Administrative data
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According to: OECD Guideline for the testing of Chemicals 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method (Original Guideline adopted July 26, 2013), and as described in detail in the Protocol for: In Vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) for use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200), Rev. 1/19/2010
Attached justification
Cross-reference
Data source
Materials and methods
Test guideline
#1 - Test guideline
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Test material
In vitro test system
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Test animals
Test system
Each approximately 25 mg of the neat test item were applied to three skin equivalents. Additionally the test item treated skin equivalents were wetted with 25 µL DPBS per tissue.
The skin equivalents were exposed to the neat test item for on hour.
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Each approximately 25 mg of the neat test item and 30 µL of the positive control (5% SDS in deionised water) or the negative control (DPBS) were applied to each three skin equivalents per dose group. Additionally the test item treated skin equivalents were wetted with 25 µL DPBS per tissue. The skin equivalenst were exposed to the neat test item and the controls for 1 hour. Subsequently the test item as well as the controls were rinsed off the skin equivalents and they were further incubated for about 41 hours. Afterwatrds the viability was determined using the MTT assay.
Model and software
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Any other information on materials and methods incl. tables
Results and discussion
In vitro
Results
Compared to the relative absorbance value of the negative control the mean relative absorbance value was not reduced (100.8%) after exposure of the test item to the skin tissues. The threshold for irritancy is = 50%. Therefore, the test item is not considered to possess an irritant potential.
In vivo
Results
Additional information about applicability domain and reliability of (Q)SAR predictions
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Similar substances with experimental data
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Any other information on results incl. tables
Results after treatment with (-)-Shikimic acid and the controls
Dose Group | Treat-ment Interval | Absorbance 570 nm | Absorbance 570 nm | Absorbance 570 nm | Mean Absorbance of 3 Tissues | Rel. Absorbance [%] Tissue 1, 2 + 3** | Relative Standard Deviation [%] | Mean Rel. Absorbance [% of Negative Control]*** |
Negative Control | 60 min | 1.666 | 1.624 | 1.606 | 1.632 | 102.1 | 1.9 | 100.0 |
Positive Control | 60 min | 0.063 | 0.065 | 0.062 | 0.063 | 3.8 | 2.7 | 3.9 |
Test Item | 60 min | 1.592 | 1.599 | 1.744 | 1.645 | 97.5 | 5.2 | 100.8 |
* Mean of three replicate wells after blank correction
** relative absorbance per tissue [rounded values]:
*** relative absorbance per treatment group [rounded values]:
Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.
The mean relative absorbance value of the test item, corresponding to the cell viability, did not decrease (100.8%; threshold for irritancy:=50%), consequently the test item was non irritant to skin.
Overall remarks, attachments
Attachments
Applicant's summary and conclusion
In conclusion, it can be stated that in this study and under the experimental conditions reported, (-)-Shikimic acid is not irritant to skin.
This in vitro study was performed to assess the irritation potential of (-)-Shikimic acid by means of the Human Skin Model Test.
Each three tissues of the human skin model EpiDerm™were treated with the test item, the negative or the positive control for 60 minutes.
Each about 25 mg of the test item was applied to each tissue.
30 µL of either the negative control (DPBS) or the positive control (5% SLS) were applied to each tissue.
The test item and the positive and negative controls were washed off the skin tissues after 60 minutes treatment. After further incubation for about 41 hours the tissues were treated with the MTT solution for 3 hours following approximately 70 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.
After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD equal or greater than 0.8 and less or equal than 2.8 for the 60 minutes treatment interval thus showing the quality of the tissues.
Treatment with the positive control induced a decrease in the relative absorbance compared to the negative control to 3.9% thus ensuring the validity of the test system.
The rel. standard deviations between the % variabilities of the test item, the positive and negative controls were below 6% (threshold of the "OECD Guideline for the Testing of Chemicals 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”: 18%), thus ensuring the validity of the study.
Compared to the relative absorbance value of the negative controlthe mean relative absorbance value was not reduced (100.8%) after exposure of the test item to the skin tissues. The threshold for irritancy is = 50%. Therefore, the test item is not considered to possess an irritant potential.