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Diss Factsheets

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:
From March 27, 2017 to April 06, 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 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
Updated Guideline adopted July 29, 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
morpholin-4-ium morpholine 6-(4-methylbenzenesulfonamido)hexanoate
EC Number:
944-090-8
Molecular formula:
C13H19NO4S.C4H9NO/C4H9NO/H2O
IUPAC Name:
morpholin-4-ium morpholine 6-(4-methylbenzenesulfonamido)hexanoate
Test material form:
liquid

In vitro test system

Test system:
human skin model
Source species:
other: Human Skin Model Test with EpiDermTM tissues models
Cell type:
other: Normal, human-derived epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis
Justification for test system used:
The In vitro Skin Corrosion: Human Skin Model Test is based on the observation that skin corrosion (necrotic damage of viable skin cells) shows a high correlation with skin cell cytotoxicity, occurring rapidly after brief exposure of the skin barrier (stratum corneum) to a corrosive chemical. It is designed to predict and classify the skin corrosivity potential of a chemical by using a three-dimensional human epidermis model. Ultrastructurally, the skin models closely parallel human skin
Vehicle:
unchanged (no vehicle)
Details on test system:
HUMAN SKIN MODEL TEST WITH EPIDERMTM TISSUES MODEL
- Model used: Human epidermis model derived from human keratinocytes and consists of normal, human-derived epidermal keratinocytes (NHEK)
- Date of initiation of testing: March 27, 2017

TEMPERATURE USED FOR TEST SYSTEM
- Pre-incubation period: EpiDermTM tissues were pre-incubated at least 1 - 24 hours before dosing. Inserts were transferred from the refrigerator into 6-well plates containing pre-warmed assay medium (temperature not specified)
- Temperature used during treatment / exposure: EpiDermTM tissues were treated with the test item and a positive and negative control and prepared in duplicate. During treatment, EpiDermTM tissues within a set of well plates were kept at room temperature for a 3 ± 0.5 minute exposure period. The second replicate of well plates underwent a 60 ± 5 minute exposure period in an incubator (37 ± 1.5 °C, 5 ± 0.5 % CO2)
- Temperature of post-treatment incubation: As part of the MTT-100 assay (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazoliumbromide), tissues were incubated for 3 hours (37 ± 1.5 °C, 5 ± 0.5 % CO2)

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 50 µL (79.4 µL/cm2 according to guideline) of the test item and controls was dispensed directly onto duplicate EpiDermTM tissue surfaces. After the exposure periods, the tissues were removed from the 6-well plate and gently
rinsed using a wash bottle / multi-pipette containing 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
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: Yes, at the end of the exposure period the tissues were rinsed using DPBS instead of PBS specified in the guideline

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: The MTT-solution was prepared freshly on day of use (resulting: 1 mg/mL). MTT concentrate from MatTek, MTT diluent from MatTek
- Incubation time: Well plates containing MTT solution (300 µL) were prepared in the pre-warming period and kept in an incubator (37 ± 1.5 °C, 5 ± 0.5% CO2) until required. Following rinsing after the exposure period, the tissues were transferred from the holding plates to the MTT-plates and incubated for a 3 hour period (37 ± 1.5 °C, 5 ± 0.5% CO2)
- Spectrophotometer: Microplate reader (Versamax®, Molecular Devices, SoftMax Pro Enterprise (version 4.7.1))
- Wavelength: 570 nm (OD570)
- Filter: No reference filter

NUMBER OF REPLICATE TISSUES: Tissues prepared in duplicate

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Justification: A test item may interfere with the MTT endpoint if: a) it is coloured and/or b) able to directly reduce MTT
- Pre-test for colour interference: 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
- Pre-test for direct MTT reduction: 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 must be performed

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is < 50%, or if the viability after 3 minutes exposure is ≥50 % and the viability after 1 hour exposure is <15%
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is ≥50% and the viability after 1 hour exposure is ≥15%
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL (79.4 µL/cm2 according to guideline) of the test item were dispensed directly onto duplicate EpiDermTM tissue surfaces

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL was applied to each set of duplicate tissues for the 3 min and 1 hour exposure periods

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL was applied to each set of duplicate tissues for the 3 min and 1 hour exposure periods
Duration of treatment / exposure:
Two exposure groups consisting of 3 ± 0.5 and 60 ± 5 minutes. After the pre-incubation 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)
Duration of post-treatment incubation (if applicable):
Following the exposure period, tissues were rinsed and transferred from the holding plates to MMT-plates and incubated for 3 hours (37 ± 1.5 °C, 5 ± 0.5% CO2)
Number of replicates:
Duplicate

Test system

Type of coverage:
not specified
Preparation of test site:
other: At least 1 hour or maximum 24 hours before dosing, EpiDerm™ tissues were removed from the refrigerator to 6-well plates containing the pre-warmed assay medium
Vehicle:
unchanged (no vehicle)
Controls:
yes

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Remarks:
Relative absorbance (% of negative control)
Run / experiment:
3 minutes
Value:
76
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Remarks:
Relative absorbance (% of negative control)
Run / experiment:
60 minutes
Value:
47.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: Optical evaluation after 1 hour revealed no blue colour and, therefore, the test item did not reduce MTT
- Colour interference with MTT: No colour interference noted

DEMONSTRATION OF TECHNICAL PROFICIENCY:
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

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, range: 1.557 - 1.681
- Acceptance criteria met for positive control: Yes, 3.4 %
- Acceptance criteria met for variability between replicate measurements: Yes, range: 1.2 - 13.7 %

Any other information on results incl. tables

Results after treatment with the reaction mass of morpholine and 6-[(p-tosyl)amino]hexanoic acid, compound with morpholine (1:1) and the controls:

Dose Group Exposure Interval Mean Absorbance (OD) of 2 Tissues CV (%) Relative Absorbance (% of Negative Controls)
Negative Control

3 minutes

1.553 2.1 100.0
Positive Control 0.349 1.2 22.5
Test Item 1.179 2.6 76.0
Negative Control 60 minutes  1.583 5.6 100.0
Positive Control 0.054 0.2 3.4
Test Item 0.754 13.7 47.6

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The relative mean tissue viability calculated as a percentage of the negative control was 47.6% after the 60 minutes exposure and 76% after the 3 minutes exposure. In accordance with CLP Regulation (EC) No. 1272/2008, the substance can be considered as non-corrosive to the skin.
Executive summary:

An in vitro skin corrosion study was undertaken using a Human Skin Model Test with EpiDerm™ tissues model to determine the corrosive potential of the reaction mass of morpholine and 6-[(p-tosyl)amino]hexanoic acid, compound with morpholine (1:1) in accordance with the OECD Testing Guideline 431. The test was GLP-compliant.

Reaction mass of morpholine and 6-[(p-tosyl)amino]hexanoic acid, compound with morpholine (1:1) did not reduce MTT ( 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazoliumbromide), neither did it dye water in a pre-test, indicating that the substance would not interfere with the MTT endpoint.

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. The validity of the test system and the specific batch of the tissue models was confirmed according to the assay acceptability criteria. After exposure of the tissues to the test item the relative absorbance value decreased to 76.0% after 3 minutes exposure. After 1 hour exposure the relative absorbance value was reduced to 47.6%. 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 Reaction mass of morpholine and 6-[(p-tosyl)amino]hexanoic acid, compound with morpholine (1:1) is non corrosive to skin according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.