were made and any damaged eyes discarded. Three eyes were used per test substance and a negative control (untreated) eye included. The test materials were tested undiluted and at 10% (w/v) solutions in distilled water, 20 ml of test material were applied to the cornea every 10 s up to 1 min before washing. The eects of treatment were assessed at regular intervals throughout the following 4 h. Assessments of corneal opacity were made by trained assessors and graded according to the scheme of Draize et al. (1944). Corneal thickness was measured using an ultrasonic pachometer, (Teknar1 Ophthasonic1 pachometer, Mentor O&O Inc., MA, USA) and the extent to which ¯uorescein penetrates the cornea assessed visually using a Zeiss slit lamp. Percentage corneal swelling throughout the 4 h was determined using the pre-treatment thickness value. Fluorescein penetration is expressed using a graded scoring system. After 4 h of incubation, the corneas were excised and ®xed for histological assessment of epithelial and stromal responses. The numbers of layers of epithelial cells which had eroded and evidence of other histopathological changes were recorded. 2.3.1. Presentation of data 2.3.1.1. Opacity, corneal swelling and histology. From the data generated, an overall classi®cation is made on the irritancy potential (Table 3). 2.4. In vivo rabbit eye test It should be noted that data are historical; no animal tests were undertaken as part of this study. The procedure followed was a modi®cation of that described by
a The classi®cation is generally based on the weight of evidence from all three parameters, with any one triggering the inclusion of the higher classi®cation, e.g. a substance causing slight opacity, 26% corneal swelling with loss of three to six epithelial cell layers will be classi®ed as borderline slight to moderate.
Draize et al. (1944). 100 ml of the undiluted test material were instilled onto the cornea of one eye of three New Zealand White rabbits. Eyes were held closed for a period of 2 s following instillation and rinsed after a 15-s contact period with approximately 40 ml of tap water. The contralateral eye remained untreated and served as a control. The eyes were examined at 24, 48, 72 and 168 h after treatment; if there was no evidence of irritation at 72 h, the study was ended. Additional examinations were performed up to a maximum of 21 days, if persistent corneal involvement or other ocular irritation was present. Sodium ¯uorescein and UV light provided by a Spectroline, model Q-12, Long Wave UV-365 nm, 10 Magni®er were employed to reveal possible corneal injury commencing with the 24-h observation. Ocular lesions were scored according to Draize (1965) and maximum group mean scores calculated, from which a classi®cation was assigned (Kay and Callandra, 1962). 3. Results 3.1. BCOP When tested undiluted for 10, 30 and 60 min incubation, and also at 10% dilutions for 10, 30, 60 and 120 min incubation, both shampoo G the baby shampoo and shampoo D the aggressive adult shampoo showed exposure time-dependent responses in the ¯uorescein permeability endpoint, but showed minimal responses in the opacity endpoint (Table 4). Based on the time course study results, the permeability endpoint provided a reasonable resolution between the two undiluted test articles tested for 10 min, but showed even greater resolution between the two 10% dilutions of test articles tested for 60 min. During the treatment of the undiluted shampoo D, a foaming reaction was observed on several of the treated corneas. Extreme opacity values of 832 and 441 were observed in two corneas (one each from the 10 and 30 min exposures). It appears that the reaction may have penetrated the cornea and have introduced
K.J. Cooper et al. / Toxicology in Vitro 15 (2001) 95±103 Table 4 BCOP time course study summary Treatment Exposure Mean opacity Mean Mean pH of time measurement permeability in vitro dosing (min) measurement score solution 100% G
10 30 60
0.0 0.3 2.3
0.169 0.434 0.901
2.5 6.2 15.8
7.0
100% D
10 30 60
8.7a 9.2b 8.3
0.687a 0.897b 3.495
19.0 22.6 60.8
4.4
Ethanol
10
30.7
0.888
44.0
NA
10% G
10 30 60 120
0.7 1.0 1.7 4.0
0.009 0.126 0.248 0.512
0.8 2.9 5.4 11.7
Ethanol
10
35.3
1.084
51.6
10 30 60 120
0.0 1.7 5.3 4.7
0.280 0.453 0.950 2.862
4.2 8.5 19.6 47.6
10
30.3
1.613
54.5
D (10%)
Ethanol
7.5
NA 7.9
NA
a
The opacity and permeability scores for one of the three corneas treated in this group were 832 and 0.008, respectively. The results of this cornea were not included in the group mean opacity, permeability and in vitro score calculations. b The opacity and permeability scores for one of the three corneas treated in this group were 441 and 0.022, respectively. The results of this cornea were not included in the group mean opacity, permeability and in vitro score calculations.
some of the white foamy material within the cornea. In addition, uncharacteristically low permeability scores for these corneas were also observed (0.008 and 0.022 for the two corneas, respectively), suggesting that the introduction of the foam generating material within the cornea created a barrier to ¯uorescein passage in the permeability endpoint determination. The results of these corneas were not included in the group mean opacity, permeability, and in vitro score calculations presented in Table 5. Using the combined endpoints of opacity and permeability for the 10% dilution of shampoos A through D, the standard BCOP protocol failed to correctly predict the rank order of irritancy (based on in vivo classi®cations), and appeared to inversely rank irritancy based on the ¯uorescein permeability endpoint (Table 5). However, BCOP predicted the 10% dilutions of the reference controls E and F to be moderate, and the 10% dilution of G, the baby shampoo to be mild and, based on in vivo data/market history, these predictions were accurate. When tested undiluted, all seven shampoos were predicted to be mild and there was little resolution between them based on permeability data. However, the
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rank order of opacity values was in line with the in vivo predictions (Table 5). Addition of the endpoint of corneal swelling to the standard BCOP protocol generated values that failed to correlate with the predicted irritancy of the shampoos. There was, however, a high degree of variability in the individual punch weights, making swelling data dicult to interpret (variability data not presented). For both the 10% dilutions and the undiluted shampoos, the extent of histopathological change observed in the bovine corneas appeared to increase with the predicted ocular irritancy of the formulations (Table 5). Very little disruption was apparent in corneas treated with the mildest shampoo, G with only some loosening of the squamous epithelium and slight swelling in the upper wing cells; basal cells, stroma and endothelium were similar to the controls. In contrast, extensive necrosis and separation from the wing cells were observed in corneas treated with the most aggressive shampoos C and D. In addition, shampoo D, when tested both undiluted and at 10%, showed a tendency towards eliciting pyknosis in the stromal nuclei. 3.2. IRE At 10% dilution, the IRE failed to distinguish clearly between the base shampoo A and the highest level of ingredient X, shampoo D, grading both as moderate (Table 6). The baby shampoo, G, was graded as very slight to slight. When tested undiluted, the IRE classi®cation of moderate and severe for shampoos B and D, respectively, were in accordance with in vivo classi®cations (no IRE test data were available for shampoo C). The IRE corneal swelling values, derived from the increase in corneal swelling from the pre-treatment value correlated well with the irritant potential of the shampoos. 4. Discussion Within Unilever, the IRE has been used successfully for almost two decades to provide safety evaluation data on a range of ingredients and formulations pertinent to its product portfolio. Over this period there has never been a safety incident in the marketplace that has caused toxicologists to doubt the data generated from the IRE. The BCOP was evaluated as a possible alternative to the IRE, since several relevant endpoints are common to both techniques and the BCOP oers a departure from using tissue from a laboratory animal, while retaining the advantages associated with using the target organ. The aim of this investigation was to evaluate the ability of the IRE and the BCOP to predict the ocular irritancy potential of seven hair shampoos. Existing in vivo data were available for four of the shampoos; two were reference controls used routinely in
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Table 5 BCOP ®xed exposure results Mean corneal Mean In vitro Histological evaluation Treatment Exposure Mean Mean time (min) opacity permeability swelling (%) in vitro classi®cation score (OD490) 10% A
60
10.9
1.497
10
33.3
Moderate
Squamous epithelium lost, marked damage to wing cells, variable damage to basal cell layers, slight stromal swelling near Bowman's membrane.
