Download Human Ocular Response to Instillation of Surfactant Solutions

Human Ocular Response to Instillation of Surfactant
Solutions and Water Across 10,000 Subjects
Preeya Khanna 1, M. Catherine Mack 1, Vincent R. Walczak 1, Andrea Robillard 1, Matthew T. Hamilton 2,
Jordana Composto 1, Katharine M. Martin 1, and Russel M. Walters 1
1 Johnson
& Johnson Consumer Products Company, Skillman, NJ, USA; 2 Wellspring Worldwide, Pittsburgh, PA, USA
Summary
For the last three decades we have been dedicated to the development and use of in vitro alternatives to replace
and reduce animal testing. The trans-epithelial permeability (TEP) assay has been validated internally as an
alternative to the Draize rabbit eye test and has been used as part of our safety assessment program for 20
years. In parallel, we have compiled a large data set of human ocular testing with endpoints of eye stinging,
bulbar conjunctive conjunctivitis, palpebral conjunctive conjunctivitis, and lacrimation. This data set includes
ocular testing on 20,198 human eyes with 323,168 endpoints.
This large data set allows the evaluation of variations in ocular response to formulations and to control
solutions at different time points, as well as among subject populations. We observe changes in frequency
of ocular conjunctivitis across the months of the year and see differences in the age dependence of ocular
conjunctivitis between males and females. In addition, the neuronal eye sting response is not observed to
correlate with eye conjunctivitis.
Keywords: ocular exposure, surfactant, conjunctivitis, in silico
1 Introduction
Accidental eye exposure to chemicals is a common cause of eye
injury and necessitates the safety testing of many compounds.
While animal alternatives testing regimes have advanced significantly, the Draize rabbit eye test developed in 1944 (Draize
et al., 1944) is still widely used in many industries where validated test methods do not exist for specific ingredient classes or
product forms or where international regulatory agencies do not
accept in vitro data.
A modified Draize test, the low volume eye test (LVET) was
developed (Griffith et al., 1980), and researchers have worked to
correlate LVET results to the human ocular response (Freeberg
et al., 1986; Roggeband et al., 2000) to prove its sufficiency for
simulating human responses. However, the U.S. and other international regulatory agencies often still require Draize testing
(ICCVAM, 2010).
The Draize test has significant limitations, however: ethical
considerations limit the number of animals tested; the endpoints
relate to visually observable tissue damage; and the generalizability of rabbit results to humans presents additional challenges. Furthermore, endpoints related to subject perception cannot
be measured: unlike humans, rabbits do not self-report a sting
response.
These limitations reinforce the desire in the field to replace
animal testing with a combination of in vitro testing and in silico
determinations. Much work has been done recently in developing new in vitro test methods and validating these new test
methods to either Draize results or existing human ocular exposure data (Barile, 2010; McNamee et al., 2009).
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The design of relevant in vitro tests or meaningful in silico
models that can predict the human ocular response should be
based on an in-depth understanding of the human ocular response. Recent work has capitalized on advancement of biomedical measurement techniques to observe the cellular and
tissue response in order to develop a better mechanistic understanding of eye irritation (Jester et al., 2001). In vivo confocal
microcopy has been used to visualize and categorize ocular irritation and the depth of injury (Jester et al., 1996, 1998).
For the past three decades Johnson and Johnson has established dedicated research programs focused on furthering the development and use of in vitro alternatives to replace and reduce
animal testing across a variety of endpoints, including ocular
irritation. In recent years, we have incorporated bio-informatics
and knowledge management capability into these programs to
further refine our safety assessment process. These systems have
allowed further validation of in vitro test methods with clinical
endpoints and have enabled the generation of additional mechanistic understanding of ingredient-specific responses. Ocular irritation has been a priority area for this research. Incorporating
and accessing large clinical data sets into the alternative method
validation process allows us to avoid limitations with Draize
test data. In addition to the ethical considerations, there are objective limitations to correlating an in vitro method designed
to predict a clinical response with an intermediate preclinical
method that is essentially one step removed.
Here we present a very large, previously unpublished data set
of human ocular exposure to a large library of relatively mild
cosmetic formulations. This dataset includes 10,099 subjects,
with 20,198 eyes on which four biological responses were mon-
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itored over four time points, for a total of 323,168 endpoints.
