USE OF HISTOLOGICAL EXAMINATION IN BOVINE CORNEAL
OPACITY AND PERMEABILITY (BCOP) ASSAY FOR ASSESSING
THE OCULAR IRRITATION POTENTIAL OF FRAGRANCED
FORMULATIONS
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2
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J Burdick , J Merrill , T Spangler , G Moyer and J Harbell
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2
Bath and Body Works, Reynoldsburg, OH; The Institute for In Vitro Sciences,
Inc., Gaithersburg, MD
ABSTRACT
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Fragrances are complex mixtures containing numerous chemical
compounds and are referred to generically in the context of consumer
product formulations. Aside from the inherently complex contribution of
fragrance to a formulation's ocular irritation potential, it is also difficult to
elucidate the role of other components in the mixture. This study was
undertaken to evaluate the use of histological analysis to identify ocular
damage not detectable by BCOP alone. Formulations were grouped
according to base and fragrance type/concentration and comparisons
made between base-only and a series of fragranced formulations using the
same base. Ocular irritation was measured using a standard BCOP assay
with isolated corneas exposed to test material for 10 minutes, followed by
opacity and permeability determination and histological analysis. Ethanol
was used as a concurrent positive control. Within a series, BCOP scores
varied significantly across fragrance type, ranging from 0.1 to 44.8, due
mainly to changes in permeability. Histological analysis confirmed the
relative degree of damage. For a given base, predicted irritation potential
was dependent on the particular fragrance mixture. This study reinforces
the need to determine the ocular irritation potential of formulations and not
rely solely on component information. Understanding the nature and
degree of ocular irritation potential to complex mixtures may help to
improve the selection process for base and fragrance components of
consumer products.
st
Presented at the 41 Annual Meeting of the Society of Toxicology
Nashville, Tennessee
March 17-21, 2002
H I S T O L O G Y
OBJECTIVES
Control
Ø To evaluate eye irritation potential of two different formulation types and their
respective components using the BCOP assay including histological examination.
Epithelium
Ø To evaluate whether fragrance oil at different concentrations and in different
formula matrices plays a role in eye irritation as assessed by BCOP in an effort to
evaluate the necessity of testing all fragrance variants of a formulation.
Ø To assess whether histological evaluation has utility in providing additional
understanding of ocular irritation that is not detectable by BCOP alone (Curren,
2000).
Stroma
Bowman’s
layer
Histological Evaluation
Stroma
Endothelium
Keratocytes
Representative corneal section
showing normal epithelium,
Bowman’s layer and stroma
MATERIALS AND METHODS
Representative corneal section
showing normal stroma and
keratocytes
Representative corneal section
showing normal lower stroma and
endothelium
The BCOP protocol was based on the procedure of Sina et al. (1995).
Sample Preparation and In Vitro Scoring Procedure: Excised bovine eyes were
mounted in holders between culture reservoirs filled with Minimum Essential
Medium Eagle. After a 1 h equilibration at 32 ± 1 ºC the opacity value was
determined for each cornea using an opacitometer. The anterior side of the
corneas was treated with 750 µl of either the neat test article, positive control
(100% ethanol), or negative control (deionized water) for 10 min at 32 ± 1 ºC.
Following extensive rinsing, the corneas were incubated for 120 min before the
final opacity reading. Fluorescein was added to the anterior chamber and
corneas were incubated (anterior side up) for 90 min. The passage of fluorescein
into the posterior chamber was determined spectrophotometrically at 490 nm and
used as an indicator of permeability.
Aqueous-Based Formulations
CMV: Collagen matrix vacuolization (swelling)
KE: Keratocyte cytoplasmic eosinophilia
Opacity Measurement: The change in opacity for each cornea was calculated by
subtracting the pre-treatment opacity readings from the final 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 values of each treatment group were then
calculated.
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 values of each treatment group were then
calculated.
A1
Opacity
0.9
Permeability
0.002
I.V. Score
0.9
A3
Opacity
7
Permeability
0.679
I.V. Score
17.2
A2
Opacity
1.3
Permeability
0.027
I.V. Score
1.7
In Vitro score was determined using the following formula:
In Vitro Score = Mean Opacity Value + (15 x Mean OD490 Value)
Histological Preparation and Evaluation
Corneas were fixed in 10% buffered formalin prior to embedding, sectioning and
staining which was done at Pathology Associates- A Charles River Company
(Frederick, MD). Sections were stained with hematoxylin and eosin and corneal
lesions were examined and described.
CONCLUSIONS
Ø Base formulations (Group A and B) varied with respect to eye irritation profile via
differences in in vitro scores and histology.
Ø Histological findings corroborated in vitro score and provides an additional
dimension to the analysis.
STUDY FORMULATIONS
Ø Addition of either high level fragrance oil (10-20%) or low level fragrance oil (12%) increased the in vitro score relative to respective base formulations primarily by
increasing the permeability component.
GROUP A: Aqueous-based
Ø Addition of either high level fragrance oil (10-20%) or low level fragrance oil (12%) generally produced increased depth of damage to the cornea relative to base
formulations. These lesions are consistent with increases in the permeability
component of the in vitro score.
Ø Differences in in vitro score and histology among fragrance type was evident
across both data sets owing to the complexity of the fragrance mixtures.
Ø This data set demonstrates the importance of testing mixtures and not relying
solely on component information (Swanson and Harbell [2000]).
REFERENCES
Curren, R., Evans, M.., Raabe, H., Ruppalt, R., Harbell, J. (2000) Correlation of
histopathology, opacity, and permeability of bovine corneas exposed in vitro to
known ocular irritants. Veterinary Pathology 37(5):557.
A4
Opacity
9.0
Permeability
1.698
I.V. Score
34.5
Hydroalcoholic-Based Formulations
A1: Base formulation: (minus fragrance)
A2: Base + fragrance A (10-20%)
A3: Base + fragrance B (10-20%)
A4: Base + fragrance C (10-20%)
GROUP B: Hydroalcoholic-based
B1: Pre-base formulation: (water/alcohol only)
B2: Base formulation:(water/alcohol/solvent/surfactant)
B3: Base + fragrance D (1-2%)
B4: Base + fragrance E (1-2%)
B1
Opacity
1.7
Permeability
0.007
I.V. Score
1.8
B3
Opacity
16.8
Permeability
0.776
I.V. Score
28.4
B2
Opacity
17.7
Permeability
0.322
I.V. Score
22.5
Sina, J.F., Galer, D.M., Sussman, R.G., Gautheron, P.D., Sargent, E.V., Leong,
B., Shah, P.V., Curren, R.D., and 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.
Swanson, J.E. and Harbell, J.W. (2000) Evaluating the eye irritancy potential of
ethanolic test materials with the bovine corneal opacity and permeability assay.
The Toxicologist 54(1):188-189.
B4
Opacity
15.5
Permeability
0.969
I.V. Score
30.1