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Workshop 5.11
Mechanisms of chemically-induced
ocular injury and recovery: Current
understanding and research needs

Poster
The assessment of the oral irritation potency of
dentifrices with and without sodium lauryl sulphate as
evaluated with the Slug Mucosal Irritation assay
Els Adriaens 1, Bart Vande Vannet 2, Bart De Wever 3 and Jean-Paul Remon 1
1
Ghent University, Lab Pharmaceutical Technology, Ghent, Belgium; 2 Free University of Brussels, Dentistry, Brussels, Belgium;
3
De Wever Consulting bvba, Oud-Turnhout, Belgium

Ingredients such as sodium lauryl sulfate often used in denti- 30% dilutions resulted in significantly increased mucus produc-
frices can induce oral irritation. In this study the mucosal irrita- tion. Formulation B induced an increased protein and LDH
tion potency of dentifrices with and without SLS was assessed release whereas formulation A induced no tissue damage. The
using the Slug Mucosal Irritation test. The concentration- SLS (C, D, E) containing dentifrices induced an increased
response effect of 5 OTC dentifrices (A, B: containing no SLS; mucus production already at 3% dilutions. Higher concentra-
C, D, E: containing SLS) on the mucosal tissue was evaluated by tions induced mild to moderate tissue damage as was detected
placing the slugs two times for 60 min each on the diluted by the increased protein (>100 µg/ml.g) and LDH release
dentifrice (1%, 3%, 10% and 30% w/v in PBS). The mucus pro- (>1 U/l.g). None of the dentifrices induced ALP release.
duced during each 60 min treatment is a measure for irritation. According to the SMI assay the following rank order of increas-
After the 60 min treatments, the protein and enzyme release ing irritation potency was established: A<B<<C~D<E. These
(LDH, ALP) from the slug mucosa was measured. results are consistent with other in vitro data and confirm clini-
Concentrations up to 10% of dentifrice A and B resulted in a cal inflammatory effects of SLS in oral care products reported in
mucus production (< 2%) and protein release (<50 µg/ml.g) that the literature.
was comparable with the negative controls (PBS). However, the

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Lecture
Ocular toxicology in vitro – cell based assays
Monica Berry and Marcus Radburn-Smith
University of Bristol, Academic Unit of Ophthalmology, Bristol, UK

Background and aims: Interactions between the three cell Results: Stratification of epithelium, compared to monolayer
types in the cornea control cell differentiation and responses cultures, did not modify responses to toxicants probed by classi-
to stimuli. We have sequentially added cell types in a 3- cal toxicology assays. Co-cultured cell types displayed patterns
dimensional construct to assess the minimal requirements for of cytokines different from the single cell-type 3D models, sug-
a representative model of the human cornea to be used in tox- gesting interactions between the different cells of the construct.
icology tests. Following exposure to toxicants there were marked changes in
Methods: We used single applications of toxicants from 3 cytokine profiles, that could be related to the toxicant used.
different classes on cell cultures in defined medium. For a These changes were, however, markedly influenced by the
number of human corneal epithelial cell lines we assessed epithelial cell-line used.
whether stratification modifies responses to toxicants. Primary Conclusions: To rationalise the choice of cell lines for com-
corneal stromal cells were grown in collagen gels, keeping plex corneal constructs, their steady-state immune signal
activation to a minimum. We assessed the influence of three- molecule patterns should be compared to those observed in nor-
dimensional co-culture of the two cell types on cell differenti- mal human preocular fluid.
ation, cytokine production and recovery from exposure to the
chosen toxicants. This process was repeated after the addition Supported by Colipa.
of an endothelial layer.

Lecture
Can toxicogenomics be used to identify chemicals
which cause ocular injury?
Mike Boulton and Mike Wride
School of Optometry and Vision Sciences, Cardiff University, Cardiff, USA

