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Description of key information

Skin corrosion/ irritation: The substance was observed to be corrosive in the in vitro skin corrosion test (Reconstructed Human Epidermis RHE test method, performed according to OECD guideline 431, Envigo Research Limited, 2017)

Eye irritation: this study does not need to be conducted as the substance is classified as skin corrosive, leading to classification as serious eye damage (cat 1).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-8-09 - 2017-08-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
28 July 2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Method B.40bis of Commission Regulation (EC) No 440/2008
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Batch Number: 903-2016
- Chemical Name: 3,3’-Ethylenedioxybis(propylamine)
- CAS-No.: 2997-01-5
- Purity: 99.8 %
- Molecular Formula: C8H20N2O2
- Molecular Weight: 176.26 g/mol
- Total Organic Carbon (calculated by the Study Director): 54.5 %
- Appearance: Colourless to pale yellow liquid with slight haze permitted
- Expiration Date: June 27, 2018
- Storage Conditions: In the closed original container, in a well-ventilated place at room temperature, dry and in the dark.
Test system:
human skin model
Source species:
human
Cell type:
other: epithelial, derived from human skin, and formed into a stratified, cornified epithelium.
Details on animal used as source of test system:
Not applicable
Justification for test system used:
EpiDerm™ Human Skin Model: This model incorporates several features, which make it advantageous in the study of potential dermal corrosivity. The target cells are epithelial, derived from human skin, and formed into a stratified, cornified epithelium. Test items are applied to the culture surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly.
Vehicle:
unchanged (no vehicle)
Details on test system:
- EpiDerm™ Reconstructed Human Epidermis Model Kit
- Supplier: MatTek
- Date received: 08 August 2017
- EpiDermTM Tissues (0.63cm2) lot number: 25835
- Assay Medium lot number: 080317TMA
Upon receipt of the EpidermTM tissues, the sealed 24 well plate was stored in a refrigerator until use.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
50 µL test item and controls
Duration of treatment / exposure:
3 minutes and 60 minutes
Duration of post-treatment incubation (if applicable):
3 hours
Number of replicates:
2
Details on study design:
Study Design
Pre-Test Procedure

Assessment of Direct Test Item Reduction of MTT

MTT Dye Metabolism, Cell Viability Assay
The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan dye by mitochondrial succinate dehydrogenase in viable cells.
One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem if at the time of the MTT test (after rinsing) there is still a sufficient amount of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified.

Test for Direct MTT Reduction
As specified, a test item may interfere with the MTT endpoint, if it was able to directly reduce MTT and at the same time was present on or in the tissues when the MTT viability test was performed. To identify this possible interference, the test item was checked for the ability to directly reduce MTT according to the procedure below:
50 µL of the test item was added to 1 mL of a freshly prepared 1.0 mg/mL MTT solution. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 60 minutes. Untreated MTT solution was tested concurrently to act as a control.
If the MTT solution containing the test item turns blue/purple relative to the control, the test item was presumed to have reduced the MTT.
The test item was shown to directly reduce MTT in the direct MTT reduction test. There was a possibility that if the test item could not be totally rinsed off the tissues, any residual test item present on or in the tissue may directly reduce MTT and could have given rise to a false negative result. Therefore, the determination of skin corrosion potential was performed in parallel on viable and freeze- killed tissues.
This step was a functional check which employs freeze-killed tissues that possess no metabolic activity but absorb and bind the test item like viable tissues.
Freeze-killed tissues were prepared prior to the study by placing untreated EPIDERMTM tissues in an empty 12 well plate and storing in a freezer (-14 to -30 °C) for a minimum of 24 hours. Before use each tissue was thawed by placing in 0.9 mL of assay medium for approximately 1 hour at room temperature.
In addition to the normal test procedure, the MTT reducing test item was applied to two freeze killed tissues per exposure period. In addition, two freeze killed tissues per exposure period remained untreated. The untreated freeze killed control showed a small amount of MTT reduction due to residual reducing enzymes within the killed tissues.