10% B
60
2.7
0.899
5
16.1
Mild
Necrosis in squamous/upper wing layers, included basal cells in some areas. Complete separation of epithelium from basal lamina in some areas.
10% C
60
5.0
0.793
13
16.9
Mild
Squamous/wing cell layers showed necrosis in some areas. Variable damage in basal cells, but all showed weakened attachments to basal lamina.
10% D
60
5.4
0.526
16
13.3
Mild
Squamous/upper wing cell layers necrotic, lower wing/basal cell layers generally necrotic. Large areas of separation between basal cells and basal lamina. A few pyknotic nuclei in stroma.
10% E
60
7.3
2.866
12
50.3
Moderate
Large patches of epithelium lost, upper stromal cells showed vacuolated nuclei.
10% F
60
6.4
1.474
5
28.5
Moderate
Squamous epithelium and most of wing cell layers lost. Basal cells disrupted in some areas.
10% G
60
1.5
0.128
3
0.4
Mild
Some loosening of squamous epithelium and slight swelling in upper wing cells.
100% A
10
2.9
0.617
5
12.1
Mild
Squamous epithelium separated/lost. Separation between margins of basal cells. A few degenerated nuclei in stromal cells near Bowman's membrane.
100% B
10
3.3
0.450
3
10.0
Mild
Squamous epithelium necrotic and separated from wing cells, upper wing cell layers also necrotic. Basal cell attachment to the basal lamina weakened.
100% C
10
4.1
0.511
6
11.7
Mild
Squamous epithelium necrotic and separated from wing cells. Upper wing cells also necrotic. Basal cell attachment to the basal cell lamina weakened.
100% D
10
9.4
0.527
6
17.3
Mild
Squamous epithelium and upper wing cell layers necrotic. Necrosis variable in lower wing/basal cells. Upper stromal cell nuclei pyknotic.
100% E
10
2.0
0.576
11
10.6
Mild
Most squamous epithelium lost, necrosis of some wing cells and slight degeneration of the basal cell nuclei in some areas.
100% F
10
2.8
0.618
8
12.1
Mild
Squamous epithelium lost in many areas, some loss of upper wing cell layer and necrosis in remaining wing cells.
100% G
10
2.3
0.127
3
0.4
Mild
Some loosening of squamous epithelium and slight swelling in upper wing cells.
the IRE and the other a leading brand baby shampoo known from market use to be a very mild eye irritant. The BCOP and IRE both oer multiple endpoint alternatives to in vivo eye irritation testing. They include the measurement of the most important aspect of ocular irritation, corneal opacity, which in terms of human exposure is the most relevant component. The IRE uses a subjective graded scoring system for the assessment of both opacity and ¯uorescein permeability, while the BCOP uses a continuous scale, measured by instrumentation which, by eliminating human variation in interpretation, allows the method to be easily trans-
ferable between laboratories. In the IRE, visual assessment does, however, enable the description of discrete areas of the cornea. The exposure to the test material is short term for both methods and the practical work can be performed within a day. Exposure to the test material in the BCOP is to the entire corneal surface, which ensures even exposure across the tissue. Solids and liquid test materials can be tested in the IRE; however, more liquid materials sometimes run o the cornea, resulting in inadequate exposure. In the BCOP, solids are tested as solutions or suspensions, but compounds that ¯oat
K.J. Cooper et al. / Toxicology in Vitro 15 (2001) 95±103
cannot be delivered eectively to the tissue (Sina and Gautheron, 1998). Both techniques are fairly time consuming with low throughput. Only a small number of shampoos were tested in this study; however, based on existing in vivo data and market use, the major irritant categories were represented. At 10% dilutions, both standard methods failed to detect a dose±response relationship for shampoos A through D; however, when tested undiluted, IRE showed an apparent dose±response relationship from A through D, such that corneal swelling values were in accordance with in vivo data (Table 6). When tested undiluted, the standard BCOP method classi®ed all seven shampoos as mild (Table 5). Based on market use, the leading brand baby shampoo, G, was correctly classi®ed by both the IRE (10% dilution) and BCOP (undiluted and 10% dilution) (Table 7). It is worth noting that shampoos A through D shared the same base and surfactant system, and while the surfactant system may have exacerbated the eect of ingredient X by increasing its penetration into the corneal stroma, the dierences in the in vivo irritancy were not attributable to the surfactant system per se, but to the increasing concentration of the ocular irritant, ingredient X. Surfactants used in personal care products, such as shampoos, are thought to induce corneal opacity in vivo through damage to the epithelial and stromal elements, which leads to stromal swelling and in¯ammatory in®ltration (Maurer et al, 1997). This secondary in¯ammatory in®ltration process is not available in the isolated cornea or enucleated eye. Corneal opacity in the ex vivo cornea is postulated to arise from stromal swelling (changing the spacing of the collagen ®bers) and/or protein coagulation (Harbell et al., 1999). Numerous studies have shown that the milder surfactants (e.g. anionics and nonionics) induce little opacity in the ex vivo corneas even when extensive damage is done to the epithelium (Curren and Harbell, 1998). Other assay endpoints, such as corneal swelling and epithelial cell
101
loss (IRE) or ¯uorescein permeability (BCOP), are used to evaluate the irritation potential of these materials. The data from both test systems in this report are consistent with this pattern of responses. Neither corneal opacity nor stromal swelling was appreciably increased in the bovine corneas. Marked corneal swelling was measured in several of the IRE treatment groups (Table 4) without a proportional increase in opacity. These data suggest that the link between corneal swelling and opacity may be more complex than previously expected and show the importance of considering all endpoints in the ®nal evaluation of irritancy potential. The results show the standard BCOP protocol to be less sensitive than the IRE. This may be attributed at least in part to the robustness of the bovine cornea, which consists of 10±14 layers of epithelia, whereas the rabbit cornea has ®ve to seven, giving a closer model of the human cornea which on average has ®ve layers (Ara, 1997). The BCOP and the IRE were both designed for the testing of severe eye irritants, and certainly from this study the robustness of the bovine cornea was re¯ected in the inability of the standard method to distinguish between materials of dierent irritant potential. However, the results from the time course study show that the sensitivity of the BCOP assay can be increased by extending the incubation time (Table 4). The foaming reaction elicited by undiluted shampoo D was most likely caused by the non-surfactant ingredient X, and illustrates the need for a more comprehensive knowledge of the mode of action of dierent chemical classes and how chemicals within a mixture interact. Furthermore, while the Sina et al. (1995) classi®cation system provides a good initial guide to the interpretation of BCOP data, the speci®c ranges are not applicable to all classes of materials or to the extended exposure times. The scope for optimizing the standard BCOP protocol for milder materials has been investigated previously. In a study by Bruner et al. (1998), bovine corneas were exposed to cosmetic formulations containing increasing
Table 6 IRE results Treatmenta
Opacity
Mean corneal swelling (%)
Layers of epithelium lost
IRE classi®cation
10% A 10% B 10% C 10% D 10% E 10% F 10% G
Slight Slight Slight Slight Slight Slight Very slight
30 25 29 37 27 26 5
4±6 3±6 3±5 3±6 3±6 3±6 1±3
Moderate Slight/moderate Moderate Moderate Slight/moderate Slight/moderate Very slight/ slight
100% A 100% B 100% D 100% E 100% F
Slight Very slight/slight Slight/moderate Very slight/slight Slight
41 73 >92 12 33
3±5 3±6 5±7 2±5 3±6
Moderate Moderate Severe Slight Moderate
a
Exposure time was 1 min for each treatment.
