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Evaluation of MTT reducers and strongly colored substances in the Short Time Exposure test method for assessing eye
Takayuki Abo1, Yuuki Deguchi2, Takuo Yuki1
1Kao Corporation, Safety Science Research Laboratories.
Abstract:
The Short Time Exposure (STE) test evaluates eye irritation potential using a 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. MTT assays may underpredict results for some substances that directly reduce MTT (i.e., MTT reducers) or interfere with absorbance because of their strong color (i.e., strongly colored substances). Based on previous research, we selected 25 substances as MTT reducers. Of these, 13 were expected to be MTT reducers at 5% dilution (5% MTT reducers) of the STE test condition. These 13 substances were then tested to determine whether the results were interfered from direct MTT reduction. Those 5% MTT reducers that were classified as irritants based on in vivo data were identified as irritants by the STE test. In addition, the low cell viability results at 5% dilution suggested that direct MTT reduction had not occurred. Next, the remaining 5% MTT reducers that were classified as non-irritants based on in vivo data were identified as non-irritants by the STE test. We then examined two strongly colored substances. One was classified as an irritant based on in vivo data and was confirmed as an irritant by the STE test. The other was classified as a non-irritant by the STE test. This was further evaluated using a medium that did not contain MTT; the result indicated that it was a non-irritant correctly. In conclusion, the STE test is useful for evaluating eye irritation potential without the drawback of underprediction for MTT reducers and strongly colored substances.
Insights
The Short Time Exposure (STE) test accurately assesses eye irritation potential, even for substances that interfere with standard assays. This method avoids underpredicting results for MTT reducers and colored compounds.
Area of Science:
- In vitro toxicology
- Ocular safety assessment
- Chemical hazard identification
Background:
- Standard 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays may inaccurately assess eye irritation potential.
- Underprediction can occur with MTT reducers or strongly colored substances, compromising safety evaluations.
Purpose of the Study:
- To validate the Short Time Exposure (STE) test's efficacy in evaluating eye irritation potential.
- To determine if the STE test accurately classifies substances that interfere with MTT assays.
Main Methods:
- The STE test was employed using a 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
- Twenty-five known MTT reducers and two strongly colored substances were tested.
- Interference from direct MTT reduction and color was assessed, comparing results with in vivo data.
Main Results:
- The STE test correctly identified irritants and non-irritants among 13 substances with potential MTT reduction interference.
- Strongly colored substances were accurately classified as irritants or non-irritants by the STE test.
- Further testing confirmed the accuracy for a non-irritant colored substance.
Conclusions:
- The Short Time Exposure (STE) test reliably evaluates eye irritation potential.
- The STE test overcomes limitations of standard MTT assays, preventing underprediction for problematic substances.
