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![](https://echa.europa.eu/o/diss-blank-theme/images/factsheets/A-REACH/factsheet/print_toxicological-information.png)
Eye irritation
Administrative data
- Endpoint:
- eye irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2019-01-29 to 2019-01-31
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 019
- Report date:
- 2019
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- other: OECD Guideline for the Testing of Chemicals No. 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:
- 25 June 2018
- Qualifier:
- according to guideline
- Guideline:
- other: EURL ECVAM DB-ALM Method Summary No. 164: EpiOcular™ Eye Irritation Test - Summary
- Version / remarks:
- 22 July 2015
- Qualifier:
- according to guideline
- Guideline:
- other: EpiOcular™ Eye Irritation Test (OCL-200-EIT) For the prediction of acute ocular irritation of chemicals For use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model
- Version / remarks:
- 29 June 2015
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- (Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)
Test material
- Test material form:
- liquid
Constituent 1
Test animals / tissue source
- Species:
- human
- Details on test animals or tissues and environmental conditions:
- The test was carried out with the EpiOcular™ reconstructed human cornea-line epithelium (RhCE) model (MatTek). The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified, highly differentiated squamous epithelium morphologically similar to that found in a human cornea. The EpiOcular™ RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinized surface, showing a cornea-like structure analogous to that found in vivo.
Test system
- Vehicle:
- unchanged (no vehicle)
- Controls:
- yes, concurrent positive control
- yes, concurrent negative control
- Amount / concentration applied:
- 1. Negative Control 50 μL Aqua dest.
2. Positive Control 50 μL methyl acetate
3. Test Item 50 μL (undiluted) - Duration of treatment / exposure:
- Incubation: 30 +/- 2 min.
- Duration of post- treatment incubation (in vitro):
- Post soak incubation: 12 +/- 2 min
Post-treatment incubation: 120 +/- 15 min
MTT-incubation: 3h - Number of animals or in vitro replicates:
- 2
- Details on study design:
- The test was performed on EpiOcular, a reconstituted three-dimensional human corneal epithelium model. Hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 30 min exposure period and 120 min post-treatment period and compared to those of the concurrent negative controls.
Results and discussion
In vitro
Results
- Irritation parameter:
- other: mean relative tissue viability
- Remarks:
- NSMTT, NSC living, NSC killed corrected
- Run / experiment:
- 1
- Value:
- 94.8
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: value in %
- Other effects / acceptance of results:
- Value Cut off pass/fail
Mean Absolute OD570 nm NK 1.624 0.8 < NK < 2.5 pass
Mean Relative Viability PC [%] 39.9 < 50% pass
Max. Difference of % Viability [%] 6.7 < 20% pass
Any other information on results incl. tables
The mixture of 50 μL test item per 1 mL MTT medium showed reduction of MTT as compared to the solvent. The mixture turned blue/purple. Since the mean relative tissue viability of the test item treated tissues (TM) was above the 60% threshold value killed tissue controls were performed for quantitative correction of results.
NSMTT [%] = [(ODKT - ODKU)/ODNC] * 100 = [(0.026 – 0.027)/1.580]*100 = - 0.06% ≈ -0.1%
Difference of NSMTT of the two duplicate tissues must be < 20%, otherwise not accepted.
NSMTT1 [%] = [meanODKT1 - ODKU)/ODNC] * 100 = [(0.026 – 0.027)/1.580]*100 = - 0.06%
NSMTT2 [%] = [meanODKT2 - ODKU)/ODNC] * 100 = [(0.026 – 0.027)/1.580]*100 = - 0.06%
NSMTT1 - NSMTT2 = ± 0%
NSMTT was ≤ 60% relative to the negative control of living epidermis and could therefore be used for determination of the killed control corrected viability (KCCV) according to the following formula:
KCCV [%] = viabilityTM – NSMTT = 94.8 – (-0.1) = 94.9%
The mixture of 50 μL test item per 2 mL isopropanol showed no colouring but per 1 mL Aqua dest colouring was observed as compared to the solvent. Since the mean relative tissue viability of the test item treated tissues (TM) was above the 60% threshold value coloured tissue controls were performed for quantitative correction of results.
NSCliving [%] = [ODTVT/ODNC] * 100 = [0.003/1.580] = 0.19 % ≈ 0.2%
Difference of NSCliving of the two duplicate tissues must be < 20%, otherwise not accepted.
NSC1 [%] = [ODTVT1 /ODNC] * 100 = [0.003/1.580] = 0.19 % ≈ 0.2%
NSC2 [%] = [ODTVT2 /ODNC] * 100 = [0.003/1.580] = 0.19 % ≈ 0.2%
NSC1 – NSC2 = ± 0.0%
NSCliving was ≤ 60% (0.2%) relative to the negative control of living epidermis and could therefore be used for determination of the NSC-corrected mean relative tissue viability (NSCCV) according to the following formula:
NSCCV [%] = viabilityTM [%] – NSCliving [%] = 94.8% - 0.2% = 94.6%
Since the test item showed non-specific MTT-reduction and non-specific colouring of living tissues, a third control for non-specific colour in killed tissues (NSCkilled) was performed to avoid a possible double-correction for colour interference.