Chemical injury to the eye can be severe, moderate or mild print directory can be used to identify mild/moderate chemical
dependent on the type of chemical (e.g. acid, alkali, surfactant), preparations, which are toxic to the eye. This can be achieved by
concentration, duration of exposure and the ability of the eye to gene expression profiling of bioengineered human corneas using
repair itself. Severe chemical injuries to the cornea can result in microarray analysis. Proof of principle is confirmed by exposure
tissue coagulation, degradation of the stroma, acute inflamma- to generic chemicals/preparations with well documented ocular
tion, angiogenesis, fibrosis, and recurrent corneal ulcers. By injury characteristics. Clustering and pathway analysis of the
contrast, mild chemical injury can present as irritation, pain, gene profiles will lead to the development of diagnostic arrays to
inflammation, red eye and cell loss, which usually subsides fol- identify key genes/pathways differentially regulated by various
lowing treatment and tissue repair. The Draize test in rabbits is chemicals. The diagnostic arrays can then be used for high
currently the “gold standard” for identifying ocular hazards throughput testing of new chemical preparations. Thus, this two-
associated with chemicals and a variety of household formula- pronged approach; the combination of human bio-engineered
tions (e.g. cleaners and cosmetics). However, political necessity corneas and microarray analysis will provide a cheap, effective
and public concern require an alternative to animal testing. One and rigorous alternative to the Draize test.
option is the toxicogenomic approach, whereby a gene finger-

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Poster
Evaluating the eye irritancy of solvents in a
simple fragrance mixture with the Bovine Corneal
Opacity and Permeability (BCOP) assay
Nicole Cuellar 1, Paul Lloyd 2, Judith Swanson 1, Greg Mun 3, John Harbell 3 and Kim Bonnette 4
1
SC Johnson & Son, Inc., Product Safety, Racine, USA; 2 SCJ EURAFNE Ltd., Product Toxicology, Egham, UK;
3
Institute for In Vitro Sciences, Inc., Gaithersburg, USA; 4 Charles River Laboratories, Inc., Spencerville, USA

Fragrances are complex mixtures used in many consumer then the eyes taken for histology. Individual solvents in both
products. Organic solvents, such as ethanol, are major compo- assays impacted the level of irritation of these formulations. In
nents of fragrance formulations functioning mainly as solubilis- vivo, certain solvents increased the rate of lesion development
ers and fragrance delivery mechanisms. The BCOP assay and but not the overall intensity or duration compared to the fra-
primary eye irritation study (EPA-OPPTS 870.2400) were con- grance alone. Other solvents decreased the overall intensity and
ducted using simple fragrance mixtures containing six com- duration. The BCOP assay showed a generally similar pattern of
monly used solvents. The corneal depth of injury was assessed lesion development as seen in vivo. Those combinations that
histologically both in vitro and in vivo. In the BCOP assay, showed opacity at 4 hours in vivo, showed epithelial and stromal
corneas were exposed for 3 minutes, rinsed and incubated for 2, lesion in the BCOP by 4 hours post-exposure. Fragrance alone
4 and 20 hours before the opacity and permeability endpoints was slower to develop opacity in vivo and required the 20 hour
were assessed. Thus, the time course of lesion development was post-exposure to produce appreciable lesions in vitro. These data
determined. The early lesions (2 and 4 hours after exposure) suggest that the standard post-exposure (2 hour) can be predic-
were compared to damage observed after 20+ hours in vitro and tive of irritation potential of fragrance/solvent mixtures.
in vivo. In vivo, animals were scored at 1, 4, and 24 hours and

Lecture
In vitro models for ocular injury:
Current and potential biomarkers
Chantra Eskes 1, David Allen 2, Raymond Tice 2, Neepa Choksi 2, James Truax 2, Wiley Chambers 3,
William Stokes 4 and Leonard Schechtman 3
1
ECVAM, European Commission DG Joint Research Centre, Ispra, Italy; 2 Intergrated Laboratory Systems, Inc., Research
Triangle Park, North Carolina, USA; 3 US Food and Drug Administration, Rockville, Maryland, USA; 4 National Toxicology
Program Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM), NIEHS, Research Triangle
Park, North Carolina, USA