Assessment of Color Interference with the MTT Endpoint
A test item may interfere with the MTT endpoint if it is colored. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed.
50 µL of test item was added to 300 µL of sterile water. The solution was incubated in the dark at 37 oC, 5% CO2 in air for 60 minutes. A visual assessment of the color was then made.

Main Test
Pre-Incubation
The assay medium was pre-warmed before use. 0.9 mL of this assay medium was pipetted into the appropriate wells of two pre-labeled 6-well plates for both the 3 Minute and 60 Minute exposure periods. EpiDerm™ tissues were transferred into the 6 well plates containing the assay medium. The 6 well plates containing the EpiDerm™ samples were pre-incubated (37 °C, 5% CO2) for approximately 1 hour before dosing.

Application of Test Item and Rinsing
Before pre-incubation was complete, a 24 well plate was prepared for use as a “holding plate” for both the 3 Minute and 60 Minute exposure periods. This plate was used to maintain the viability of the tissue inserts between rinsing following chemical exposure and MTT loading. Another 24 well plate was prepared for the MTT loading. 300 µL of either pre warmed assay medium (holding plate) or MTT medium (MTT loading plate) was dispensed into each well. The two plates were placed into the incubator until required.
After pre incubation of the EpiDerm™ tissues, the medium was aspirated and replaced with 0.9 mL of fresh assay medium. The 6-well plate for the 3 Minute exposure period was returned to the incubator, while the other was being dosed for the 60 Minute exposure. For the 60 Minute exposure period, 50 µL of sterile distilled water (negative control) was added to the first two tissues. The tissues were dosed at regular intervals to allow for the time taken to rinse each tissue following exposure and to ensure that each tissue gets an equal exposure time. 50 µL of the test item and 50 µL of 8.0 N Potassium Hydroxide (positive control) were also applied to the corresponding tissues in turn. The plate was returned to the incubator (37 °C, 5% CO2) for the 60 Minute exposure period.
When dosing for the 60 Minute exposure period was complete, the same procedure was repeated for the 3 Minute exposure period. Because the exposure time was so short, the tissues were dosed at regular intervals to ensure that each tissue received an equal exposure time and to allow for the time taken to rinse each tissue following exposure. Rinsing was achieved by filling and emptying each tissue under a constant soft stream of DPBS to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper. Each tissue was placed into the prepared holding plate until all tissues were rinsed. They were then blotted and transferred to the 24 well plate prepared for MTT loading. The plate was incubated (37 °C, 5% CO2) for 3 hours. Once the 60 Minute exposure period was complete, the same rinsing and MTT loading procedure was repeated.
After the 3 Hour MTT incubation was complete, the inserts were blotted and transferred to labeled 24 well plates for MTT extraction. 2 mL of MTT extractant (isopropanol) was used to completely immerse each insert and the plate was covered with plate sealer to prevent Isopropanol evaporation. The plates stood overnight at room temperature, to allow extraction to proceed.

Absorbance/Optical Density Measurements
After extraction, each tissue was pierced with a pipette fitted with a 1000 µL tip and the extraction solution was forced vigorously up and down to form a homogenous solution. 3 x 200 µL aliquots of the extract were transferred to the appropriate wells of a pre labeled 96 well plate. 200 µL of isopropanol alone was added to the three wells designated as blanks. Absorbency at 570nm (OD570) of each well was measured using the Labtech LT 4500 microplate reader.

Irritation / corrosion parameter:
% tissue viability
Run / experiment:
exposure period 3 minutes
Value:
46.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
% viability: 100%
Positive controls validity:
valid
Remarks:
% viability: 4.9%
Remarks on result:
other: corrosive
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
exposure period 60 minutes
Value:
14.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
% viability: 100%
Positive controls validity:
valid
Remarks:
% viability: 3.5%
Remarks on result:
other: corrosive
Other effects / acceptance of results:
Quality criteria: The quality criteria required for acceptance of results in the test were satisfied.
The test item was considered to be corrosive: UN GHS H314 Combination of sub-categories 1B and 1C.