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Table 7 Comparison of IRE and BCOP with in vivo data Treatment
IRE classi®cation
BCOP classi®cation (score)
In vivo classi®cation (MAS)
10% A 10% B 10% C 10% D 10% E 10% F 10% G 100% A 100% B 100% C 100% D 100% E 100% F 100%G
Moderate Slight/moderate Moderate Moderate Slight/moderate Slight/moderate Very slight/slight Moderate Moderate ±a Severe Mild Moderate ±a
Moderate (33.3) Mild (16.1) Mild (16.9) Mild (13.3) Moderate (50.3) Moderate (28.5) Mild (0.4) Mild (12.1) Mild (10.0) Mild (11.7) Mild (17.3) Mild (10.6) Mild (12.1) Mild (-0.4)
±a ±a ±a ±a ±a ±a Mild (predicted) Mild (14.3) Moderate (30.0) Extreme (59.0) Extreme (77.0) Moderate (predicted) Moderate (predicted) Mild (predicted)
a
No test data.
levels of organic acid for 24 h, during this time the test articles were washed from the cornea at 6-h intervals, the opacity measured followed by re-application of the test article. Results showed a well de®ned dose-response between the level of organic acid and corneal damage, and also illustrated how this extended, multiple exposure technique could be used to investigate the eects of ingredient interactions, an area in which little is known. In this study, the additional endpoint of histological evaluation to the BCOP was found to be a useful descriptor of corneal damage (Table 5). Histological evaluation is integral to the IRE test and a scoring system is employed to grade epithelial and stromal responses to treatment, the results of which are used to determine the overall classi®cation. A system of quantifying and comparing histopathological change would be a valuable addition to the existing BCOP protocol. The way in which the histological evaluation could be used as a semi-quantitative means of grading corneal damage would need to be investigated in a larger study. The BCOP corneal swelling values, based on tissue weight gain compared to untreated controls, showed no correlation with the irritant potential of the shampoos (Table 5). The variability in punch weight values probably accounts for this (variability data not presented). Improvement in the methodology is required before determining the value of bovine corneal swelling data. In the IRE, an ultrasonic pachometer is used to take corneal thickness readings at regular intervals over the 4 h post-treatment; measurements are made by placing a probe on the corneal surface. An audible signal is emitted when the probe is positioned in the centre of the cornea. The post-treatment values are then compared with pretreatment values. This method of measuring corneal swelling is simple and may be worth investigating to enhance the BCOP assay, since it is based on the increase in the pre-treatment value (as opposed to the
increase in weight compared to the untreated controls, which may vary greatly) and therefore provides a more representative metric of corneal swelling. Alternatively, the dierence between wet and dry weights for individual punch biopsies may give more accurate data. 5. Conclusion The results of this investigation demonstrated that both the IRE and the BCOP provide biologically relevant information to assist in the evaluation of eye irritation potential. However, the IRE appeared to be a slightly better predictor of eye irritant potential as measured in the Draize rabbit eye test than the standard BCOP assay for the range of prototype shampoos investigated, which is consistent with the origin of the organ used. Optimisation of the standard BCOP protocol may be achieved by using prolonged exposure times and incorporation of the additional endpoint of histological evaluation. In addition, corneal swelling, based on increased corneal thickness, is worthy of further investigation and may be used to augment the BCOP assay, as it has done for the IRE. As such, there is scope to further develop the standard BCOP assay, to achieve a better correlation with in vivo data. Acknowledgements The authors gratefully acknowledge the practical contributions of Ms. Angela Sizemore and Mr. Andy Minney in conducting the studies and the intellectual contribution of Dr. Julia Fentem in the writing of this paper. Unilever Home & Personal Care- USA (Rolling Meadows) is also acknowledged for providing some of the test materials and the historical eye irritation data used for comparative purposes.
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References Ara, R.C., 1997. In: Craven, L. (Ed.), Diseases of the Cornea, 4th Edition. Mosby, pp. 7±8. Balls, M., Botham, P.A., Bruner, L.H., Spielmann, H., 1995. The EC/ HO international validation study on alternatives to the Draize eye irritation test. Toxicology in Vitro 9, 871±929. Bruner, L.H., Evans, M.G., McPherson, J.P., Williamson, P.S., 1998. Investigation of ingredient interactions in cosmetic formulations using isolated bovine corneas. Toxicology in Vitro 12, 669± 690. Burton, A.B.G., York, M., Lawrence, R.S., 1981. The in vitro assessment of severe eye irritants. Food and Cosmetics Toxicology 19, 471±481. Commission of the European Communities, 1991. Collaborative Study on the Evaluation of Alternative Methods to the Eye Irritation Test. EC Document XI/632/91, V/E/1/131/91. Curren, R.D., Harbell, J.W. (Eds.), 1998. Report from the Bovine Corneal Opacity and Permeability Technical Workshop, 3±4 November 1997. In Vitro & Molecular Toxicology 11, 315± 351. Curren, R., Evans, M., Raabe, H., Dobson, T., Harbell, J., 1999. Optimization of the bovine corneal opacity and permeability assay: histopathology aids understanding of the EC/HO false negative materials. ATLA 27, 344. Draize, J., Woodard, G., Calvery, H., 1944. Methods for the study of irritation and toxicity of substances applied topically to the skin and mucous membranes. Journal of Pharmacology and Experimental Therapy 82, 377±390.
