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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
This study was conducted between 11 April 2017 and 11 May 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:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
29 July 2016
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Qualifier:
according to
Guideline:
other: MatTek test protocol “In vitro EpiDermTM Skin Corrosion Test (EPI-200-SCT)”, 07 November 2014.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Identification: MEDOL-10
Chemical Name: (2-Ethyl-2-methyl-1,3-dioxolan-4-yl)methyl acrylate CAS No.: 69701-99-1
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 EpiDerm™ tissue consisted of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis
Source strain:
not specified
Details on test system:
Epi-200 Kit Components Needed for the Assay
EpiDerm™ Kit Lot No.: 25811
1 Sealed 24-well plate Contains 24 inserts with EpiDerm™ tissues on agarose
2 24-well plates For MTT viability assay
4 6-well plates For storing inserts, or for topically applying test agents
1 bottle Serum-Free Assay Medium DMEM-based medium
1 bottle DPBS Rinse Solution For rinsing the inserts in MTT assay

MTT-100 Assay Kit Components
1 vial, 2 mL MTT concentrate
1 vial, 8 mL MTT diluent (supplemented DMEM) For diluting MTT concentrate prior to use in the MTT assay
1 bottle, 60 mL Extractant Solution (Isopropanol) For extraction of formazan crystals
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
Test Item Preparation
50 µL (79.4 µL/cm2 according to guideline) of the test item were dispensed directly onto duplicate EpiDermTM tissue surface
Duration of treatment / exposure:
3 minutes and 60 Minutes
Number of replicates:
2, duplicate tissues were treated with: test substance, positive control or negative control.

Test system

Details on study design:
MTT-Solution
The MTT-solution was prepared freshly on day of use (resulting: 1 mg/mL).
For use in the pre-test (step 3): MTT from Sigma, Germany, DMEM from Gibco, Germany
For use in the main experiment: MTT concentrate from MatTek, MTT diluent from MatTek.

Cell Culture
Epi-200 kits and MTT-100 assays were purchased from MatTek Corporation (Bratislava, Slovakia). The EpiDerm™ tissue consisted of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consisted of organised 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 EpiDerm™ tissues (surface 0.63 cm²) are cultured on specially prepared cell culture inserts (MILLICELL, 10 mm diam).
EpiDerm™ tissues were shipped at 4 °C on medium-supplemented agarose gels in a 24-well plate and reached Envigo CRS GmbH on 10 May 2017. On day of receipt the pre-incubation phase of the EpiDerm™ tissues started.

Test for Direct MTT Reduction and Colour Interference
A test item may interfere with the MTT endpoint if: a) it is coloured and/or b) able to directly reduce MTT. The MTT assay is affected only if the test item is present in the tissues when the MTT viability test is performed.
Some non-coloured test items may change into coloured test items in wet or aqueous conditions and thus stain tissues during the 60 min exposure. Therefore, before exposure, a functional check for this possibility should be performed (step 1).
Step 1
50 µL of the test item were added to 0.3 ml of deionised water (transparent glass test-tube). The mixture was incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5 % CO2) for 60 min. At the end of the exposure time, the mixture was shaken and the presence and intensity of the staining (if any) was evaluated. If the solution changed colour significantly, the test item is presumed to have the potential to stain the tissue. An additional test on viable tissues (without MTT addition) should be performed (step 2).
Since the test item did not dye water when mixed with it, step 2 did not have to be performed.
Step 3
All test items (including those already evaluated in step 1 and step 2) should be further evaluated for their potential to interfere with MTT. To test if an item directly reduces MTT, 50 µL of the test item were added to 1 ml of a MTT/DMEM solution (1 mg/mL) and were incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5 CO2) for 60 minutes. Untreated MTT/DMEM solution (1 mg/mL) medium was used as control. If the MTT/DMEM solution (1 mg/mL) turns blue/purple, the test item reduces MTT and an additional test on freeze-killed tissues (step 4) must be performed.
Since the test item did not prove to be a MTT reducer, step 4 did not have to be performed.

Experimental Performance
Pre-warming of EpiDerm™ Tissues
One hour before dosing, EpiDerm™ tissues were removed from the refrigerator, and the inserts were transferred into 6-well plates containing the pre-warmed assay medium under sterile conditions using sterile forceps. A 24-well plate was prepared as holding plate containing 300 µL assay medium. The holding plate was pre warmed in an incubator (37 ± 1.5 °C, 5 ± 0.5 % CO2) until use.

