Download Ocular Irritation Assay

In vitro Toxicology
Ocular Irritation
Background Information
• Cyprotex’s ocular irritation test is
performed using MatTek’s in vitro 3D
EpiOcular™ model. Cyprotex are a
MatTek-approved laboratory for performing
these studies.
‘The EpiOcular-EIT, together
with EpiOcular’s long history
of reproducibility and proven
utility for ultra-mildness testing,
make EpiOcular a useful
model for addressing current
legislation related to animal use
in the testing of potential ocular
irritants.’
Kaluzhey Y et al., (2011) Altern
Lab Anim 39(4); 339-364
3
• The EpiOcular™ tissue model consists
of normal, human-derived epidermal
keratinocytes that have been cultured to
form a stratified, squamous epithelium
which closely mimics the human corneal
epithelium.
•EpiOcularTM was developed by MatTek to
create an in vitro (non-animal) alternative
to the animal-based Draize Eye Irritation
Test used in the cosmetics, personal
care, household products, chemical,
pharmaceutical, and biotech industries.
• The EpiOcular™ eye irritation test model
is currently undergoing ECVAM validation.
Peer-reviewed publications have shown
the EpiOcularTM eye irritation model
achieves greater than 80% accuracy when
compared with the Draize eye irritation
test1.
• The EpiOcular™ model is metabolically
and mitotically active and produces the
same cytokines involved in irritation and
inflammation in vivo2.
• A range of different test articles can be
assessed including liquids, solids, semisolids, pastes, gels, creams and waxes.
Related Services
Skin corrosion
Skin irritation
Phototoxicity
Skin absorption
• Cyprotex’s ocular irritation test is based
upon assessment of the cytotoxicity
following exposure to a test chemical,
typically at three time points. Cytotoxicity is
expressed as a decrease in mitochondrial
conversion of MTT to formazan.
To find out more contact [email protected]
Protocol
Method Available
EpiOcularTM Eye Irritation Test (ET50 determination)
Model Used
MatTek EpiOcularTM 3D human tissue model
(others available on request)
Number of Concentrations
Typically 1 (depending on client needs)
Number of Replicates
1 - 3 (depending on assay and client needs)
Exposure Times
3 - 5 (depending on assay and client needs)
Controls
Positive Control = between 1-10% Benzalkonium
chloride
Negative control = Sterile ultrapure water
Reference control = 10% Baby shampoo (optional)
Endpoints
MTT
IL-1α release (optional)
Histology (optional)
Additional endpoints available:
• cytokine release (IL-6, IL-8, TNFα)
• oxidative stress
• glutathione levels
• mitochondrial function
‘Damage to the corneal epithelium resulting from exposure to chemicals and mixtures of
chemicals may compromise tissue function, and can result in various effects ranging from mild
irritation, to the loss of cornea transparency or blindness3.’
Graph illustrating the cell viability over time using the
EpiOcularTM EIT model. Error bars represent ± SEM
(n=3).
Cell Viability (% of Control)
Figure 1
Water
10% Baby shampoo
10% Benzalkonium
chloride
1% Triton X-100
100
50
0
0
50
100
150
200
250
Exposure Time (min)
Table 1
The ET50 and estimated Draize modified maximum
average scores (MMAS) were determined using
the cell viability data. Based on these results, the
Predicted Potency was determined and compared
to actual potency from various published reports.
Table 2
Proposed method for classification of ocular
irritation potency.
ET50 (min)
Estimated Draize
(MMAS)
Predicted
Potency
Actual
Potency
10% Baby shampoo
58.9
8.5
Non- to Minimal
Non-Irritant
10% Benzalkonium chloride
0.2
>110
Severe/Extreme
Severe
1% Triton X-100
7.0
33.7
Moderate
Moderate
Chemical
ET50 (min)
Score Range
Examples
Draize Score
> 26.5
Non / Minimal
PEG-75 Lanolin, Tween 20
0 - 15
26.5 - 11.7
Mild
3% Sodium dodecyl sulphate
15.1 - 25
11.7 - 3.45
Moderate
5% Triton X-100
25.1 - 50
< 3.45
Severe / Extreme
5% Benzalkonium chloride
50 - 110
ET50 = Time taken for test article to reduce cell viability to 50% of negative control
Figure 2
Structural similarity between EpiOcularTM 3D tissue
model and the rabbit cornea.
EpiOcularTM
Magnification: 360x
Rabbit Cornea
Magnification: 360x
References
Pfannebecker U et al., (2013) Cosmetics Europe multi-laboratory pre-validation of the EpiOcularTM reconstituted human tissue test
method for the prediction of eye irritation. Toxicology In Vitro 27(2); 619-626
2
Thakur A et al., (1997) Modulation of cytokine production from an EpiOcular corneal cell culture model in response to Staphylococcus
aureus superantigen Aust N Z J Opthalmol 25(4); 43-45
2
Kaluzhey Y et al., (2011) Development of the EpiOcularTM eye irritation test for hazard identification and labelling of eye irritating chemicals
in response to the requirements of the EU cosmetics directive and REACH legistlation Altern Lab Anim 39(4); 339-364
1