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Draize, J., 1965. Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics. Dermal Toxicity. Association of Food and Drug Ocials of the US, Topeka, KS pp.49±52. Frazier, J.M., Gad, S.C., Goldberg, A.M., McCulley, J.P., 1987. A critical evaluation of alternatives to acute ocular irritation testing. In: Goldberg, A.M. (Ed.), Alternative Methods in Toxicology, Vol. 4. M.A. Liebert, New York, pp. 1±136. Gautheron, P., Dukic, M., Alix, D., Sina, J.F., 1992. Bovine corneal opacity and permeability test: An in vitro assay of ocular irritancy. Fundamental and Applied Toxicology 18, 442±449. Gautheron, P., Giroux, J., Cottin, M. et al., 1994. Interlaboratory assessment of the bovine corneal opacity and permeability (BCOP) assay. Toxicology in Vitro 8, 381±392. Harbell, J.W., Raabe, H.A., Evans, M.G., Curren, R.D., 1999. Histopathology associated with opacity and permeability changes in bovine corneas in vitro. Toxicologist 48, 336±337. Kay, J.H., Callandra, J.C., 1962. Interpretation of eye irritation tests. Journal of the Society of Cosmetic Chemists 13, 281±289. Maurer, J.K., Li, H.F., Petroll, W.M., Parker, R.D., Cavanagh, H.D., Jester, J.V., 1997. Confocal microscopic characterization in initial corneal changes of surfactant-induced eye irritation in the rabbit. Toxicology and Applied Pharmacology 143, 291±300. Sina, J.F., Galer, D.M., Sussman, R.G., Gautheron, P.D., Sargent, E.V., Leong, B., Shah, P.V., Curren, R.D., Miller, K., 1995. A collaborative evaluation of seven alternatives to the Draize eye irritation test using pharmaceutical intermediates. Fundamental and Applied Toxicology 26, 20±31. Sina, J.F., Gautheron, P., 1998. Report from the Bovine Corneal Opacity and Permeability Technical Workshop November 3±4, 1997. In Vitro & Molecular Toxicology 11, 316±326.
www.elsevier.com/locate/toxinvit
Prediction of ocular irritancy of prototype shampoo formulations by the isolated rabbit eye (IRE) test and bovine corneal opacity and permeability (BCOP) assay K.J. Cooper a,*, L.K. Earl a, J. Harbell b, H. Raabe b a
Safety and Environment Assurance Centre, Toxicology Unit, Unilever Research, Colworth House, Sharnbrook, Bedfordshire MK44 1LQ, UK b Institute for In Vitro Sciences, Inc., Gaithersburg, Maryland, USA Accepted 11 October 2000
Abstract The isolated rabbit eye (IRE) test and bovine corneal opacity and permeability (BCOP) assay were evaluated for their ability to predict the eye irritation potential of a range of hair shampoo formulations, some containing a novel non-surfactant ingredient known to be an ocular irritant. The additional endpoints of corneal swelling and histological examination were incorporated into the standard BCOP protocol. Historic Draize data were available for several of the formulations and served as a reference. The standard BCOP assay (without histology) failed to distinguish between shampoos of low and high irritant potential, when exposure times of 10 and 60 min were employed (for undiluted and 10% dilution of the shampoos, respectively) and the in vitro score classi®ed the majority of formulations as mild. The incorporation of the histological endpoint to the BCOP protocol allowed discrimination between formulations of diering irritancy, and should be included to augment the standard BCOP protocol. Corneal swelling values did not, however, correlate with the irritant potential of the shampoos tested. The IRE which includes the endpoints of corneal swelling and histopathological scoring produced classi®cations of irritancy that were fairly consistent with in vivo data and distinguished between the high and low irritant potential shampoos. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Ocular irritation; Bovine corneal opacity and permeability assay; Isolated rabbit eye test; Shampoo formulations
1. Introduction In an eort to identify a replacement for the in vivo eye test (Draize et al., 1944), a number of alternative in vitro methods have been devised (Frazier et al., 1987). Many of these methods are based on cytotoxic endpoints; however, a model comprising a multicellular penetration barrier is more likely to have a closer anity with both in vivo results and ultimately with accidental human exposure. The isolated rabbit eye (IRE) test was ®rst proposed by Burton et al. (1981), as a means of screening for severe irritants without using live animals. A good correlation was obtained with in vivo results for a range of Abbreviations: IRE, isolated rabbit eye; BCOP, bovine corneal opacity and permeability; HBSS, Hanks' balanced salt solution; MEM, minimum essential medium; * Corresponding author. Tel.: +44-1234-222018; fax: +44-1234222122. E-mail address: [email protected] (K.J. Cooper).
chemicals with dierent in vivo irritancies. In addition, the method is capable of distinguishing between mild and moderate eye irritants, such as `baby' and normal `adult' shampoos, and is used in-house as part of a tiered safety evaluation strategy for materials and formulations of interest to Unilever, in order to obviate the need for in vivo testing. A collaborative study on the evaluation of alternative methods to the eye irritation test, sponsored by the Commission of the European Communities (1991) found the IRE to be an accurate predictor of ocular irritancy, based on in vivo classi®cations for 21 chemicals tested. The IRE confers the advantage that animals are not bred exclusively for the purpose, and have previously been used in dermal irritation studies; however, the use of laboratory animals in any guise remains an emotive issue. The BCOP assay was introduced by Gautheron et al. (1992) as a method for screening process intermediates for worker safety. In a multinational interlaboratory study (Gautheron et al., 1994) using Draize data as the reference, BCOP data correctly predicted whether a
0887-2333/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0887-2333(00)00060-6
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compound would be irritating or non-irritating for 44 of the 52 compounds. The method was also found to be easily transferable between participating laboratories, and confers the ethical advantage of using abattoir waste, as well as the practical advantage of the high throughput of slaughterhouse animals. In the European Community/Home Oce (EC/HO) study (Balls et al., 1995), for a group of 59 chemicals, concordance with in vivo data was, however, relatively poor for both the IRE and the BCOP. Several of the chemicals for which the ocular irritation was signi®cantly under-predicted by the opacity and permeability endpoints in the original BCOP studies, were recently re-evaluated using histology as an additional endpoint. Appreciable pathologic changes were observed in both the epithelium and stroma, showing that the tissue was damaged by the exposure to the chemicals. However, these changes were not re¯ected in proportionally increased opacity or permeability. These data suggest that histological examination can provide important supplemental data to the opacity and permeability endpoints in assessing the degree and depth of injury (Curren et al., 1999). Both methods use the metric of corneal opacity, which in relation to accidental human exposure to an irritant is arguably the most important parameter, since it provides information on how visual acuity may be impaired following accidental exposure. In this investigation, seven shampoos were tested in the IRE and the BCOP. For the BCOP assay the additional endpoints of corneal swelling (assessed by water uptake of treated corneas) and histological evaluation were included. The shampoo formulations diered in irritant potential, ranging from a fairly mild, leading brand baby shampoo to a relatively aggressive adult shampoo formulation. 2. Materials and methods 2.1. Test materials The seven shampoo formulations were assigned the arbitrary codes A through G (Table 1); based on historic data the major irritant categories were represented by these materials. In vivo (modi®ed Draize) data were available for shampoos A through D, shampoos E and F were standard IRE reference controls which, based on original in vivo (modi®ed Draize) data and market history, were considered to be moderate ocular irritants; shampoo G was a branded baby shampoo known from market history to be mild. Shampoo A consisted of a base only, common also to shampoos B±D, but with increasing levels of an oxidising agent, ingredient `X', which is non-surfactant and known to be an ocular irritant, with B containing the lowest concentration and D the highest. It should be noted that the same surfactant system was common to shampoos A through D.
