Ethanol, 2,2'-iminobis-, N-tallow alkyl derivs., N-oxides

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

05 April - 23 May, 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:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

29 July 201

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Ethanol, 2,2'-iminobis-, N-tallow alkyl derivs., N-oxides

EC Number:

263-179-6

EC Name:

Ethanol, 2,2'-iminobis-, N-tallow alkyl derivs., N-oxides

Cas Number:

61791-46-6

Molecular formula:

n.a

IUPAC Name:

Ethanol, 2,2'-iminobis-, N-tallow alkyl derivs., N-oxides

Test material form:

semi-solid (amorphous): gel

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The Reconstructed Human Epidermal Model EpiDerm™ (EPI-200-SCT) was selected as test system to assess the skin corrosion potential of the test item as it represents a recommended in vitro test system according to OECD Guideline No. 431.

Vehicle:

water

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Reconstructed Human Epidermal Model - EpiDerm™ (EPI-200-SCT)
- Tissue lot number: 25892
- Date of initiation of testing: 05-04-2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during exposure:
3 min: Room temersture
1 h: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After completion of 1 hour period of test item exposure, the tissues were rinsed with sterile PBS (fill and empty insert 20 times in a constant soft stream of PBS) to remove any residual test item/NC/PC.
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: 96-well plate reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
see: " Any other information on materials and methods"

NUMBER OF REPLICATE TISSUES: 2

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
A test item is considered to be “corrosive” to skin in accordance with UN GHS Category 1, if;
Step 1
The tissue viability after 3 minutes exposure is < 50%.
The tissue viability after 3 minutes exposure is ≥ 50% and < 15% after 1 hour exposure.
The test item is considered as non-corrosive to skin in accordance with UN GHS No Category, if the tissue viability after 3 minutes exposure is ≥ 50% and ≥1 5% after 1 hour incubation exposure.
Step 2
A test item identified as being corrosive in step 1 is further subcategorised in accordance with UN GHS based on the following:
The tissue viability after 3 minutes exposure is < 25%: optional subcategory 1A.
The tissue viability after 3 minutes exposure is ≥ 25 %: a combination of optional Sub-Categories 1B-and-1C.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 25 mg + 25 µL water

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL

Duration of treatment / exposure:

3 min or 1 h

Number of replicates:

2

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min treatment

Value:

96.5

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 h treatment

Value:

79.6

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

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

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 2: Summary of the 3 min treatment

Treatment		OD	Viability (%)	Classification
Negative Control (Sterile water)	Mean	1.748	100.0	NC
	±SD	0.046	3.8
	n	2	2
Positive Control (Glacial acetic acid)	Mean	0.027	1.6	C (Category 1A)
	±SD	0.003	0.3
	n	2	2
Test Item (Humectol LYS Mod )	Mean	1.656	96.5	NC
	±SD	0.183	15.1
	n	2	2

Table 3: Summary of the 1 h treatment

Treatment		OD	Viability (%)	Classification
Negative Control (Sterile water)	Mean	1.871	100.00	NC
	±SD	0.024	1.8
	n	2	2
Positive Control (Glacial acetic acid)	Mean	0.025	1.3	C (Category 1A)
	±SD	0.005	0.4
	n	2	2
Test Item (Humectol LYS Mod )	Mean	1.490	79.6	NC
	±SD	0.268	20.2
	n	2	2

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In an in vitro skin corrosion assay (RhE) according to OECD guideline 431, the thest item did not show skin corrosive properties.

Executive summary:

The skin corrosive potential of the test item was evaluated in an in vitro skin corrosion assay (RhE) according to OECD guideline 431. In an pre-test the test item did not develope 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.

Test items were exposed for 1 hour and 3 minutes separately. All the treatments were maintained in duplicates. For 3 minutes and 1 h treatment, 50 µL of sterile distilled water (NC) was dispensed into the inserts atop the tissue. Tissues were treated with 25 mg test item + 25 µL distilled water or 50 µL of positive control (glacial acetic acid). Tissues were incubated for 3 min at room temperature or 1 h at 37 °C. At the end of treatment time tissue inserts were rinsed with sterile PBS. In an MTT assay, the viability of the tissues was calculated. The optical density of the extracted formazan was measured in 96-well plate spectrophotometer at 570 nm.

For 3 minutes exposure, percentage viability of negative control, positive control and test item was 100 ± 3.8%, 1.6 ± 0.3% or 96.5 ± 15.1%, respectively. As the percentage viability of test item was greater than 50% of negative control, the test item is considered as non-corrosive, whereas the percentage viability of positive control (PC) is less than 50% of negative control and clearly represents the irritation potential of positive control. For 1 hour exposure, percentage viability of negative control, positive control and test item was 100 ± 1.8, 1.3 ± 0.4 and 79.6 ± 20.2 respectively. As the percentage viability of test item was greater than 15% of negative control, the test item is considered as non-corrosive, whereas the percentage viability of positive control (PC) is less than 50% of negative control clearly represents the irritation potential of positive control.

Based on the results obtained under the laboratory testing conditions, the test item is categorized as non-corrosive to reconstructed human epidermis (RhE) (UN GHS No category), as the mean percentage tissue viability was greater than 50% after 3 minutes exposure and greater than 15% after 1 hour exposure of the negative control.