With this data set we can better understand the ocular response
in humans across four unique responses.
This enhanced understanding of the human ocular response
will assist our future development of an in silico model of the
ingredient effects on the ocular response. Ingredient effects of
surfactant solution will be modeled from this data set, and human clinical results are predicted.
2 Materials and methods
Human ocular test
Each study consisted of ten adult subjects aged 18 to 70 years.
Subjects were prescreened by an ophthalmologist, and only
subjects with baseline sting, lacrimation, bulbar, and palpebral
irritation scores of 0 (within normal limits) were permitted to
participate. The test and control (distilled water) solutions were
maintained at 37-38°C before one drop of the appropriate material was instilled into the right eye followed by the other solution into the left eye. At time points of 30 sec, 15 min, and
60 min an ophthalmologist examined and graded the subjects’
eyes regarding bulbar conjunctiva, palpebral conjunctiva, and
lacrimation. The following grading scales were used for bulbar
conjunctiva and palpebral conjunctiva: 0 (within normal limits),
1 (mildly pink), 2 (moderately pink with some vessel dilation),
3 (intense red, dilated vessels), and for lacrimation: 0 (within
normal limits), 1 (excessive wetness, no distinct tears), 2 (a few
formed tears, contained in orbit), 3 (severe intense tearing). The
ocular sting response was self-reported by the subject, with the
scale of: 0 (within normal limits), 1 (mild, very slight), 2 (moderate), 3 (severe).
Test articles
All formulations tested were liquid formulations that were
mostly surfactant-based cleansing systems consisting primarily of amphoteric surfactants, non-ionic surfactants, and often
anionic surfactants. Additionally, nearly all formulas contained
preservatives, cationic polymers, and many contained fragrance
with a pH between 4.5 and 6.8. All the liquid surfactant formulas were designed to be mild (Walters et al., 2008) and were
confirmed as such by in vitro testing: Transepithelial permeation
is also known as the fluorescein dye leakage, and it assesses
the aggressiveness of surfactant systems by quantifying the dye
Fig. 1: Global averages of human ocular response to water and test article
Averages across 10,099 subjects in Ocular Irritation test at 30 sec, 15 min, and 60 sec in the test eye and water eye for endpoints (A)
Palpebral Conjunctivitis (B) Bulbar Conjunctivitis (C) Eye Sting and (D) Lacrimation.
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leakage across a monomer of epithelial cells after treatment
with surfactant system (Cottin and Zanvit, 1997).
3 Results and discussion
Clinical grading and self-reported eye sting after ocular instillation of a test solution or DI water were compiled from 10,099
subjects. Scores for each parameter were averaged across the
entire population to evaluate both frequency of observation and
severity of the effect. As seen in Figure 1, the average scores
across all endpoints measured were greater for the test solution compared to DI water. Palpebral and bulbar conjunctivitis
scores were similar to each other and, on average, higher than
scores for lacrimation and stinging. Conjunctivitis is very common at 30 sec for both the test and DI water ocular exposure,
and it decreases with increasing time after the exposure. While
lacrimation and conjunctivitis rapidly decline after 30 sec, the
ocular sting response is unique in its persistence from 30 sec to
15 min. Lacrimation was observed to be infrequent for both the
test eye and the control eye; it was negligible after the 30 sec
time point.
Each individual subject is presented with a test solution in one
eye and DI water in the other eye. Figure 2 shows the ocular responses on a per subject basis to the test article and the DI water
at the 30 sec and 15 min time points; any given subject can have
a response in the water eye only, test eye only, both eyes, or
neither eye. For all endpoints measured, a majority of subjects
exhibited no reactions in either eye (Fig. 2, blue regions). As
observed in Figure 1, subjects frequently display conjunctivitis
with both DI water and the test solutions, whereas lacrimation is
an uncommon response to both DI water and test solutions.
For sting at both time points, lacrimation at 30 sec and conjunctivitis at 15 min, among the test results where a reaction
was observed, the reaction was observed most commonly in the
test eye alone. However, for both conjunctivitis endpoints at 30
sec, reactions in both the test eye and control eye were more
common than reactions in the test eye alone, suggesting that the
response is a general response to anything being placed into the
eye. It is interesting to note that for both bulbar and palpebral
conjunctivitis, the number of test eye only reactions is greater at
15 min than it is at 30 sec, even though the total number of test
eye reactions is greater at 30 sec.