Table 1 Test shampoo formulations and in vivo scores (MAS) Code
Formulation
In vivo score (MAS)
Classi®cationa
A B C D E F G
Base Base+1.5% ingredient X Base+3.0% ingredient X Base+6.0% ingredient X Reference control Reference control Baby shampoo
14.3 30.0 59.0 77.0 No data No data No data
Mildb Moderateb Extremeb Extremeb Moderate (predicted)b,c Moderate (predicted)b,c Mild (predicted)c
a b c
Classi®cations derived from: In vivo (modi®ed Draize) data Market history.
Shampoos E±G did not contain ingredient `X' and were based on standard shampoo surfactant systems. 2.2. BCOP assay 2.2.1. Preparation of corneas and treatment The BCOP assay was performed according to modi®cations of the procedures described by Gautheron et al. (1992), the additional endpoints of corneal swelling and histological evaluation were also included. Bovine eyes were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised and then placed in Hanks' balanced salt solution (HBSS), supplemented with penicillin/streptomycin, and transported to the laboratory on ice packs. The corneas were grossly examined for damage and those exhibiting defects were discarded. The connective tissue surrounding the eyeball was carefully removed and the cornea excised such that a 2±3 mm rim of sclera was present around the cornea. The isolated corneas were then stored in a petri dish containing HBSS until they were mounted in a corneal holder. Each cornea was mounted in a holder with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was then positioned on top of the cornea and the screws were tightened. Starting with the posterior compartment, the two compartments of the corneal holder were then ®lled with Eagle's minimum essential medium (MEM) without phenol red, with 1% fetal bovine serum (complete MEM). The corneal holders were incubated at 321 C for a minimum of 1 h. Following incubation, the corneal holders were removed from the incubator and the medium from both compartments was replaced with fresh medium. The opacity was determined for each cornea using a Spectro Designs OP-KIT opacitometer. Three corneas whose opacity readings were approximately the median of all the corneas were selected as the negative control corneas. The medium was then removed from the anterior part of the holder and replaced with an aliquot of 750 ml
K.J. Cooper et al. / Toxicology in Vitro 15 (2001) 95±103
of test material, dilution of test material or positive control. Five corneas were used per treatment. The concentrations and exposure times employed were derived from the work of Gautheron et al. (1992), in which surfactants were tested at up to 10% (w/v) for 30 min exposure. When selecting the test regimen, consideration was also given to the fact that the degree of corneal injury elicited by ingredient `X' could not be predicted. Consequently, the test materials were tested undiluted, and at a dilution of 10% (w/v) prepared in complete MEM, for incubation times of 10 and 60 min, respectively. In a separate, time course study using three corneas per treatment, the baby shampoo, G and shampoo D (highest level of active), were tested neat for 10, 30 and 60 min and also at 10% solutions for 10, 30, 60 and 120 min incubation. All treated corneas were incubated at 321 C. The concurrent positive control, ethanol, was tested for 10 min in two corneas. When the test material was removed, the epithelial side of the cornea was washed at least three times with complete MEM containing phenol red to ensure total removal of the test material, the corneas were then given a ®nal rinse with complete MEM (without phenol red). The anterior compartment was re®lled with complete MEM and the opacity determined. The corneas were then incubated for a total of approximately 2 h at 321 C. The 2 h included the actual exposure time and postexposure incubation. Thus, the 120-min exposure group did not receive a post-rinsing incubation and only one post-exposure opacity reading was taken. At the completion of the incubation period, a second measure of opacity (except the 120-min exposure group) was performed. The values obtained at this second opacity measurement were used in calculating the corneal opacity. 2.2.2. Permeability measurements After the ®nal opacity measurement was performed, the medium was removed from both chambers of the holder. The posterior compartment was re®lled with fresh complete MEM. 1 ml of a 4 mg/ml ¯uorescein solution was added to the anterior compartment. The corneas were then incubated in a horizontal position, anterior side up for approximately 90 min at 321 C. An aliquot of medium was removed from the posterior chamber and placed into tubes. A volume of 360 ml were placed into designated wells of a 96-well plate. The optical density at 490 nm (OD490) was determined using a Molecular Devices Vmax kinetic microplater reader. If the OD490 of a test or control sample was greater than 2000, a 1 in 5 dilution of the sample in MEM was made. Samples of the 1 in 5 dilution were transferred to speci®ed wells on the 96-well plate. The plate was reread and the ®nal reading recorded. 2.2.3. Corneal swelling measurements Once the opacity measurements (or the permeability measurements for the time course study) were completed
97
the last cornea from each treatment group was removed from the chamber and three 8-mm punches were taken from around the edge of the cornea. The punches were taken towards the corneal centre and transferred onto a mesh screen in a petri dish above a moistened paper towel (to prevent corneal dehydration). The punches were then weighed. Treated tissue weights were compared to control tissue weights to assess the uptake of water (swelling) by the corneas. 2.2.4. Histology After determination of ¯uorescein permeability, the corneas were ®xed for 12 h in Davidson's Fixative. Sections were made from the paran blocks, from which haematoxylin and eosin stained slides were made. Corneal sections were examined for the presence of changes in the epithelial, stromal and endothelial components of the tissue. 2.2.5. Presentation of data 2.2.5.1. Opacity measurement. The change in opacity for each cornea was calculated by subtracting the pretreatment opacity readings from the ®nal opacity readings. The corrected opacity value of each cornea was calculated by subtracting the average change in opacity of the negative control corneas from that of each treated cornea. The mean opacity value of each treatment group was calculated by averaging the mean corrected opacity values of the treated corneas for each treatment condition. 2.2.5.2. Permeability measurement. The corrected OD490 was calculated by subtracting the mean OD490 of the negative control corneas from the OD490 value of each treated cornea. The mean OD490 value of each treatment group was calculated by averaging the corrected OD490 values of the treated corneas for that treatment condition. The following formula was used to determine the in vitro score: In vitro Score Mean Opacity Value 15 Mean OD490 Value 2.2.5.3. Relative corneal swelling. Calculated by comparing the combined weights of the three punches from the test article treated cornea at each exposure time with the combined weights of the three punches from a negative (solvent) control cornea. Data were expressed as percent increase in weight of the treated corneas to the negative control. 2.2.5.4. Irritancy classification from opacity and permeability data. The following system (Table 2) was established by Sina et al. (1995), based on 10 min exposure data from a wide range of liquid pharmaceutical intermediates.
