1-Propanaminium, 2-hydroxy-N-(2-hydroxyethyl)-N-methyl-N-[2-[(1-oxo-9-octadecen-1-yl)amino]ethyl]-3-sulfo-, methyl sulfate, sodium salt (1:1:1)

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

4 April 2018 - 8 April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

28 July 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

6th July 2012

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: SOP: In Vitro EpiDerm™ Skin Irritation Test (EPI-200-SIT) for use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm™ (EPI-200-SIT).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of fatty acids, C16 and C18 (unsaturated) and 2-(2-aminoethylamino)ethanol, subsequently reacted with 1-chloro-2,3-epoxypropane and sodium hydrogensulfite, partially quaternized with dimethyl sulfate, sodium salts

EC Number:

701-473-2

Cas Number:

73791-61-4

Molecular formula:

C26H53N2O6S.CH3O4S.Na

IUPAC Name:

Reaction products of fatty acids, C16 and C18 (unsaturated) and 2-(2-aminoethylamino)ethanol, subsequently reacted with 1-chloro-2,3-epoxypropane and sodium hydrogensulfite, partially quaternized with dimethyl sulfate, sodium salts

Test material form:

other: paste

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

The Reconstructed Human Epidermal Model - EpiDerm™ (EPI-200-SIT) was used as test system (MatTek In Vitro Life Science Laboratories, s.r.o, MlynskéNivy 73, 821 05, Bratislava II, Slovak Republic).

Justification for test system used:

As recommended in OECD Guideline No. 439, Reconstructed Human Epidermal Model EpiDerm™ (EPI-200-SIT) has been selected as test system for in vitro skin irritation. The RhE test system uses human derived non-transformed keratinocytes as cell source to reconstruct an epidermal model with representative histology and cytoarchitecture.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ (EPI-200-SIT), MatTek In Vitro Life Science Laboratories, s.r.o, MlynskéNivy 73, 821 05, Bratislava II, Slovak Republic
- Tissue batch number: 25891
- Date of initiation of testing: 4th April 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 35 min at 37 °C, 25 min at room temperature
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: tissues were rinsed with sterile DPBS by filling and emptying the tissue insert for 15 times to remove any residual test item. The constant stream of DPBS was applied from the nearest distance from the tissue surface. After the 15th rinse with washing bottle, the inserts were completely submerged 3 times in approximately 50 mL of DPBS and shaken to remove all traces of test item/NC/PC.
- Observable damage in the tissue due to washing: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: plate reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS
- Viability: 1.577
- Barrier function: ET50= 5.34 hours
- Morphology: proven presence of a functional stratum corneum, a viable basal cell layer and intermediate spinous and granular layers
- Contamination: sterile


NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
None - The test substance did not directly reduce MTT.

DECISION CRITERIA
- The test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1) If the tissue viability after exposure and post-treatment incubation is ≤ 50%.
- The test item is considered as non-irritant to skin in accordance with UN GHS No Category, if the tissue viability after exposure and post-treatment incubation is > 50%.

ACCEPTANCE CRITERIA
1. The assay meets the acceptance criterion if the mean OD570 of the NC tissues is ≥ 0.8 and ≤ 2.8.
2. The assay meets the acceptance criterion if the mean viability of PC tissues expressed as % of the negative control tissues should be ≤ 20%. If the mean viability of PC tissues is not within the ≤ 20% and the SD of the three tissues replicates above 18%, the assay will be repeated.
3. The assay meets the acceptance criterion if the standard deviation (SD) calculated from individual % tissue viabilities of the 3 identically treated replicates is < 18%.
Note: Test items that provide tissue viabilities in a range of 30 to 70% may provide high SD. If the high SD (above acceptance limits) is typical for the test item and the classification of the test item is consistent in all independent runs, it is recommended to accept this result, although the Assay Acceptance Criterion 3 is not met.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 25 mg

NEGATIVE CONTROL
- Amount applied: 30 µL

POSITIVE CONTROL
- Amount applied: 30 µL
- Concentration: 5%

Duration of treatment / exposure:

60 min

Duration of post-treatment incubation (if applicable):

39 hours and 45 minutes

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1st run

Value:

9.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

3.3%

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No
- Colour interference with MTT: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Any other information on results incl. tables

Table 1: Summary of optical density (OD) and viability (%)

Treatment		OD	Viability (%)	Classification
Negative Control (DPBS)	Mean	1.766	100	NI
	±SD	0.011	0.62
	n	3	3
Positive Control (5% Sodium Dodecyl Sulphate)	Mean	0.059	3.3	I
	±SD	0.005	0.26
	n	3	3
Test Item	Mean	0.165	9.4	I
	±SD	0.001	0.03
	n	3	3

NI = Non Irritant; I = Irritant; n = No. of tissues; SD = Standard Deviation

Applicant's summary and conclusion

Interpretation of results:

other: study cannot be used to decide on classification alone, further information necessary

Conclusions:

Based on the results obtained under the laboratory testing conditions, the test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), as the mean percentage tissue viability was less than 50% of the negative control. Since the test guideline cannot resolve between UN GHS Categories 1 and 2, further information on skin corrosion will be required to decide on its final classification.

Executive summary:

A study was conducted to assess the skin irritation potential of the test item according to OECD Guideline No. 439 using Reconstructed Human Epidermal Model - EpiDerm™ (EPI-200-SIT).

Tissues were topically exposed to 30 µL of DPBS (negative control: NC), 30 µL of 5% aq. SDS solution (positive control: PC) or 25 mg of test item. All the treatments were maintained in triplicates. After 60 minutes of exposure the tissues were washed using DPBS. Later, the tissue inserts were blotted and transferred to fresh medium and incubated in CO2 incubator at 37°C for 22 hours and 57 minutes. After incubation period (Day 1), tissue inserts were shifted from upper wells to lower wells of 6-well plates prefilled with 0.9 mL of assay medium. After the media change, tissues were incubated for an additional 16 hours and 48 minutes in CO2 incubator at 37°C. After the post-incubation period, the bottom of the tissue inserts was blotted and transferred into an MTT solution and incubated for 3 hours. The resultant purple-blue formazan salt, formed mainly by mitochondrial metabolism, was extracted for 2 hours using extraction solvent and the OD was measured at 570 nm.

The test item did not develop any colour when dissolved in distilled water/isopropanol and was considered as non-reducer of MTT as no purple colour was developed when mixed and incubated with MTT solution.

Percentage viability of negative control, positive control and test item was 100±0.62, 3.3±0.26 and 9.4±0.03 respectively. As the percentage viability of the test item was not greater than 50% of the negative control, the test item is considered as “irritant”. The percentage of viability in the positive control (PC) was less than 50%, which shows the irritative potential of the positive control and the suitability of the test method. As the test method does not allow to distinguish between Category 1 and 2, further testing is required to exclude or confirm a corrosive property.