The non-specific colour of additional killed tissues (NSCkilled) was calculated according to the following formula:
NSCkilled [%] = [ODTKT/ODNC]*100 = [0.002/1.580] = ≈ 0.1%
The true tissue viability was then calculated as the percent tissue viability obtained with living tissues minus NSMTT minus NSCliving plus NSCkilled.
True Tissue Viability = [%] mean Tissue viability – NSMTT - NSCliving + NSCkilled = (94.8 + 0.1 – 0.2 + 0.1) = 94.8%
Result of the Test Item
Name |
Negative Control |
Positive Control |
Test Item |
|||
Replicate Tissue |
1 |
2 |
1 |
2 |
1 |
2 |
Absolute OD570 |
1.574 |
1.684 |
0.667 |
0.676 |
1.487 |
1.569 |
1.569 |
1.669 |
0.663 |
0.689 |
1.516 |
1.593 |
|
Mean Absolute OD570 |
1.624**** |
0.674 |
1.541 |
|||
OD570(Blank Corrected) |
1.531 |
1.640 |
0.624 |
0.632 |
1.443 |
1.525 |
1.525 |
1.626 |
0.619 |
0.646 |
1.473 |
1.550 |
|
Mean OD570of the Duplicates |
1.528 |
1.633 |
0.621 |
0.639 |
1.458 |
1.537 |
Total Mean OD570of the 2 Replicate Tissues (Blank Corrected) |
1.580* |
0.630 |
1.498 |
|||
TODTT- NSMTT |
- |
- |
1.499 |
|||
TODTTNSMTT and NSCliving |
- |
- |
1.496 |
|||
SD of Mean OD570of the Duplicates (Blank Corrected) |
0.074 |
0.012 |
0.056 |
|||
Relative Tissue Viability [%] |
96.7 |
103.3 |
39.3 |
40.4 |
92.3 |
97.3 |
Relative Tissue Viability |
6.7 |
1.1 |
5.0 |
|||
Mean Relative Tissue Viability [%] |
100.0 |
39.9** |
94.8 |
|||
Mean Tissue Viability [%] |
- |
- |
94.9, |
|||
Mean Tissue Viability [%] |
- |
- |
94.7 |
|||
True Tissue Viability [%] |
- |
- |
94.8 |
* Corrected mean OD570of the negative control corresponds to 100% absolute tissue viability
** Mean relative tissue viability of the positive control is < 50%
*** Relative tissue viability difference of replicate tissues is < 20%
**** Mean absolute OD570of the negative control is ≥ 0.8 and ≤ 2.5
Result of the NSMTT control
NSMTT |
KU |
KT |
Negative Control |
||||
Replicate Tissue |
1 |
2 |
1 |
2 |
1 |
2 |
|
Absolute OD570 |
0.070 |
0.070 |
0.069 |
0.070 |
1.574 |
1.684 |
|
0.072 |
0.072 |
0.070 |
0.070 |
1.569 |
1.669 |
||
OD570(Blank Corrected) |
0.026 |
0.027 |
0.025 |
0.026 |
1.531 |
1.640 |
|
0.028 |
0.028 |
0.026 |
0.026 |
1.525 |
1.626 |
||
Mean OD570of the Duplicates |
0.027 |
0.027 |
0.026 |
0.026 |
1.528 |
1.633 |
|
Total Mean OD570 of the 2 Replicate Tissues (Blank Corrected) |
0.027 |
0.026 |
1.580 |
||||
SD of Mean OD570of the Duplicates (Blank Corrected) |
0.000 |
0.000 |
0.074 |
||||
NSMTT [%] |
-0.1 |
- |
|||||
Relative Tissue Viability [%] |
- |
96.7 |
103.3 |
||||
Relative Tissue Viability |
- |
6.7 |
|||||
Mean Relative Tissue Viability [%] |
- |
100.0 |
Result of the NSCliving control
NSCliving |
TVT |
Negative Control |
|||
Replicate Tissue |
1 |
2 |
1 |
2 |
|
Absolute OD570 |
0.045 |
0.045 |
1.574 |
1.684 |
|
0.048 |
0.048 |
1.569 |
1.669 |
||
OD570(Blank Corrected) |
0.001 |
0.002 |
1.531 |
1.640 |
|
0.005 |
0.004 |
1.525 |
1.626 |
||
Mean OD570of the Duplicates |
0.003 |
0.003 |
1.528 |
1.633 |
|
Total Mean OD570 of the 2 Replicate Tissues (Blank Corrected) |
0.003 |
1.580 |
|||
SD of Mean OD570of the Duplicates (Blank Corrected) |
0.000 |
0.074 |
|||
NSCliving[%] |
0.2 |
- |
|||
Relative Tissue Viability [%] |
- |
96.7 |
103.3 |
||
Relative Tissue Viability |
- |
6.7 |
|||
Mean Relative Tissue Viability [%] |
- |
100.0 |
Result of the NSCkilledcontrol
NSCkilled |
TKT |
Negative Control |
|||
Replicate Tissue |
1 |
2 |
1 |
2 |
|
Absolute OD570 |
0.045 |
0.046 |
1.574 |
1.684 |
|
0.046 |
0.047 |
1.569 |
1.669 |
||
OD570(Blank Corrected) |
0.001 |
0.002 |
1.531 |
1.640 |
|
0.003 |
0.004 |
1.525 |
1.626 |
||
Mean OD570of the Duplicates |
0.002 |
0.003 |
1.528 |
1.633 |
|
Total Mean OD570 of the 2 Replicate Tissues (Blank Corrected) |
0.002 |
1.580 |
|||
SD of Mean OD570of the Duplicates (Blank Corrected) |
0.001 |
0.074 |
|||
NSCkilled[%] |
0.1 |
- |
|||
Relative Tissue Viability [%] |
- |
96.7 |
103.3 |
||
Relative Tissue Viability |
- |
6.7 |
|||
Mean Relative Tissue Viability [%] |
- |
100.0 |
Applicant's summary and conclusion
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- In this study under the given conditions the test item showed no irritant effects. The test item is classified as “non-irritant“ in accordance with UN GHS “No Category”.