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Table 2 BCOP standard protocol irritancy classi®cation scheme
Table 3 IRE classi®cation scheme
In vitro score
Irritancy classi®cation
Classi®cationa
Opacity (score)
0±25 25.1±55 55.1 and above
Mild Moderate Severe
Corneal swelling (%)
Epithelial cell layers eroded
Very slight Slight Moderate Severe
None (0) Slight opacity (1±2) Slight±moderate (2±3) Moderate±severe (3±4)
11% 12±25 26±35 >35
0±2 3±4 5±6 7±8
2.3. Isolated rabbit eye (IRE) test The isolated rabbit eye test was ®rst proposed by Burton et al. (1981). Rabbits are not bred exclusively for this purpose, eyes are enucleated from animals which have already been used for other purposes at a nearby laboratory and then transported to the testing facility with minimum delay. On arrival, eyes were placed in clamps and mounted in a maintenance chamber, which provides optimum conditions; water sleeving maintains the temperature at 31 C and a saline drip irrigates the anterior corneal surface. The eyes were left in the maintenance chamber for a short period to stabilise. Observations of the eyes during this period were made and any damaged eyes discarded. Three eyes were used per test substance and a negative control (untreated) eye included. The test materials were tested undiluted and at 10% (w/v) solutions in distilled water, 20 ml of test material were applied to the cornea every 10 s up to 1 min before washing. The eects of treatment were assessed at regular intervals throughout the following 4 h. Assessments of corneal opacity were made by trained assessors and graded according to the scheme of Draize et al. (1944). Corneal thickness was measured using an ultrasonic pachometer, (Teknar1 Ophthasonic1 pachometer, Mentor O&O Inc., MA, USA) and the extent to which ¯uorescein penetrates the cornea assessed visually using a Zeiss slit lamp. Percentage corneal swelling throughout the 4 h was determined using the pre-treatment thickness value. Fluorescein penetration is expressed using a graded scoring system. After 4 h of incubation, the corneas were excised and ®xed for histological assessment of epithelial and stromal responses. The numbers of layers of epithelial cells which had eroded and evidence of other histopathological changes were recorded. 2.3.1. Presentation of data 2.3.1.1. Opacity, corneal swelling and histology. From the data generated, an overall classi®cation is made on the irritancy potential (Table 3). 2.4. In vivo rabbit eye test It should be noted that data are historical; no animal tests were undertaken as part of this study. The procedure followed was a modi®cation of that described by
a The classi®cation is generally based on the weight of evidence from all three parameters, with any one triggering the inclusion of the higher classi®cation, e.g. a substance causing slight opacity, 26% corneal swelling with loss of three to six epithelial cell layers will be classi®ed as borderline slight to moderate.
Draize et al. (1944). 100 ml of the undiluted test material were instilled onto the cornea of one eye of three New Zealand White rabbits. Eyes were held closed for a period of 2 s following instillation and rinsed after a 15-s contact period with approximately 40 ml of tap water. The contralateral eye remained untreated and served as a control. The eyes were examined at 24, 48, 72 and 168 h after treatment; if there was no evidence of irritation at 72 h, the study was ended. Additional examinations were performed up to a maximum of 21 days, if persistent corneal involvement or other ocular irritation was present. Sodium ¯uorescein and UV light provided by a Spectroline, model Q-12, Long Wave UV-365 nm, 10 Magni®er were employed to reveal possible corneal injury commencing with the 24-h observation. Ocular lesions were scored according to Draize (1965) and maximum group mean scores calculated, from which a classi®cation was assigned (Kay and Callandra, 1962). 3. Results 3.1. BCOP When tested undiluted for 10, 30 and 60 min incubation, and also at 10% dilutions for 10, 30, 60 and 120 min incubation, both shampoo G the baby shampoo and shampoo D the aggressive adult shampoo showed exposure time-dependent responses in the ¯uorescein permeability endpoint, but showed minimal responses in the opacity endpoint (Table 4). Based on the time course study results, the permeability endpoint provided a reasonable resolution between the two undiluted test articles tested for 10 min, but showed even greater resolution between the two 10% dilutions of test articles tested for 60 min. During the treatment of the undiluted shampoo D, a foaming reaction was observed on several of the treated corneas. Extreme opacity values of 832 and 441 were observed in two corneas (one each from the 10 and 30 min exposures). It appears that the reaction may have penetrated the cornea and have introduced
K.J. Cooper et al. / Toxicology in Vitro 15 (2001) 95±103 Table 4 BCOP time course study summary Treatment Exposure Mean opacity Mean Mean pH of time measurement permeability in vitro dosing (min) measurement score solution 100% G
10 30 60
0.0 0.3 2.3
0.169 0.434 0.901
2.5 6.2 15.8
7.0
100% D
10 30 60
8.7a 9.2b 8.3
0.687a 0.897b 3.495
19.0 22.6 60.8
4.4
Ethanol
10
30.7
0.888
44.0
NA
10% G
10 30 60 120
0.7 1.0 1.7 4.0
0.009 0.126 0.248 0.512
0.8 2.9 5.4 11.7
Ethanol
10
35.3
1.084
51.6
10 30 60 120
0.0 1.7 5.3 4.7
0.280 0.453 0.950 2.862
4.2 8.5 19.6 47.6
10
30.3
1.613
54.5
D (10%)
Ethanol
7.5
NA 7.9
NA
a
The opacity and permeability scores for one of the three corneas treated in this group were 832 and 0.008, respectively. The results of this cornea were not included in the group mean opacity, permeability and in vitro score calculations. b The opacity and permeability scores for one of the three corneas treated in this group were 441 and 0.022, respectively. The results of this cornea were not included in the group mean opacity, permeability and in vitro score calculations.