The DI water control eye frequently exhibited conjunctivitis
at 30 sec, and this was further investigated with respect to the
seasonality of the ocular exposure. The seasonality of eye redness is well studied, and it is caused primarily by seasonal allergies with a peak in June and July (Wolffsohn et al., 2011; Singh
et al., 2010). Figure 3 shows the seasonality of the conjunctivitis
response where the abscissa marks the months, with the width
of each bar proportional to the number of subjects tested in that
month. While the conjunctivitis score range is 0, 1, 2, 3, nearly
all responses are either 0 or 1, so each bar consists of a red and
blue bar, where the relative length of each bar is respectively
proportional to the fraction of 0 (within normal limits) and1
(mildly pink).
A two-tailed t-test assuming equal variance was performed on
the responses in each month compared to the overall population.
Fig. 2: Within subject global correlation between water and test
The number of subjects reporting reactions in their water eye, test eye, both eyes, or neither eye at time points 30 sec and 15 min for
Bulbar Conjunctivitis (Bulb), Palpebral Conjunctivitis (Palp), Eye Sting (Sting), and Lacrimation (Lacr).
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As can be seen in Figure 3A, in the test eye, the months displaying a significantly (α = 0.05) higher fraction of conjunctivitis
responses are April (p < 0.05), June (p < 0.001), and October (p
< 0.001), while significantly lower responses are seen in May
(p < 0.001) and July (p < 0.05), exhibiting lower response fractions. In the DI water eye, Figure 3B, the months displaying
significantly higher conjunctivitis response compared to the total subject set are October (p < 0.01), November (p < 0.05), and
December (p < 0.01), while significantly lower conjunctivitis
responses are observed in May (p < 0.001). A clear seasonality
trend is not observed, and certainly not one that corresponds
to allergy season. Perhaps in the water eye slightly increased
conjunctivitis is observed during the winter months (October
to February). The other endpoints were evaluated for seasonal
effects, but similarly inconclusive results were found. Subjects
are prescreened, and subjects suffering conjunctivitis from allergies, or any other cause, would not be selected for study.
To evaluate potential effects due to population variability over
test panels, the effects of age and gender on the four endpoints
(bulbar and palpebral conjunctivitis, eye sting, and lacrimation)
are shown in Figure 4 at the 30 sec and 15 min time points.
In Figure 4, the width of the columns represents the relative
number of subjects within that age/gender grouping. With this
large data set, even the smaller columns represent 100-300 subjects and the larger columns represent ~2000 subjects.
The fraction of subjects that exhibit ocular sting is not observed to be a function of age or gender; there is no age or gender
effect observed for the sting at either 30 sec or 15 min. Lacrima-
tion at 30 sec in both females and males tends to decrease with
age. Lacrimation at 15 min is infrequent for all populations.
The bulbar and palpebral conjunctivitis responses at both 30
sec and 15 min time points have similar trends between the demographic groups. For females, both bulbar and palpebral conjunctivitis response at 30 sec does not display a strong effect of
age; at 15 min, however, the conjunctivitis response increases with female age. A different response is observed among
men. Interestingly, both the bulbar and palpebral conjunctivitis response at 30 sec does display an age effect for men, with
men age 25-45 displaying significantly more conjunctivitis. In
contrast to women, an age effect is not observed among men.
Across most age groups and time points, men generally exhibit
more conjunctivitis than women.
4 Conclusions
While surfactant interaction with skin is generally well understood (Saad et al., 2011), the interaction with the eye has not
been as well studied, due to various limitations. Many factors
complicate the development of an in silico model of the human
ocular response to chemical exposure. A strong understanding
of the human response with respect to a variety of endpoints
is necessary if we are eventually to replace the Draize test and
human ocular testing. Perhaps not surprisingly, the human ocular response to exposure is complicated, and it is found to be a
function of time after exposure, gender, and age, with interac-
Fig. 3: Seasonality of human ocular response
The seasonal effect on bulbar eye conjunctivitis was investigated by showing the subject data distribution (of 1 sec and 0 sec) for
conjunctivitis at 30 sec for the test eye.