- Executive summary:
In the present study the eye irritating potential of dialkyl-methyldihydro-heteropolycycle was analysed. Since irritant substances are cytotoxic to the corneal epithelium after a short time exposure the cytotoxic effects of the test item on EpiOcularTM, a reconstituted three-dimensional human corneal epithelium model, were determined.
Hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 30 min exposure period and 120 min post-treatment period and compared to those of the concurrent negative controls.
The mixture of 50 μL test item per 1 mL MTT medium showed reduction of MTT as compared to the solvent. The mixture turned blue/purple. Since the mean relative tissue viability of the test item treated tissues (TM) was above the 60% threshold value killed tissue controls were performed for quantitative correction of results.
NSMTT [%] = [(ODKT - ODKU)/ODNC] * 100 = [(0.026 – 0.027)/1.580]*100 = - 0.06% ≈ -0.1%
Difference of NSMTT of the two duplicate tissues must be < 20%, otherwise not accepted.
NSMTT1 [%] = [meanODKT1 - ODKU)/ODNC] * 100 = [(0.026 – 0.027)/1.580]*100 = - 0.06%
NSMTT2 [%] = [meanODKT2 - ODKU)/ODNC] * 100 = [(0.026 – 0.027)/1.580]*100 = - 0.06%
NSMTT1 - NSMTT2 = ± 0%
NSMTT was ≤ 60% relative to the negative control of living epidermis and could therefore be used for determination of the killed control corrected viability (KCCV) according to the following formula:
KCCV [%] = viabilityTM – NSMTT = 94.8 – (-0.1) = 94.9%
The mixture of 50 μL test item per 2 mL isopropanol showed no colouring but per 1 mL Aqua dest colouring was observed as compared to the solvent. Since the mean relative tissue viability of the test item treated tissues (TM) was above the 60% threshold value coloured tissue controls were performed for quantitative correction of results.
NSCliving [%] = [ODTVT/ODNC] * 100 = [0.003/1.580] = 0.19 % ≈ 0.2%
Difference of NSCliving of the two duplicate tissues must be < 20%, otherwise not accepted.
NSC1 [%] = [ODTVT1 /ODNC] * 100 = [0.003/1.580] = 0.19 % ≈ 0.2%
NSC2 [%] = [ODTVT2 /ODNC] * 100 = [0.003/1.580] = 0.19 % ≈ 0.2%
NSC1 – NSC2 = ± 0.0%
NSCliving was ≤ 60% (0.2%) relative to the negative control of living epidermis and could therefore be used for determination of the NSC-corrected mean relative tissue viability (NSCCV) according to the following formula:
NSCCV [%] = viabilityTM [%] – NSCliving [%] = 94.8% - 0.2% = 94.6%
Since the test item showed non-specific MTT-reduction and non-specific colouring of living tissues, a third control for non-specific colour in killed tissues (NSCkilled) was performed to avoid a possible double-correction for colour interference.
The non-specific colour of additional killed tissues (NSCkilled) was calculated according to the following formula:
NSCkilled [%] = [ODTKT/ODNC]*100 = [0.002/1.580] = ≈ 0.1%
The true tissue viability was then calculated as the percent tissue viability obtained with living tissues minus NSMTT minus NSCliving plus NSCkilled.
True Tissue Viability = [%] mean Tissue viability – NSMTT - NSCliving + NSCkilled = (94.8 + 0.1 – 0.2 + 0.1) = 94.8%
The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 60% (94.8% NSMTT-, NSCliving-,NSCkilled- corrected).
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
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