some of the white foamy material within the cornea. In addition, uncharacteristically low permeability scores for these corneas were also observed (0.008 and 0.022 for the two corneas, respectively), suggesting that the introduction of the foam generating material within the cornea created a barrier to ¯uorescein passage in the permeability endpoint determination. The results of these corneas were not included in the group mean opacity, permeability, and in vitro score calculations presented in Table 5. Using the combined endpoints of opacity and permeability for the 10% dilution of shampoos A through D, the standard BCOP protocol failed to correctly predict the rank order of irritancy (based on in vivo classi®cations), and appeared to inversely rank irritancy based on the ¯uorescein permeability endpoint (Table 5). However, BCOP predicted the 10% dilutions of the reference controls E and F to be moderate, and the 10% dilution of G, the baby shampoo to be mild and, based on in vivo data/market history, these predictions were accurate. When tested undiluted, all seven shampoos were predicted to be mild and there was little resolution between them based on permeability data. However, the
99
rank order of opacity values was in line with the in vivo predictions (Table 5). Addition of the endpoint of corneal swelling to the standard BCOP protocol generated values that failed to correlate with the predicted irritancy of the shampoos. There was, however, a high degree of variability in the individual punch weights, making swelling data dicult to interpret (variability data not presented). For both the 10% dilutions and the undiluted shampoos, the extent of histopathological change observed in the bovine corneas appeared to increase with the predicted ocular irritancy of the formulations (Table 5). Very little disruption was apparent in corneas treated with the mildest shampoo, G with only some loosening of the squamous epithelium and slight swelling in the upper wing cells; basal cells, stroma and endothelium were similar to the controls. In contrast, extensive necrosis and separation from the wing cells were observed in corneas treated with the most aggressive shampoos C and D. In addition, shampoo D, when tested both undiluted and at 10%, showed a tendency towards eliciting pyknosis in the stromal nuclei. 3.2. IRE At 10% dilution, the IRE failed to distinguish clearly between the base shampoo A and the highest level of ingredient X, shampoo D, grading both as moderate (Table 6). The baby shampoo, G, was graded as very slight to slight. When tested undiluted, the IRE classi®cation of moderate and severe for shampoos B and D, respectively, were in accordance with in vivo classi®cations (no IRE test data were available for shampoo C). The IRE corneal swelling values, derived from the increase in corneal swelling from the pre-treatment value correlated well with the irritant potential of the shampoos. 4. Discussion Within Unilever, the IRE has been used successfully for almost two decades to provide safety evaluation data on a range of ingredients and formulations pertinent to its product portfolio. Over this period there has never been a safety incident in the marketplace that has caused toxicologists to doubt the data generated from the IRE. The BCOP was evaluated as a possible alternative to the IRE, since several relevant endpoints are common to both techniques and the BCOP oers a departure from using tissue from a laboratory animal, while retaining the advantages associated with using the target organ. The aim of this investigation was to evaluate the ability of the IRE and the BCOP to predict the ocular irritancy potential of seven hair shampoos. Existing in vivo data were available for four of the shampoos; two were reference controls used routinely in
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Table 5 BCOP ®xed exposure results Mean corneal Mean In vitro Histological evaluation Treatment Exposure Mean Mean time (min) opacity permeability swelling (%) in vitro classi®cation score (OD490) 10% A
60
10.9
1.497
10
33.3
Moderate
Squamous epithelium lost, marked damage to wing cells, variable damage to basal cell layers, slight stromal swelling near Bowman's membrane.
10% B
60
2.7
0.899
5
16.1
Mild
Necrosis in squamous/upper wing layers, included basal cells in some areas. Complete separation of epithelium from basal lamina in some areas.
10% C
60
5.0
0.793
13
16.9
Mild
Squamous/wing cell layers showed necrosis in some areas. Variable damage in basal cells, but all showed weakened attachments to basal lamina.
10% D
60
5.4
0.526
16
13.3
Mild
Squamous/upper wing cell layers necrotic, lower wing/basal cell layers generally necrotic. Large areas of separation between basal cells and basal lamina. A few pyknotic nuclei in stroma.
10% E
60
7.3
2.866
12
50.3
Moderate
Large patches of epithelium lost, upper stromal cells showed vacuolated nuclei.
10% F
60
6.4
1.474
5
28.5
Moderate
Squamous epithelium and most of wing cell layers lost. Basal cells disrupted in some areas.
10% G
60
1.5
0.128
3
0.4
Mild
Some loosening of squamous epithelium and slight swelling in upper wing cells.
100% A
10
2.9
0.617
5
12.1
Mild
Squamous epithelium separated/lost. Separation between margins of basal cells. A few degenerated nuclei in stromal cells near Bowman's membrane.
100% B
10
3.3
0.450
3
10.0
Mild
Squamous epithelium necrotic and separated from wing cells, upper wing cell layers also necrotic. Basal cell attachment to the basal lamina weakened.
100% C
10
4.1
0.511
6
11.7
Mild
Squamous epithelium necrotic and separated from wing cells. Upper wing cells also necrotic. Basal cell attachment to the basal cell lamina weakened.
100% D
10
9.4
0.527
6
17.3
Mild
Squamous epithelium and upper wing cell layers necrotic. Necrosis variable in lower wing/basal cells. Upper stromal cell nuclei pyknotic.
100% E
10
2.0
0.576
11
10.6
Mild
Most squamous epithelium lost, necrosis of some wing cells and slight degeneration of the basal cell nuclei in some areas.
100% F
10
2.8
0.618
8
12.1
Mild
Squamous epithelium lost in many areas, some loss of upper wing cell layer and necrosis in remaining wing cells.
100% G
10
2.3
0.127
3
0.4
Mild
Some loosening of squamous epithelium and slight swelling in upper wing cells.
the IRE and the other a leading brand baby shampoo known from market use to be a very mild eye irritant. The BCOP and IRE both oer multiple endpoint alternatives to in vivo eye irritation testing. They include the measurement of the most important aspect of ocular irritation, corneal opacity, which in terms of human exposure is the most relevant component. The IRE uses a subjective graded scoring system for the assessment of both opacity and ¯uorescein permeability, while the BCOP uses a continuous scale, measured by instrumentation which, by eliminating human variation in interpretation, allows the method to be easily trans-
ferable between laboratories. In the IRE, visual assessment does, however, enable the description of discrete areas of the cornea. The exposure to the test material is short term for both methods and the practical work can be performed within a day. Exposure to the test material in the BCOP is to the entire corneal surface, which ensures even exposure across the tissue. Solids and liquid test materials can be tested in the IRE; however, more liquid materials sometimes run o the cornea, resulting in inadequate exposure. In the BCOP, solids are tested as solutions or suspensions, but compounds that ¯oat
K.J. Cooper et al. / Toxicology in Vitro 15 (2001) 95±103