Quality Criteria
The mean OD570for the negative control treated tissues was 1.425 for the 3‑Minute exposure period and 1.636 for the 60‑Minute exposure period. The negative control acceptance criteria were therefore satisfied.
The relative mean tissue viability for the positive control treated tissues was 3.5% relative to the negative control following the 60‑Minute exposure period. The positive control acceptance criterion was therefore satisfied.
In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied

Direct MTT Reduction

An assessment found the test item was able to directly reduce MTT. Therefore, an additional procedure using freeze‑killed tissues was performed. The results of the freeze‑killed tissues were subtracted from the mean OD of the test item treated viable tissues to obtain the true amount of MTT reduction that reflects metabolic conversion only.

Assessment of Color Interference with the MTT endpoint

The solution containing the test item did not become colored. This was taken to indicate the test item did not have the potential to cause color interference.

Test Item, Positive Control Item and Negative Control Item

Mean OD570values and viabilities for the negative control, positive control and test item are given in Table1.

The relative mean viabilities for each treatment group were as follows:

3 minutes exposure: 46.6%

60 minutes exposure: 14.8%

     

Table 1 Mean OD570Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Tissue

Exposure Period

MeanOD570of individual tissues

Mean OD570of duplicate tissues (tvt)

Corrected Mean OD570(tvt-[tkt-ukt])

Standard Deviation

Coefficient of Variation

(%)

Relative Mean Viability (%)

Negative Control

3 Minutes

1.548

1.425

 

0.175

12.3

100*

1.301

 

60 Minutes

1.626

1.636

 

0.014

0.9

1.646

 

Positive Control

3 Minutes

0.082

0.071

 

0.016

na

4.9

0.059

 

60 Minutes

0.061

0.057

 

0.006

na

3.5

0.052

 

Test Item

3 Minutes

0.567

0.701

0.664

0.189

27.0

46.6

0.834

60 Minutes

0.247

0.254

0.242

0.009

3.6

14.8

0.260


OD= Optical density

tvt =       Treated killed tissues

tkt=        Untreated killed tissues

ukt =       Untreated killed tissues

*=   The mean % viability of the negative control tissue is set at 100%

na=  Not applicable

Interpretation of results:
Category 1B (corrosive) based on GHS criteria
Conclusions:
The test item was considered to be corrosive according to UN GHS H314 (category 1B).
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (corrosive)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
the study does not need to be conducted because the substance is classified as skin corrosion, leading to classification as serious eye damage (Category 1)
Endpoint:
eye irritation: in vivo
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
the study does not need to be conducted because the substance is classified as skin corrosion, leading to classification as serious eye damage (Category 1)
Endpoint conclusion
Endpoint conclusion:
no study available

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation/corrosion:

The purpose of this test is to evaluate the corrosivity potential of the test item using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes.

Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item.

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. The test item was found to directly reduce MTT and therefore additional non-viable tissues were incorporated into the testing for correction purposes. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT‑loading. After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction.

At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 mL samples were transferred to the appropriate wells of a pre-labeled 96‑well plate. The optical density (OD) was measured at 570 nm (OD570). Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The relative mean viabilities for each treatment group were as follows:

Exposure Period

Percentage Viability

Negative Control

Positive Control

Test Item

3 minute

100*

4.9

46.6

60 minute

100*

3.5

14.8

*The mean viability of the negative control tissues is set at 100%

The quality criteria required for acceptance of results in the test were satisfied.

The test item was considered to be corrosive: UN GHS H314 category 1B.

Justification for classification or non-classification

Based on the available data and the criteria of the CLP Regulation, the substance is classified as corrosive to the skin category 1B.

Therefore, the substance is also classified for serious eye damage category 1, based on the criteria of the CLP regulation.