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Fig. 4: Demographic of R, R, S, L (30 sec and 15 min)
Age and gender distributions for bulbar conjunctivitis at (A) 30 sec and (B) 15 min, palpebral conjunctivitis at (C) 30 sec and (D) 15 min,
lacrimation at (E) 30 sec and (F) 15 min, and eye sting at (G) 30 sec and (H) 15 min. Each column is the fraction of subjects that
exhibit an ocular response, with the column width proportional to the number of subjects in that that category. The left side of the graph is
the female response over different age groupings and the right side is the male response.
tion among the variables. No prominent seasonal trends in the
human ocular response are apparent.
In the future, we will explore the correlations between these
different endpoints and ingredients in test articles in order to
further our understanding of the ocular response and to work
towards the development of a meaningful in silico model.
References
Barile, F. A. (2010). Validating and troubleshooting ocular in
vitro toxicology tests. J. Pharmacol. Toxicol. Methods 61,
136-145.
Altex Proceedings, 1/12, Proceedings of WC8
Cottin, M. and Zanvit, A. (1997). Fluorescein leakage test: a
useful tool in ocular safety assessment. Toxicol. In Vitro 11,
399-405.
Draize, J., Woodard, G., and Calvery, H. (1944). Methods for
the study of irritation and toxicity of substances applied topically to the skin and mucous membranes. J. Pharm. Exp. Ther.
82, 377-390.
Freeberg, F. E., Nixon, G. A., Reer, P. J., et al. (1986). Human
and rabbit eye responses to chemical insult. Fundam. Appl.
Toxicol. 7, 626-634.
Griffith, J. F., Nixon, G. A., Bruce, R. D., et al. (1980). Doseresponse studies with chemical irritants in the albino rabbit
131
Khanna et al.
eye as a basis for selecting optimum testing conditions for
predicting hazard to the human eye. Toxicol. Appl. Pharmacol. 55, 501-513.
ICCVAM (2010). ICCVAM test method evaluation report: Recommendation to discontinue use of the low volume eye test
for ocular safety testing. NIH Publication No. 10-7515. Research Triangle Park, NC: National Institute of Environmental Health Services.
Jester, J. V., Maurer, J. K., Petroll, W. M., et al. (1996). Application of in vivo confocal microscopy to the understanding of
surfactant-induced ocular irritation. Toxicol. Pathol. 24, 412428.
Jester, J. V., Li, H. F., Petroll, W. M., et al. (1998). Area and
depth of surfactant-induced corneal injury correlates with cell
death. Invest. Ophthalmol. Vis. Sci. 39, 922-936.
Jester, J. V., Li, L., Molai, A., and Maurer, J. K. (2001). Extent
of initial corneal injury as a basis for alternative eye irritation
tests. Toxicol. In Vitro 15, 115-130.
McNamee, P., Hibatallah, J., and Costabel-Farkas, M. (2009).
A tiered approach to the use of alternatives to animal testing
for the safety assessment of cosmetics: eye irritation. Regul.
Toxicol. Pharmacol. 54, 197-209.
Roggeband, R., York, M., Pericol, M., et al. (2000). Eye irritation responses in rabbit and man after single applications of
132
equal volumes of undiluted model liquid detergent products.
Food Chem. Toxicol. 38, 727-734.
Saad, P., Flach, C. R., Walters, R. M., and Mendelsohn, R.
(2011). Infrared spectroscopic studies of sodium dodecyl suphate permeation and interaction with stratum corneum lipids
in skin. Int. J. Cosmet. Sci. 1, 1-8.
Singh, K., Axelrod, S., and Bielory, L. (2010). The epidemiology of ocular and nasal allergy in the United States, 19881994. J. Allergy Clin. Immunol. 126, 778-783.
Walters, R., Fevola, M., LiBrizzi, J., et al. (2008). Designing
cleansers for the unique needs of baby skin. Cosmet. Toiletries 123, 53-60.
Wolffsohn, J. S., Naroo, S. A., Gupta, N., and Emberlin, J.
(2011). Prevalence and impact of ocular allergy in the population attending UK optometric practice. Cont. Lens Anterior
Eye 34, 133-138.
Correspondence to
Russel M. Walters
199 Grandview Rd.
Skillman, NJ 08558
USA
e-mail: [email protected]
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