Numerous in vitro/ex vivo methods for eye irritation have been existing assays. During the ICCVAM-NICEATM-ECVAM
developed and are currently being used within industry for spe- symposium on Mechanisms of Chemically-Induced Ocular
cific purposes. In vitro model systems for eye irritation can be Injury and Recovery (May 11-12, 2005), novel and existing
divided into four major categories: organotypic models, human biomarkers were identified that may allow further mechanistic
corneal epithelium models, cell cytotoxicity assays and cell insight into the ocular irritancy potential of a test substance.
function assays. The biomarkers and mechanisms usually Discussions addressed the potential in vitro test systems and
addressed range from simple cytotoxicity to more complex func- biomarkers that may allow adequate prediction of the mecha-
tional endpoints such as corneal light transmission and barrier nisms of chemically-induced ocular injury and lesion persis-
functions. However, the range of criteria for injury, inflamma- tence versus reversibility. Finally, novel in vitro biomarkers or
tion and reversibility covered by the Draize rabbit eye test was test systems were identified where further research and develop-
found to be unlikely to be replaced by a single in vitro test. One ment is recommended to investigate the correlation with the in
of the recommendations to achieve full animal replacement is to vivo test.
support the development of mechanistically-based models in
order to address the mechanisms not currently covered by the ILS staff supported by NIEHS contract N01-ES 35504.

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Poster
Using histological evaluation to enhance the
Bovine Corneal Opacity and Permeability (BCOP) assay
John Harbell and Rodger Curren
Institute for In Vitro Sciences, Inc., Gaithersburg, USA

The BCOP assay was developed by Pierre Gautheron and Joe precipitation, have proven more difficult to identify without the
Sina to address ocular irritation potential of pharmaceutical addition of histological evaluation of the treated corneas.
intermediates and is now widely applied across industries and Histological evaluation is performed on the epithelial, stromal
chemical/formulation classes. For many, if not most, of these and endothelial layers of the cornea and can identify lesions not
chemical/formulation classes, the mode of action(s) of the test revealed by opacity or permeability. It also provides a direct
material is known. Membrane lysis, protein coagulation, and measure of the depth of injury which Maurer et al. (2002) have
saponification are common modes of action that lead to ocular shown to be predictive of the degree and duration of eye irrita-
irritation. In our experience, the opacity and permeability end- tion. Thus, understanding the depth of injury to the treated
points (generally combined into an “in vitro score”) have been corneas (especially relative to the injury from known benchmark
able to identify the epithelial and stromal changes associated materials) through histological evaluation of the bovine corneal
with this type of damage. However, chemicals that react with tissue, can be crucial to interpreting the actual ocular irritation
nucleic acids, mitochondrial proteins, or other cellular targets, potential of novel materials or formulations. Data from reference
that do not lead to immediate loss of cellular integrity or protein compounds will be used to illustrate the approach.

Lecture
An overview of the COLIPA eye irritation
research programme
Penny Jones 1, Dan Bagley 2, Claudine Faller 3, Beatrice Le Varlet 4, Pauline McNamee 5,
Irene Manou 6, Wolfgang Pape 7, Christine Van den Berghe 8 and Freddy Van Goethem 9
1
Unilever-Safety and Environmental Assurance Centre, Colworth Laboratory, Sharnbrook, UK; 2 Colgate-Palmolive, Piscataway,
NJ, USA; 3 Cosmital Wella, Marly, Switzerland; 4 LVMH Recherche, Saint Jean de Braye, France; 5 The Procter & Gamble
Company, Egham, Surrey, UK; 6 COLIPA, Brussels, Belgium; 7 Beiersdorf, Hamburg, Germany; 8 L’Oréal, Aulnay Sous Bois,
France; 9 Johnson & Johnson, Pharmaceutical Research & Development, Beerse, Belgium

The COLIPA eye irritation programme for the development of val and corneal constructs and 3) a genomics project using a pat-
in vitro methods for eye irritation incorporates integrated tern recognition approach to identify new endpoints for injury
research projects and collaborative activities with external part- and repair that build on corneal models being evaluated in pro-
ners. The integrated research projects focus on understanding jects 1 and 2 for potential use in current/future in vitro assays.
mechanisms of eye injury and identification of new in vitro end- Equally important to achieve validated in vitro methods is col-
points that are more predictive of the in vivo human response to laboration of industry, academia, external scientific organisa-
chemical injury. This is expected to result in new or improved in tions and regulators. COLIPA is working with ECVAM by
vitro methods that would proceed to formal validation. There are actively participating in its Eye Irritation Task Force and provid-
three projects: 1) investigation of whether kinetics/patterns of ing support for post-hoc statistical analysis of current in vitro
change in physiological function and signals of injury released methods. The presentation describes these different projects and
from the cornea in vitro can predict a chemical’s potential to activities and how they are combined into the overall COLIPA
damage the eye with a focus on recovery; 2) identification of strategy to address the development of in vitro alternatives for
endpoints related to magnitude of injury and quality of repair in eye irritation.
human immortalised cells and 3-dimensional human conjuncti-

