Reaction mass of C18 (unsatd.) fatty acid amides/esters of diethanolamine, C16-18 (even-numbered) fatty amine ethoxylates, castor oil ethoxylates and sulfosuccinates of C18 (unsatd.) fatty acid diethanolamide, sodium salt

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From August 3, 2015 to August 7, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of C18 (unsatd.) fatty acid amides/esters of diethanolamine, C16-18 (even-numbered) fatty amine ethoxylates, castor oil ethoxylates and sulfosuccinates of C18 (unsatd.) fatty acid diethanolamide, sodium salt

EC Number:

943-172-0

IUPAC Name:

Reaction mass of C18 (unsatd.) fatty acid amides/esters of diethanolamine, C16-18 (even-numbered) fatty amine ethoxylates, castor oil ethoxylates and sulfosuccinates of C18 (unsatd.) fatty acid diethanolamide, sodium salt

Test material form:

semi-solid (amorphous): gel

Remarks:

Paste

Details on test material:

- Name of test material (as cited in study report): Vegetable or animal oil, hydrogenated, reaction products with diethanolamine, maleic anhydride and sodium sulphite
- Lot No.: CH 192435/01
- Purity: Not applicable (UVCB)
- Organic Carbon: 30.2%
- Hydrotox No.: 15/5241
- Origin: DACC
- Stability: Stable within the durability and with appropriate storage conditions
- Solubility in water: ~1 g/L
- Appearance: Light beige liquid/paste
- Storage conditions: Ambient temperature, dark, dry
- Expiration date: July 15, 2015
- Safety directions: H315, H319. Use protective clothing: gloves and protection goggles.

Test animals

Details on test animals or test system and environmental conditions:

TEST SYSTEM
EpiDerm Skin Model (EPI-200, Lot no.: 22625 kit U) consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized 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 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.

SOURCE
EpiDerm Skin Model were procured from MatTek Corporation, Ashland MA, U.S.A.

CELL CULTURE
Medium: DMEM (Dulbecco’s Modified Eagle’s Medium); Supplemented DMEM medium, serum-free supplied by MatTek Corporation
MTT medium: MTT concentrate (5 mg/mL) diluted (1:5) with MTT diluent (supplemented DMEM). Both supplied by MatTek Corporation
Environmental conditions: Humid atmosphere- 80-100% (actual range 67 - 91%), containing 5.0 ± 0.5% CO2 in air in the dark,
Temperature- 37.0 ± 1.0°C (actual range 36.7 - 37.4°C).

Test system

Duration of treatment / exposure:

2 tissues per test substance- 3 min exposure
2 tissues per test substance- 1 h exposure

Details on study design:

Application/Treatment of the test substance
The skin tissues were kept in the refrigerator the day they were received. The next day, at least 1 h before the assay was started the tissues were transferred to 6-well plates containing 0.9 mL DMEM medium per well. The level of the DMEM medium was just beneath the tissue. The plates were incubated for 1.5 h at 37.0 ± 1.0⁰C. The medium was replaced with fresh DMEM medium just before test substance was applied.

The test was performed on a total of 4 tissues per test substance together with a negative control and positive control. Two tissues were used for a 3 min exposure to the test substance and two for a 1 h exposure. The skin was moistened with 25 μL Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test substance to the tissue and an excessive amount was added into the 6-well plates on top of the skin tissues using a curved spatula and gauze patch. The remaining tissues were treated with 50 μL Milli-Q water (negative control) and with 50 μL 8N KOH (positive control), respectively. After the exposure period, the tissues were washed with phosphate buffered saline (Invitrogen Corporation, Breda, Netherlands) to remove residual test substance. Rinsed tissues were kept in 24 well plates on 300 μL DMEM medium until 6 tissues (= one application time) were dosed and rinsed.

The DMEM medium was replaced by 300 μL MTT-medium and tissues were incubated for 3 h at 37°C in air containing 5% CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 mL isopropanol (MatTek Corporation) over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.

Cell viability was calculated for each tissue as percentage of the mean of the negative control tissues. Skin corrosion potential of the test substance was classified according to remaining cell viability following exposure of the test substance with either of the two exposure times.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: other: mean relative tissue viability

Run / experiment:

3 minutes

Value:

ca. 89

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

other: other: mean relative tissue viability

Run / experiment:

1 hour

Value:

ca. 91

Remarks on result:

no indication of irritation

Any other information on results incl. tables

Table 1: Mean absorption in the in vitro skin corrosion test

	3 min application					1 h application
	A (OD570)	B (OD570)	Mean (OD570)		SD	A (OD570)	B (OD570)		Mean (OD570)		SD
Negative control	1.781	1.917	1.849	±	0.096	1.927		1.872	1.899	±	0.039
Test substance	1.770	1.528	1.649	±	0.171	1.748		1.703	1.725	±	0.031
Positive control	0.138	0.156	0.147	±	0.013	0.131		0.138	0.134	±	0.005

SD- Standard deviation

OD- Optical density

Table 2: Mean tissue viability in the in vitro skin corrosion test

	3 min application viability (percentage of control)	1 h application viability (percentage of control)
Negative control	100	100
Test substance	89	91
Positive control	8	7

The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The mean relative tissue viability following 3 min exposure to the positive control was 8%. Because the mean relative tissue viability for the test substance was not below 50% after 3 min treatment and not below 15% after 1 h treatment, test substance is considered to be not corrosive.

Applicant's summary and conclusion

Interpretation of results:

other: CLP criteria not met

Conclusions:

The test substance was not corrosive to the skin.

Executive summary:

A study was conducted to evaluate the skin corrosion potential of the test substance in an in vitro human three dimensional epidermal model (EpiDerm (EPI-200)) according to OECD Guideline 431 and EU method B.40, in compliance with GLP. Skin tissue was moistened with 25 μL of Milli-Q water and an excessive amount of the test substance was applied directly on its surface. Skin corrosion is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 3 min and 1 h treatments with the test substance compared to the negative control tissues was 89 and 91%, respectively. Because the mean relative tissue viability was not below 50% after the 3 min treatment and not below 15% after the 1 h treatment, the test substance was considered to be not corrosive. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The mean relative tissue viability following 3 min exposure to the positive control was 8%. Under the study conditions, the test substance was not corrosive to skin (Eurlings IMJ, 2015a).