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Diss Factsheets

Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 Apr 2017 - 02 Jun 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: UN GHS
Version / remarks:
published 2003, last (6th) revision 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
(2-ethyl-2-methyl-1,3-dioxolan-4-yl)methyl prop-2-enoate
EC Number:
807-159-2
Cas Number:
69701-99-1
Molecular formula:
C10H16O4
IUPAC Name:
(2-ethyl-2-methyl-1,3-dioxolan-4-yl)methyl prop-2-enoate
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch: 09892701
- Purity: 99.9%
- Appearance: Colourless liquid
- Expiry Date: 06 March 2018
- Storage Conditions: At room temperature, under protection of sunlight
- Stability in Solvent: Not indicated by the Sponsor
- Purpose of Use: Industrial chemical

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: The EpiSkin™ tissue consists of NHEK, which have been cultured to form a multilayered, highly differentiated model of the human epidermis.
Source strain:
not specified
Details on animal used as source of test system:
N/A
Justification for test system used:
Because systemic reactions play a minor role in modulating local skin toxicity potential of chemicals, skin irritation potential may be predicted by in vitro systems, provided they are sufficiently complex to mimic human skin barrier and cell reactivity. In an international validation study performed by ECVAM, the in vitro skin irritation test using the human skin models EpiSkin™ and EpiDerm™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for skin irritancy potential .

The test consists of a topical exposure of the neat test item to a human reconstructed epidermis model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazoliumbromide], in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison with untreated negative controls is used to predict skin irritation potential (see OECD TG 439) and is used for the purpose of classification as irritating or non-irritating according to chemicals law (EU CLP, UN GHS). The test chemical is considered to be irritant to skin in accordance with UN GHS and EU CLP Category 2 if the tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50%.
Vehicle:
unchanged (no vehicle)
Details on test system:
EpiSkin™ Kit Components Needed for the Assay

EpiSkin™ Kit Lot No.: 17-EKIN-022
1 Sealed 12-well plate; Contains 12 inserts with EpiSkin™ tissues on agarose
1 12-well plate; For MTT viability assay
1 bottle; Assay Medium; Basic medium for use in MTT assays
1 bottle; EpiSkin™ Maintenance Medium; Basic medium for incubations

MTT-Solution
The MTT-solution was prepared freshly on day of use with assay medium (concentration: 0.3 mg/mL).

Cell Culture
EpiSkin™ kits are purchased from SkinEthic Laboratories (69007 Lyon, France). The EpiSkin™ tissue consists of NHEK, which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiSkin™ tissues (surface 0.38 cm²) are cultured on specially prepared cell culture inserts.

EpiSkin™ tissues were shipped at ambient temperature on medium-supplemented agarose gels in a 12-well plate and reached Envigo CRS GmbH on 30 May 2017.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 10 µL

NEGATIVE CONTROL
- Amount(s) applied: 10 µL

POSITIVE CONTROL
- Amount(s) applied: 10 µL

TEST ITEM PREPARATION
10 µL (26.3 µL/cm^2) of the undiluted test item were applied to each of the triplicate tissues
Duration of treatment / exposure:
15 minutes
Number of replicates:
3, triplicate tissues were treated with test substance, positive control or negative control.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: Relative absorbance
Run / experiment:
15 Minute exposure
Value:
7.1
Negative controls validity:
valid
Remarks:
Set to 100%
Positive controls validity:
valid
Remarks:
5.3
Remarks on result:
positive indication of irritation

Any other information on results incl. tables

Results after treatment with MEDOL-10 and the controls

Dose Group

Absorbance 570nm
Tissue

Well 1

Absorbance 570 nm
Tissue

Well 2

Mean Absorbance 570nm

Mean Absorbance

570 nm

*

Mean Absorbance (%) of 3 Tissues

Relative Absorbance [%] Tissue 1, 2 + 3

**

Relative Standard Deviation
[%]

Rel. Absorbance [% of Negative Control]**

 Blank  0.038  0.038  0..38  0.000        
 Negative Control  0.945  0.905  0.925  0.887  0.880  100.8  6.6  100.0
   0.983  0.960  0.972  0.934    106.2    
   0.872  0.840  0.856  0.818    93.0    
 Positive Control  0.073  0.083  0.078  0.040  0.046  4.5  12.7  5.3
   0.091  0.080  0.086  0.048    5.4    
   0.087  0.091  0.089  0.051    5.8    
 Test Item  0.098  0.096  0.097  0.059  0.062  6.7  6.4  7.1
   0.102  0.096  0.099  0.061    6.9    
   0.102  0.107  0.105  0.067    7.6    

* Mean of two replicate wells after blank correction

** relative absorbance per tissue [rounded values]:  

*** relative absorbance per treatment group [rounded values]:  

The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not lead to a change in colour.  

Optical evaluation of the MTT-reducing capacity of the test item after 3 hour incubation with MTT-reagent did not show blue colour.

The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 7.1% (threshold for irritancy: ≤ 50%), consequently the test item was irritant to skin.

The acceptance criteria were met:

• the mean OD of the three negative control exposed tissues is ≥ 0.6 till ≤ 1.5 (range: 0.856 to 0.972).

• the rel. standard deviations between tissues of the same treatment group was ≤ 18% (range: 6.4 to 12.7%).

• the mean relative tissue viability of the positive control was ≤ 40% (5.3%).

• the acceptance limit of the IC50 of the respective EpiSkin™ lot was between 1.5 and 3.0 mg/mL after 18 hours treatment with SLS (1.7 mg/mL).

• the results for the positive control and negative control (deionized water; data for PBS are in process) are within the historical data (means, rel. standard deviation, and ranges) of Envigo CRS GmbH

Applicant's summary and conclusion

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
Under the experimental conditions reported, MEDOL-10 is irritant to skin according to UN GHS and EU CLP regulation.
Executive summary:

This in vitro study was performed to assess the irritation potential of MEDOL-10 by means of the Human Skin Model Test according to OECD TG 439.
The test item did not reduce MTT (pre-test for direct MTT reduction), and it did not dye water, when mixed with it (pre-test for colour interference). Consequently, additional tests with freeze-killed tissues or viable tissues (without MTT addition) were not necessary.
Three tissues of the human skin model EpiSkin™ were treated with the test item, the negative control (PBS) or the positive control (5% sodium lauryl sulfate) for 15 minutes.
After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD ≥ 0.6 till ≤ 1.5 thus showing the quality of the tissues.
Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control thus ensuring the validity of the test system.
After treatment with the test item the mean relative absorbance value decreased to 7.1%. This value is below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered to possess an irritant potential.


 


In conclusion, it can be stated that in this study and under the experimental conditions reported, MEDOL-10 is irritant to skin according to UN GHS and EU CLP regulation.