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Poster
The COLIPA strategy for the development of
in vitro alternatives: Eye irritation
Pauline McNamee 1, Dan Bagley 2, Claudine Faller 3, Penny Jones 4, Beatrice Le Varlet 5, Irene Manou 6,
Wolfgang Pape 7, Christine Van den Berghe 8 and Freddy Van Goethem 9
1
The Procter & Gamble Company, Egham, Surrey, UK; 2 Colgate-Palmolive, Piscataway, NJ, USA; 3 Cosmital Wella, Marly,
Switzerland; 4 Unilever, Safety and Environmental Assurance Centre, Sharnbrook, UK; 5 LVMH Recherche, Saint Jean de Braye,
France; 6 COLIPA, Brussels, Belgium; 7 Beiersdorf, Hamburg, Germany; 8 L’Oreal, Aulnay Sous Bois, France; 9 Johnson &
Johnson, Pharmaceutical Research and Development, Beerse, Belgium

The standard regulatory approved test to evaluate eye irrita- quality of repair in human immortalised cells and 3-dimensional
tion is the Draize Test. Success in fully replacing it with in vitro human conjunctival and corneal constructs and 3) a genomics
methods has not occurred. This is in part attributed to lack of project using a pattern recognition approach to identify new end-
understanding of underlying physiological mechanisms of eye points for injury and repair that build on corneal models being
irritation. evaluated in projects 1 and 2 for potential use in current/future
To address this, COLIPA research is focused on understand- in vitro assays.
ing mechanisms of eye injury and identification of new in vitro Equally important to achieve validated in vitro methods is
endpoints that are more predictive of the human response to collaboration of industry, academia, external scientific organisa-
chemical injury resulting in new or improved in vitro methods tions and regulators. COLIPA is working with ECVAM
that would proceed to formal validation. The programme has by actively participating in its Eye Irritation Task Force and
three integrated projects: 1) investigation of whether providing support for post-hoc statistical analysis of current in
kinetics/patterns of change in physiological function and signals vitro methods.
of injury released from the cornea in vitro can predict a chemi- This poster provides a detailed overview of the integrated
cal’s potential to damage the eye with a focus on recovery; 2) elements of the COLIPA eye irritation programme.
identification of endpoints related to magnitude of injury and

Poster
The cytokine response of a wounded corneal model
Marcus Radburn-Smith and Monica Berry
University of Bristol, Academic Unit of Ophthalmology, Bristol, UK

Background and aims: Interactions between epithelial cells Results: IL-8 and IL-6 were detected in the Araki-Sasaki cell
and keratocytes maintain a healthy cornea and its ability to line, whilst IL-10, 8, 6 and 12p70 was produced by the USA
respond to an insult. Following chemical insults, we assessed line. The cytokine responses were different with different tox-
cytokine production, as an indication of intercellular communi- icants. The stromal constructs did not produce any measurable
cation, on addition of a stromal construct to stratified corneal cytokines. In co-cultures of stroma and USA epithelium IL-8
epithelia. production increased ten-fold, whilst there was a four-fold
Methods: Immortalised human corneal epithelial cell models increase in IL-6. On stratified epithelia the non-ionic surfactant
were built and stratified at the air-liquid interface in a fully caused an increase in IL8, while SLS and NaOH did not. In
defined medium. Models were also built with the epithelium epithelium-stroma models it is the latter which cause an
stratified on top of a collagen gel seeded with primary human increase in IL8 and trigger IL6 production, whilst the non-
keratocytes. The stratified constructs were treated for 10 min ionic surfactant did not.
with NaOH, sodium dodecyl sulfate and Tomadol™ 45-7 at a Conclusion: Upon addition of further layers to the constructs
concentration of 0.66%. Cytokine production was assessed as cytokine patterns altered, implying communication between the
well as fluorescein leakage, LDH release, protein and layers.
metabolic assays.
Supported by Colipa.