Treatment
Duplicate EpiDermTM tissues were treated with the test item, positive control or negative control for the following exposure times:
• Test Item: 3 ± 0.5 minutes, 60 ± 5 minutes
• Negative Control: 3 ± 0.5 minutes, 60 ± 5 minutes
• Positive Control: 3 ± 0.5 minutes, 60 ± 5 minutes
After the pre-incubation of the EpiDermTM tissues was completed the DMEM-based medium in each well was replaced with 0.9 mL fresh assay medium. The 6-well plates for the 3 ± 0.5 minutes exposure periods stayed at room temperature in the sterile bench, the 6-well plates for the 60 ± 5 minutes exposure period were placed into an incubator (37 ± 1.5 °C, 5 ± 0.5% CO2).
At the end of the exposure period the tissues were removed from the 6-well plate and gently rinsed with DPBS to remove any residual test material (20 times). Excess DPBS was removed by gently shaking the tissue insert and blotting the lower surface with blotting paper. The tissues were placed in the prepared holding plate until all tissues were rinsed. 

MTT Assay
Two 24-well plates were prepared prior to the end of the tissue pre-warming period. MTT solution (300 µL) was added to each well and the plates were kept in an incubator (37 ± 1.5 °C, 5 ± 0.5% CO2) until required.
Following rinsing, the tissues were transferred from the holding plates to the MTT-plates. After a 3 hour incubation period (37 ± 1.5 °C, 5 ± 0.5% CO2) the tissues were rinsed three times with DPBS and carefully dried with blotting paper. The inserts were transferred into new 24-well plates. The tissues were each immersed in 2 mL of extractant solution (isopropanol) pipetted in each well ensuring that the tissues were completely covered. The 24-well plates were sealed to minimise isopropanol evaporation. The formazan salt was extracted for 17.6 hours without shaking at room temperature.
After the extraction period the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken, and the insert was discarded. 24-well plates were then placed on a shaker for 15 minutes until the solution was homogeneous in colour.
3 x 200 µL aliquots of the blue formazan solution were transferred from each tissue into a 96-well flat bottom microtiter plate. The OD was determined in a microplate reader (Versamax®, Molecular Devices, SoftMax Pro Enterprise (version 4.7.1)) at 570 nm (OD570) without reference filter. The mean values were calculated for each set of 3 wells per tissue.

Data Recording
The data generated were recorded in the laboratory protocol. The results are presented in tabular form, including experimental groups with the test item, negative, and positive controls.

Evaluation
The mean OD of the duplicate negative control tissues was calculated after blank correction. This value corresponds to 100% tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated according to the following formula:

Relative viability (%) = (Mean OD test item/positive control / mean OD negative control) x 100

Interpretation of Results
For the test item and the positive control the mean relative viability  rel. standard deviation of the two individual tissues for both exposure periods was calculated and used for classification according to the following prediction model:
Viability measured after exposure time points Prediction to be considered
< 50% after 3 minutes exposure Corrosive
≥ 50% after 3 minutes exposure AND
< 15% after 60 minutes exposure Corrosive
≥ 50% after 3 minutes exposure AND
≥ 15% after 60 minutes exposure Non-corrosive

Test Item Identified as Corrosive
< 25% after 3 minutes exposure Optional Sub-category 1A*
≥ 25% after 3 minutes exposure A combination of optional Sub-categories 1B and 1C

* According to the data generated in view of assessing the usefulness of the RhE test methods for supporting sub-categorisation, it was shown that around 29%, 31% and 33% of the Sub-category 1A results of the EpiDERMTM test method may actually constitute Sub-category 1B or Sub-category 1C substances/mixtures (i.e. over-classifications).

Acceptability of the Assay
An assay met the acceptance criteria if
• the mean OD of the tissue replicates treated with the negative control is ≥ 0.8 and ≤ 2.8 for every exposure time
• the mean viability of the tissue replicates treated with the positive control for 1 hour, is <15% compared to the negative control
• the Coefficient of Variation (CV) in the range 20 – 100% viability between tissue replicates is ≤ 30%
The quality certificate of the supplier of the test kit demonstrating its robustness (treatment with 1% Triton X-100: 4.77 hours ≤ ET50 ≤ 8.72 hours) is annexed to the report.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
other: Relative absorbance
Run / experiment:
3 Minute exposure
Value:
100.9
Negative controls validity:
valid
Remarks:
Set to 100%
Positive controls validity:
valid
Remarks:
21.4
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
other: Relative absorbance
Run / experiment:
1 hour incubation
Value:
81.1
Negative controls validity:
valid
Remarks:
Set to 100%
Positive controls validity:
valid
Remarks:
4.3%
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not led to a change in colour.
Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour

The test item is considered to be non-corrosive to skin:
• since the viability after 3 minutes exposure is greater than 50% and
• the viability after 1 hour exposure is greater than 15%


The acceptance criteria are met:
• the mean OD of the tissue replicates treated with the negative control is ≥ 0.8 and ≤ 2.8 for every exposure time (range: 1.412 to 1.455)
• the mean viability of the tissue replicates treated with the positive control for 1 hour, is <15% compared to the negative control (4.3%)
• the Coefficient of Variation (CV) in the range 20 – 100% viability between tissue replicates is ≤ 30% (range: 1.0% to 9.8%)



Any other information on results incl. tables

Results after treatment with MEDOL-10 and the controls

Dose Group

Ex-posure Inter-val

Absor-bance
Well 1
(Tissue 1/2)

Absor-bance
Well 2 (Tissue 1/2)

Absor-bance
Well 3 (Tissue 1/2)

Mean Absor-bance (Tissue 1/2)

Mean Ab-sorbance (OD) of 3 Wells minus Blank

Mean Ab-sorbance (OD) of 2 Tissues

Absor-bance [% of Negative Control]*

CV
[%]

Rel. Absor-bance [% of Negative Control]*

Blank

 

0.037

0.037

0.036

0.037

0.000

 

Negative Control

3
minutes

1.355

1.484

1.467

1.435

1.399

1.409

99.3

1.0

100.0

1.437

1.443

1.486

1.455

1.419

100.7

Positive Control

0.362

0.359

0.354

0.358

0.322

0.301

22.8

9.8

21.4

0.316

0.312

0.322

0.317

0.280

19.9

Test Item

1.410

1.465

1.493

1.456

1.419

1.421

100.8

0.2

100.9

1.455

1.463

1.462

1.460

1.424

101.1

Blank

 

0.036

0.036

0.035

0.035

0.000

 

Negative Control

1
hour

1.467

1.448

1.441

1.452

1.417

1.397

101.4

2.0

100.0

1.405

1.428

1.403

1.412

1.377

98.6

Positive Control

0.099

0.103

0.102

0.101

0.066

0.060

4.7

13.4

4.3

0.091

0.089

0.089

0.090

0.054

3.9

Test Item

1.162

1.156

1.145

1.154

1.119

1.133

80.1

1.8

81.1

1.188

1.187

1.174

1.183

1.147

82.2

*             relative absorbance [rounded values]:

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

Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

The test item is considered to be non-corrosive to skin:

•       since the viability after 3 minutes exposure is greater than 50% and

•       the viability after 1 hour exposure is greater than 15%

The acceptance criteria are met:

•       the mean OD of the tissue replicates treated with the negative control is ≥ 0.8 and ≤ 2.8 for every exposure time (range: 1.412 to 1.455)

•       the mean viability of the tissue replicates treated with the positive control for 1 hour, is <15% compared to the negative control (4.3%)

•       the Coefficient of Variation (CV) in the range 20 – 100% viability between tissue replicates is ≤ 30% (range: 1.0% to 9.8%)

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
Criteria used for interpretation of results: EU CLP and UN GHS
Conclusions:
In conclusion, it can be stated that in this study and under the reported experimental conditions, MEDOL-10 is non corrosive to skin according to EU CLP and UN GHS.
Executive summary:

This in vitro study was performed to assess the corrosive potential of MEDOL-10 by means of the Human Skin Model Test with EpiDerm™ tissues models.

The test item did not reduce MTT (test for direct MTT reduction), and it did not change colour, when mixed with deionised water (test for colour interference). Consequently, additional tests with freeze-killed or viable tissues to determine correction factors for calculating the true viability in the main experiment were not necessary.

Independent duplicate tissues of EpiDerm™ were exposed to either the test item, the negative control (deionised water) or the positive control (8.0 N KOH) for 3 minutes and 1 hour, respectively.

After exposure to the negative control the absorbance values met the required acceptability criterion of a mean OD570 ≥ 0.8 and ≤ 2.8 for both treatment intervals thereby confirming the acceptable quality of the tissues.

Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 minutes exposure period and for the 1 hour exposure period. The 1 hour exposure caused a decrease of the cell viability < 15% of the negative control. The CV in the range 20 – 100% viability between the tissue replicates is ≤ 30%, thus the validity of the test system and the specific batch of the tissue models is confirmed.

After exposure of the tissues to the test item the relative absorbance value did not decrease (100.9%) after 3 minutes exposure. After 1 hour exposure the relative absorbance value was reduced to 81.1%. Both values did not affect the threshold for corrosivity which is defined to be 50% after the 3 minutes exposure and 15% after the 1 hour exposure. Therefore, the test item is not considered to be corrosive.

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