cannot be delivered eectively to the tissue (Sina and Gautheron, 1998). Both techniques are fairly time consuming with low throughput. Only a small number of shampoos were tested in this study; however, based on existing in vivo data and market use, the major irritant categories were represented. At 10% dilutions, both standard methods failed to detect a dose±response relationship for shampoos A through D; however, when tested undiluted, IRE showed an apparent dose±response relationship from A through D, such that corneal swelling values were in accordance with in vivo data (Table 6). When tested undiluted, the standard BCOP method classi®ed all seven shampoos as mild (Table 5). Based on market use, the leading brand baby shampoo, G, was correctly classi®ed by both the IRE (10% dilution) and BCOP (undiluted and 10% dilution) (Table 7). It is worth noting that shampoos A through D shared the same base and surfactant system, and while the surfactant system may have exacerbated the eect of ingredient X by increasing its penetration into the corneal stroma, the dierences in the in vivo irritancy were not attributable to the surfactant system per se, but to the increasing concentration of the ocular irritant, ingredient X. Surfactants used in personal care products, such as shampoos, are thought to induce corneal opacity in vivo through damage to the epithelial and stromal elements, which leads to stromal swelling and in¯ammatory in®ltration (Maurer et al, 1997). This secondary in¯ammatory in®ltration process is not available in the isolated cornea or enucleated eye. Corneal opacity in the ex vivo cornea is postulated to arise from stromal swelling (changing the spacing of the collagen ®bers) and/or protein coagulation (Harbell et al., 1999). Numerous studies have shown that the milder surfactants (e.g. anionics and nonionics) induce little opacity in the ex vivo corneas even when extensive damage is done to the epithelium (Curren and Harbell, 1998). Other assay endpoints, such as corneal swelling and epithelial cell
101
loss (IRE) or ¯uorescein permeability (BCOP), are used to evaluate the irritation potential of these materials. The data from both test systems in this report are consistent with this pattern of responses. Neither corneal opacity nor stromal swelling was appreciably increased in the bovine corneas. Marked corneal swelling was measured in several of the IRE treatment groups (Table 4) without a proportional increase in opacity. These data suggest that the link between corneal swelling and opacity may be more complex than previously expected and show the importance of considering all endpoints in the ®nal evaluation of irritancy potential. The results show the standard BCOP protocol to be less sensitive than the IRE. This may be attributed at least in part to the robustness of the bovine cornea, which consists of 10±14 layers of epithelia, whereas the rabbit cornea has ®ve to seven, giving a closer model of the human cornea which on average has ®ve layers (Ara, 1997). The BCOP and the IRE were both designed for the testing of severe eye irritants, and certainly from this study the robustness of the bovine cornea was re¯ected in the inability of the standard method to distinguish between materials of dierent irritant potential. However, the results from the time course study show that the sensitivity of the BCOP assay can be increased by extending the incubation time (Table 4). The foaming reaction elicited by undiluted shampoo D was most likely caused by the non-surfactant ingredient X, and illustrates the need for a more comprehensive knowledge of the mode of action of dierent chemical classes and how chemicals within a mixture interact. Furthermore, while the Sina et al. (1995) classi®cation system provides a good initial guide to the interpretation of BCOP data, the speci®c ranges are not applicable to all classes of materials or to the extended exposure times. The scope for optimizing the standard BCOP protocol for milder materials has been investigated previously. In a study by Bruner et al. (1998), bovine corneas were exposed to cosmetic formulations containing increasing
Table 6 IRE results Treatmenta
Opacity
Mean corneal swelling (%)
Layers of epithelium lost
IRE classi®cation
10% A 10% B 10% C 10% D 10% E 10% F 10% G
Slight Slight Slight Slight Slight Slight Very slight
30 25 29 37 27 26 5
4±6 3±6 3±5 3±6 3±6 3±6 1±3
Moderate Slight/moderate Moderate Moderate Slight/moderate Slight/moderate Very slight/ slight
100% A 100% B 100% D 100% E 100% F
Slight Very slight/slight Slight/moderate Very slight/slight Slight
41 73 >92 12 33
3±5 3±6 5±7 2±5 3±6
Moderate Moderate Severe Slight Moderate
a
Exposure time was 1 min for each treatment.
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K.J. Cooper et al. / Toxicology in Vitro 15 (2001) 95±103
Table 7 Comparison of IRE and BCOP with in vivo data Treatment
IRE classi®cation
BCOP classi®cation (score)
In vivo classi®cation (MAS)
10% A 10% B 10% C 10% D 10% E 10% F 10% G 100% A 100% B 100% C 100% D 100% E 100% F 100%G
Moderate Slight/moderate Moderate Moderate Slight/moderate Slight/moderate Very slight/slight Moderate Moderate ±a Severe Mild Moderate ±a
Moderate (33.3) Mild (16.1) Mild (16.9) Mild (13.3) Moderate (50.3) Moderate (28.5) Mild (0.4) Mild (12.1) Mild (10.0) Mild (11.7) Mild (17.3) Mild (10.6) Mild (12.1) Mild (-0.4)
±a ±a ±a ±a ±a ±a Mild (predicted) Mild (14.3) Moderate (30.0) Extreme (59.0) Extreme (77.0) Moderate (predicted) Moderate (predicted) Mild (predicted)
a
No test data.
levels of organic acid for 24 h, during this time the test articles were washed from the cornea at 6-h intervals, the opacity measured followed by re-application of the test article. Results showed a well de®ned dose-response between the level of organic acid and corneal damage, and also illustrated how this extended, multiple exposure technique could be used to investigate the eects of ingredient interactions, an area in which little is known. In this study, the additional endpoint of histological evaluation to the BCOP was found to be a useful descriptor of corneal damage (Table 5). Histological evaluation is integral to the IRE test and a scoring system is employed to grade epithelial and stromal responses to treatment, the results of which are used to determine the overall classi®cation. A system of quantifying and comparing histopathological change would be a valuable addition to the existing BCOP protocol. The way in which the histological evaluation could be used as a semi-quantitative means of grading corneal damage would need to be investigated in a larger study. The BCOP corneal swelling values, based on tissue weight gain compared to untreated controls, showed no correlation with the irritant potential of the shampoos (Table 5). The variability in punch weight values probably accounts for this (variability data not presented). Improvement in the methodology is required before determining the value of bovine corneal swelling data. In the IRE, an ultrasonic pachometer is used to take corneal thickness readings at regular intervals over the 4 h post-treatment; measurements are made by placing a probe on the corneal surface. An audible signal is emitted when the probe is positioned in the centre of the cornea. The post-treatment values are then compared with pretreatment values. This method of measuring corneal swelling is simple and may be worth investigating to enhance the BCOP assay, since it is based on the increase in the pre-treatment value (as opposed to the
increase in weight compared to the untreated controls, which may vary greatly) and therefore provides a more representative metric of corneal swelling. Alternatively, the dierence between wet and dry weights for individual punch biopsies may give more accurate data. 5. Conclusion The results of this investigation demonstrated that both the IRE and the BCOP provide biologically relevant information to assist in the evaluation of eye irritation potential. However, the IRE appeared to be a slightly better predictor of eye irritant potential as measured in the Draize rabbit eye test than the standard BCOP assay for the range of prototype shampoos investigated, which is consistent with the origin of the organ used. Optimisation of the standard BCOP protocol may be achieved by using prolonged exposure times and incorporation of the additional endpoint of histological evaluation. In addition, corneal swelling, based on increased corneal thickness, is worthy of further investigation and may be used to augment the BCOP assay, as it has done for the IRE. As such, there is scope to further develop the standard BCOP assay, to achieve a better correlation with in vivo data. Acknowledgements The authors gratefully acknowledge the practical contributions of Ms. Angela Sizemore and Mr. Andy Minney in conducting the studies and the intellectual contribution of Dr. Julia Fentem in the writing of this paper. Unilever Home & Personal Care- USA (Rolling Meadows) is also acknowledged for providing some of the test materials and the historical eye irritation data used for comparative purposes.
K.J. Cooper et al. / Toxicology in Vitro 15 (2001) 95±103
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