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Poster
Effect of environment on signaling profiles
of corneal constructs
Marcus Radburn-Smith and Monica Berry
University of Bristol, Academic Unit of Ophthalmology, Bristol, UK

Background and aims: Keratocytes play a major role in wound were not detected. Ratios of IL-6/IL-8 are different in fibroblasts
healing, transforming from a quiescent state to an activated grown on different substrates. Constructs were stable for 34 days
fibroblast or myofibroblast phenotype that acts to contract the in KGM with some low production of IL-6. Addition of endothe-
wound and release inflammatory mediators. Cytokines are such lium caused slight increase in IL-6 and production of IL-8. A
mediators, allowing interactions to occur between all cell types marked increase in both cytokines was observed after epithelial
in the cornea. Understanding the factors that influence this com- addition. Continuity or contiguity of the two cell types influence
munication is essential for building a bioengineered model that the cytokine profile. Contraction of the gel was accompanied by
can be used to study corneal physiology. a striking increase in IL6 and IL8.
Methods: Primary fibroblasts, immortalised corneal epithelia Conclusion: Cytokine patterns were influenced by the culture
and immortalised endothelial cells were grown in either serum medium, the substrate and the presence and contact with other
enhanced or serum free media. Three dimensional cultures were cell types.
grown within type I collagen gels.
Results: Production of IL-6 and IL-8 decreased with time after Supported by Colipa.
transfer to serum-free medium. IL-12p70, TNF, IL-10 and IL-1

Poster
Inflammation mediator detection in the ex vivo
Eye Irritation Test (EVEIT)
Norbert Schrage 1, Markus Frentz 2, Marting Reim 3, Brigitte Kondring 2
and Jakob Becker 3
1
Dept. of Ophthalmology Cologne Merheim, Ophthalmology, Cologne Merheim, Germany; 2 Aachen Center of
Technologytransfer in Ophthalmology (ACTO), Aachen, Germany; 3 Dept. of Ophthalmology University of
Aachen, Aachen, Germany

Objective: Eye irritations can be simulated during cultivation controls remained low. In sodiumhydroxide exposure IL 8
of isolated rabbit corneas in the EVEIT. We found early epithe- decreased within the medium and a rise from 280 +- 220 pg/ml
lial healing of the cornea after abrasion. Irritaton was give proof to 780 +- 270 pg/ml in supernatants. In abraded corneas 290 +-
by opacificaton and epithelial defect. We examine the immuno- 30 pg/ml rose to 375 +- 50 pg/ml post expositionem. Negative
logical response of the isolated cornea on specific irritations. controls reamined stable. IL-1 alpha and beta were measured
Methods: In an ex vivo culture we subjected each 8 corneas to high after sodiumhydroxide expositon and less in case of corneal
epithelial abrasio of 34 +-3% surface, a corneal burn with 2n abrasion.
NaOH in (LVET) and another 8 cultured corneas served as neg- Conclusions: We conclude that the EVEIT model gives reli-
ative control. We examined at different time points perfusion able biochemical reactions in irritation and recovery. The medi-
medium and supernatants on the content of IL 1 alpha and beta, ators released from the isolated cornea are causative for
IL 8 and FGF by means of ELISA technique. inflammation, leucocyte recruitment and ulceration. There is
Results: We found high contents of FGF 6 h after exposition strong evidence that the system might replace animal experi-
towards NaOH in supernatants but not in perfusates. FGF con- ments in future.
centrations were elevated in supernatants of corneas with epithe-
lial abrasion at day 1 significantly. The levels of FGF in negative Sponsored by COLIPA Brussels

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Poster
Replacement of the Draize test by a new
system of ex vivo cornea culture
Norbert Schrage 1, Markus Frentz 2, Martin Reim M. 3, Alice Nietgen 2 and Jakob Becker 3
1
Dept. of Ophthalmology, Cologne Merheim, Germany; 2 Aachen Center of Technologytransfer in Ophthalmology (ACTO),
Aachen, Germany; 3 Dept. of Ophthalmology at the University Aachen, Aachen, Germany

