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Toxicological information

Eye irritation

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Administrative data

eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 Feb - 17 Mar 2016
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Version / remarks:
adopted Jul 2015
GLP compliance:
yes (incl. certificate)
Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test material

Test material form:
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL- Storage condition of test material: At room temperature

Test animals / tissue source

other: EpiOcular™

Test system

unchanged (no vehicle)
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL - Amounts applied: 50 µL NEGATIVE CONTROL - Amounts applied: 50 µLPOSITIVE CONTROL - Amounts applied: 50 µL
Duration of treatment / exposure:
30 min
Duration of post- treatment incubation (in vitro):
120 min
Number of animals or in vitro replicates:
duplicates for each treatment and control group; from each tissue, 2 absorbance measurements after MTT incubation were performed.
Details on study design:
- RhCE tissue construct used, including batch number: EpiOcular™ tissue (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia), batch number: 23701- Viability: The quality of the final tissue was assessed by undertaking a MTT cell viability test.- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 µL of 0.3% Triton X-100. The ET-50 value was determined to be 15.18 min.- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: 30 min exposure, 12 min post-exposure immersion and 120 min post-exposure incubation at 37 °C- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: The ability of the test substance to directly reduce MTT and to form a blue/purple reaction product was assessed in a pre-experiment. Since the MTT solution colour did not turn blue/purple, the test substance is not presumed to have reduced the MTT. An additional test with freeze-killed tissues was not to be required.- Number of tissue replicates used per test chemical and controls: 2- Wavelength: 570 nm- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is labelled non-irritant. If the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is labelled irritant.- Reference to historical data of the RhCE tissue construct: Historical control data was used to assess the validity of the test.- Acceptance criteria:The results are acceptable if:- The negative control OD is > 0.8 and < 2.5, - The mean relative viability of the positive control is below 50% of the negative control viability. - The difference of viability between the two relating tissues of a single test item is < 20% in the same run (for positive and negative control tissues and tissues of test items). This applies also to the killed controls (items and negative killed control) and the colorant controls which are calculated as percent values related to the viability of the relating negative control. This applies also to the killed controls (single items and negative killed control) which are calculated as percent values related to the viability of the relating negative control.

Results and discussion

In vitro

Irritation parameter:
other: % tissue viability mean value of 2 tissues
Run / experiment:
30 min exposure
Vehicle controls validity:
not applicable
Negative controls validity:
Positive controls validity:
Other effects / acceptance of results:
- OTHER OBSERVATIONS:The optical pre-experiment (colour interference pre-experiment) to investigate the colour change potential of the test substance in water or isopropanol did not led to a change in colour. Optical evaluation of the MTT-reducing capacity of the test substance with MTT-reagent did not show blue colour.ACCEPTANCE OF RESULTS:- Acceptance criteria met for negative control: The negative control values were between 1.903 and 2.290.- Acceptance criteria met for positive control: Treatment with the positive control induced a decrease in the mean relative absorbance to 24.2%, compared with the negative control, thus the validity of the test system is ensured. - Acceptance criteria met for viability: The difference in mean viability between 2 duplicate tissues during the same run was 4.6-6.9%, for the positive control tissues, the negative control tissues and the test substance tissues. Please refer to Table 2 under "Any other informational results incl. Tables".

Any other information on results incl. tables

Table 1: Historical Control Data

  Positive Control Negative Control
Mean Viability 32.0%  
Rel. Standard Deviation 12.8%
Range of Viabilities 6.90 - 40.4%
Mean Absorption 0.538 1.65
Rel. Standard Deviation 0.258 0.299
Range of Absorbance 0.107 - 0.849 1.27 - 2.05

Data of 11 studies performed from July 2015 until end of February 2016

Table 2: Results after treatment for 30 min with the test substance and the controls

Test Group Absorbance 570 nm Well 1 Absorbance 570 nm Well 2 Mean Absorbance Tissue 1 and 2 Mean Absorbance* Tissue 1 and 2 minus Mean Blank Mean Absorbance of 2 Tissues* Rel. Absorbance [%] Tissue 1 and 2** Absolute Value of the Difference of the Rel. Absorbances [%] Tissue 1 and 2 Rel. Absorbance
[% of Negative Control]**
Negative Control
(Tissue 1 and 2)
2.290 2.104 2.197 2.160 2.109 102.4 4.8 100.0
1.903 2.288 2.095 2.059 97.6
Positive Control
(Tissue 1 and 2)
0.623 0.617 0.620 0.584 0.511 27.7 6.9 24.2
0.464 0.485 0.474 0.438 20.8
Test Substance
(Tissue 1 and 2)
1.181 1.194 1.187 1.151 1.103 54.5 4.6 52.3
1.081 1.102 1.091 1.054 50.0

*: Mean of two replicate wells after blank correction

**: Relative absorbance (rounded values): 100 × (absorbance test substance/positive control) / (absorbance negative control)

Applicant's summary and conclusion

Interpretation of results:
other: the results of this study as a stand-alone study are not suit able for classification according to CLP/EU GHS criteria; the results may be used for classification purposes in a weight of evidence approach
Under the conditions of the RhCE test method the test substance showed irritant properties.