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Determining the Depth of Injury in Bioengineered Tissue Models of Cornea and Conjunctiva for the Prediction of All Three Ocular GHS Categories.

Zorn-Kruppa M, Houdek P, Wladykowski E, Engelke M, Bartok M, Mewes KR, Moll I, Brandner JM - PLoS ONE (2014)

Bottom Line: Principally, the MTT-DOI test method allows distinguishing between the cytotoxic effects of the different chemicals in accordance with all 3 GHS categories for eye irritation.Using the MTT-DOI method in this model delivers comparable results as the cornea model, but does not add additional information.However, the MTT-DOI method using reconstructed cornea models already provided good predictability that was superior to the already existing established in vitro/ex vivo methods.

View Article: PubMed Central - PubMed

Affiliation: University Medical Center Hamburg-Eppendorf, Department of Dermatology and Venerology, 20246 Hamburg, Germany.

ABSTRACT
The depth of injury (DOI) is a mechanistic correlate to the ocular irritation response. Attempts to quantitatively determine the DOI in alternative tests have been limited to ex vivo animal eyes by fluorescent staining for biomarkers of cell death and viability in histological cross sections. It was the purpose of this study to assess whether DOI could also be measured by means of cell viability detected by the MTT assay using 3-dimensional (3D) reconstructed models of cornea and conjunctiva. The formazan-free area of metabolically inactive cells in the tissue after topical substance application is used as the visible correlate of the DOI. Areas of metabolically active or inactive cells are quantitatively analyzed on cryosection images with ImageJ software analysis tools. By incorporating the total tissue thickness, the relative MTT-DOI (rMTT-DOI) was calculated. Using the rMTT-DOI and human reconstructed cornea equivalents, we developed a prediction model based on suitable viability cut-off values. We tested 25 chemicals that cover the whole range of eye irritation potential based on the globally harmonized system of classification and labelling of chemicals (GHS). Principally, the MTT-DOI test method allows distinguishing between the cytotoxic effects of the different chemicals in accordance with all 3 GHS categories for eye irritation. Although the prediction model is slightly over-predictive with respect to non-irritants, it promises to be highly valuable to discriminate between severe irritants (Cat. 1), and mild to moderate irritants (Cat. 2). We also tested 3D conjunctiva models with the aim to specifically address conjunctiva-damaging substances. Using the MTT-DOI method in this model delivers comparable results as the cornea model, but does not add additional information. However, the MTT-DOI method using reconstructed cornea models already provided good predictability that was superior to the already existing established in vitro/ex vivo methods.

No MeSH data available.


Related in: MedlinePlus

Boxplots presenting rMTT-DOIs in cornea models after 10 min exposure with 12 chemicals with different eye irritating potential.The in vivo GHS categories of the selected chemicals are depicted within the figure. Medians and boxes of 3 batches (3 models per batch) for upper and lower quartiles are shown. Whiskers indicate minimum and maximum values.
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pone-0114181-g005: Boxplots presenting rMTT-DOIs in cornea models after 10 min exposure with 12 chemicals with different eye irritating potential.The in vivo GHS categories of the selected chemicals are depicted within the figure. Medians and boxes of 3 batches (3 models per batch) for upper and lower quartiles are shown. Whiskers indicate minimum and maximum values.

Mentions: To develop an adequate prediction model, 25 chemicals with different physicochemical properties were selected (Table 1). The chemicals cover all categories of eye irritation potential according to the GHS from non-irritant to severe irritant. Adopted from previous studies [40] we used a 60 min exposure times to establish a suitable protocol. In addition, for 12 selected substances also a 10 min exposure period was tested. NCs and BCs were included in each run. Means and standard deviations (SD) of the rMTT-DOIs of three independent batches were calculated (Table 2). In addition, boxplot analyses were prepared (Fig. 4 and 5) to display the characteristic distribution of the rMTT-DOI values for each single test substance in a series of experiments.