Objective: By a new construction of ex vivo corneal culture we positive staining. Defined corneal abrasions of 34 +- 3% healed
try to simulate irritation, recovery and healing on cultured ani- within 5 days completely and expositons to sodium hydroxid
mal corneas to improve predictive results on chemicals for resulted in persistent corneal erosion of 45 +- 12%. All 36
human eyes. corneas showed considerable consumption of glucose and pro-
Methods: Rabbit corneas freshly prepared from abattoir are duction of lactate. The supernatants showed less lactate in case
mounted in a perfusion system and pre-incubated for 32 hours. of epithelial damage.
After this time we expose the corneas with a modified Low Conclusions: With the presented system we are able to simu-
Volume Eye Irritation Test (LVET) or to mechanical abrasion. late the two main criteria of eye irritation in animal experiments,
We monitor the vitality of the corneas by means of continuous the acute damage and its regeneration or healing. The system is
glucose lactate measurements in medium and supernatants, close to the natural cornea and is ophthalmological evaluated
microscopic and macroscopic examination of the erosion, and proofed to replace eye irritation in animals. Additional
endothelial damage and opacification. parameters of tissue repair and inflammation are available in the
Results: Each 16 corneas were exposed to abrasion, no touch sampled media.
or 2n NaOH for 20 sec. Corneas without any touch showed sta-
ble epithelium with small rough zones of 2 +- 2% of fluorescein Sponsored by COLIPA Brussels

Lecture
Predictive eye irritation test ex vivo system
on rabbit corneas
Norbert Schrage 1, Martin Reim 2, Markus Frentz 2, Brigitte Kondring 2,
Anja Buckermann 1 and Azadeh Babaei 2
1
ACTO, Aachen, Germany: 2 University clinic of Aachen, Dept. of Ophthalmology, Aachen, Germany

The rabbit cornea is the mostly spread in vivo test system used We were able to demonstrate stability and healing of epithelium
in Draize test but also in ophthalmological experiments. Thereby of the cornea and found differential healing dependent on the
the rabbit eye has been well described and parallel diseases and amount and severity of irritative substances.
features especially in eye burns and irritation been found to be We found differential images of reactions concerning healing,
predictive for humans. This was the reason for us to search for expression of VEGF triggered by mechanical abrasion and
an ex vivo rabbit eye irritation test. We derived knowledge from hydrogen peroxide exposure as well as IL 8 releas in early phase
cornea banking and storage and built an ex vivo system for rab- of exposure.
bit corneas. In several experiments we were able to maintain the We believe that the ex vivo rabbit cornea test provides the pos-
cornea for more than 20 days in culture exposed to air with the sibility to replace Draize test by an as close to nature system as
epithelium. Perfusatees of an artificial anterior chamber and possible based on the huge amount of existing data on humans
supernatant fluids were incubated and analysed on Glucose and and rabbits.
lactate and pH and proinflammatory factors and growth factors.

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Lecture
In vivo models of ocular injury and recovery: Current
and potential biomarkers to support development
and validation of predictive in vitro models
Bill Stokes 1, Wiley Chambers 2, Meta Bonner 3, David Allen 4, Raymond Tice 4, Neepa Choksi 4,
Chantra Eskes 5, Jill Merrill 2 and Leonard Schechtman 2
1
NIEHS, NICEATM, USA; 2 US Food and Drug Administration, Rockville, USA; 3 US Environmental Protection Agency,
Washington, D. C., USA; 4 Integrated Laboratory Systems, Inc. /NICEATM, USA; 5 European Centre for the Validation of
Alternative Methods, Ispra, Italy

Significant efforts have been made during the past 25 years to routinely include slit lamp biomicroscopy, confocal microscopy,
develop and validate in vitro test methods to replace the rabbit fluorescein staining, and other measures of inflammation and
ocular irritancy and corrosivity test. Despite these efforts, there injury. Accordingly, experts have suggested that animal ocular
still is no scientifically valid test method or battery of test meth- tests, when such tests are necessary, should include similar
ods capable of completely replacing the in vivo test for the assessments as part of a new set of standard observations that
assessment of novel substances. Critical reviews of this issue by can be used to maximise the comparability of animal and human
various expert groups have generally concluded that greater data. The routine use of objective quantitative endpoints and
emphasis on mechanism-based data from in vivo and in vitro test biomarkers to assess human and animal chemically-induced
methods is essential for future meaningful progress. However, ocular injuries is expected to provide mechanistic insights that
observations in the current rabbit eye test remain unchanged will support the development and validation of more predictive
since the establishment of this test over 60 years ago. These con- in vitro methods, and improve the accuracy and reliability of
sist of visual observations and subjective numerical scores for ocular hazard assessments. Related recommendations from a
damage to the cornea, iris, and conjunctiva. In contrast, ophthal- recent ICCVAM-NICEATM-ECVAM scientific symposium will
mological assessments of human ocular injuries have evolved to be discussed.

Poster
Mechanisms of chemically-induced ocular injury and
recovery: Current understanding and knowledge gaps
W. S. Stokes 1, N. Choksi 2, D. Allen 2, J. Truax 2, R. Tice 2, C. Eskes 3, M. Wind 4 and L. M. Schechtman 5
1
NICEATM (The National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicology Methods), NIEHS,
USA; 2 ILS Inc., USA; 3 ECVAM, USA; 4 US CPSC, USA; 5 US FDA National Center for Toxicological Research, USA

A two-day Scientific Symposium on Mechanisms of theme included injury type, ocular cellular and tissue
Chemically-Induced Ocular Injury and Recovery was held in responses to chemical injury in humans and animals, and the
May 11-12, 2005 in the USA. The symposium was organised role of histopathology and depth of injury in evaluating ocular
and sponsored by NICEATM, ICCVAM and ECVAM, with injury onset, extent, severity, and recovery potential. A sum-
additional support from COLIPA. A major goal of the sympo- mary of expert speaker and panel discussions will be presented
sium was to identify research needed to advance the develop- on these topics and additional aspects, such as, the relevance of
ment of test systems necessary to meet regulatory testing species, dose, and toxicokinetics to a better understanding of
requirements that provide for human health protection while chemical-induced ocular injury-related mechanism and
reducing, refining (less pain and distress), and/or replacing the response, and, a possible future role for toxicogenomics in elu-
use of animals. Three consecutive talks will summarise the cidating processes involved in the characterisation of ocular
symposium discussions. After a brief overview of the sympo- injury and sequelae. Knowledge gaps and areas identified for
sium, this talk will focus on the part of the meeting dealing further investigation at the symposium will be highlighted.
with issues related to the present understanding of currently
known mechanisms and modes of action of chemical-related ILS staff supported by NIEHS contract N01-ES 35504.
ocular injury, persistence and recovery. Areas pertinent to this

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Poster
Comparative in vitro cytotoxicity of lens care
products with three cell lines and two assay methods
Ann Wright 1, Alana Renaud 1, Mary McKee 1 and Ruy Tchao 2
1
CibaVision; a Novartis Company, Duluth, USA; 2 University of the Sciences in Philadelphia, Department of Pharmaceutical
Sciences, Philadelphia, USA

Purpose: Compare cytotoxicity potential of contact lens care assay. The following solutions were considered cytotoxic in
products (LCP) according to the Neutral Red Uptake and comparison to the negative control for the NRUR assay: OPTI-
Release assay (NRUR), using immortalised human corneal FREE® Express® (50 & 25%) and BAC. The following solu-
epithelial (HCE-T) and murine fibroblastic cells (L929), with tions were significantly different in comparison to the negative
cytotoxicity using the Fluorescein Leakage assay with Madin- control using the MDCK assay: OPTI-FREE® Express® and
Darby canine kidney cells (MDCK). SDS. Cytotoxicity was determined by an ED5024 for NRUR.
Methods: NRUR assay: Serially dilute SOLOcare® AQUA Conclusions: The cytotoxicity results for LCPs determined
and OPTI-FREE® Express® with Aldox™ solutions on 96 well using the NRUR assay are similar to results obtained using the
plates to final test concentrations of 12.5%, 25 % and 50% for MDCK fluorescein leakage assay. The LCP cytotoxic by the
the NRUR assay for 24 hours cell exposure. Positive control: 10 NRUR assay was cytotoxic by the MDCK assay and the LCP
ppm BAC. Negative control: DPBS. MDCK assay: MDCK noncytotoxic by the NRUR assay was noncytotoxic by the
inserts exposed to neat solutions for 30 min and observed post MDCK assay. Both of these assays help differentiate subtle dif-
24 hour recovery for fluorescein leakage. Negative control: ferences in LCPs.
HBSS. Positive control: 300 µg/mL SDS.
Results: SOLOcare® AQUA was non-cytotoxic at all concen-
trations with both cell lines for the NRUR and for the MDCK

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