Determining the Depth of Injury in Bioengineered Tissue Models of Cornea and Conjunctiva for the Prediction of All Three Ocular GHS Categories.

Zorn-Kruppa M, Houdek P, Wladykowski E, Engelke M, Bartok M, Mewes KR, Moll I, Brandner JM - PLoS ONE (2014)

Boxplots presenting rMTT-DOIs in cornea models after 10 min exposure with 12 chemicals with different eye irritating potential.The in vivo GHS categories of the selected chemicals are depicted within the figure. Medians and boxes of 3 batches (3 models per batch) for upper and lower quartiles are shown. Whiskers indicate minimum and maximum values.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4262406&req=5

pone-0114181-g005: Boxplots presenting rMTT-DOIs in cornea models after 10 min exposure with 12 chemicals with different eye irritating potential.The in vivo GHS categories of the selected chemicals are depicted within the figure. Medians and boxes of 3 batches (3 models per batch) for upper and lower quartiles are shown. Whiskers indicate minimum and maximum values.
Mentions: To develop an adequate prediction model, 25 chemicals with different physicochemical properties were selected (Table 1). The chemicals cover all categories of eye irritation potential according to the GHS from non-irritant to severe irritant. Adopted from previous studies [40] we used a 60 min exposure times to establish a suitable protocol. In addition, for 12 selected substances also a 10 min exposure period was tested. NCs and BCs were included in each run. Means and standard deviations (SD) of the rMTT-DOIs of three independent batches were calculated (Table 2). In addition, boxplot analyses were prepared (Fig. 4 and 5) to display the characteristic distribution of the rMTT-DOI values for each single test substance in a series of experiments.

Bottom Line: Principally, the MTT-DOI test method allows distinguishing between the cytotoxic effects of the different chemicals in accordance with all 3 GHS categories for eye irritation.Using the MTT-DOI method in this model delivers comparable results as the cornea model, but does not add additional information.However, the MTT-DOI method using reconstructed cornea models already provided good predictability that was superior to the already existing established in vitro/ex vivo methods.

View Article: PubMed Central - PubMed

Affiliation: University Medical Center Hamburg-Eppendorf, Department of Dermatology and Venerology, 20246 Hamburg, Germany.

ABSTRACT
The depth of injury (DOI) is a mechanistic correlate to the ocular irritation response. Attempts to quantitatively determine the DOI in alternative tests have been limited to ex vivo animal eyes by fluorescent staining for biomarkers of cell death and viability in histological cross sections. It was the purpose of this study to assess whether DOI could also be measured by means of cell viability detected by the MTT assay using 3-dimensional (3D) reconstructed models of cornea and conjunctiva. The formazan-free area of metabolically inactive cells in the tissue after topical substance application is used as the visible correlate of the DOI. Areas of metabolically active or inactive cells are quantitatively analyzed on cryosection images with ImageJ software analysis tools. By incorporating the total tissue thickness, the relative MTT-DOI (rMTT-DOI) was calculated. Using the rMTT-DOI and human reconstructed cornea equivalents, we developed a prediction model based on suitable viability cut-off values. We tested 25 chemicals that cover the whole range of eye irritation potential based on the globally harmonized system of classification and labelling of chemicals (GHS). Principally, the MTT-DOI test method allows distinguishing between the cytotoxic effects of the different chemicals in accordance with all 3 GHS categories for eye irritation. Although the prediction model is slightly over-predictive with respect to non-irritants, it promises to be highly valuable to discriminate between severe irritants (Cat. 1), and mild to moderate irritants (Cat. 2). We also tested 3D conjunctiva models with the aim to specifically address conjunctiva-damaging substances. Using the MTT-DOI method in this model delivers comparable results as the cornea model, but does not add additional information. However, the MTT-DOI method using reconstructed cornea models already provided good predictability that was superior to the already existing established in vitro/ex vivo methods.

No MeSH data available.


Related in: MedlinePlus