Reaction products of 1H-Imidazole-1-ethanol, 4,5-dihydro-, 2-(C11-17 and C17 unsatd. alkyl) derivs. and sodium hydroxide and 2-propenoic acid

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-07-28 to 2015-10-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(Original Guideline adopted July 28, 2015)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

ß-Alanine, N-(2-aminoethyl)-N-(2-hydroxyethyl)-, N-(C12-C18 and C18unsatd. acyl) derivs., monosodium salts

Cas Number:

93820-52-1

Molecular formula:

C12 fatty acid based: C19H37N2NaO4 - C18 fatty acids based: C23H45N2NaO4

IUPAC Name:

ß-Alanine, N-(2-aminoethyl)-N-(2-hydroxyethyl)-, N-(C12-C18 and C18unsatd. acyl) derivs., monosodium salts

Constituent 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

dihydrogen oxide

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): Aqueous solution of beta-Alanine, N-(2-aminoethyl)-N-(2-hydroxyethyl)-, N-cocoacyl derivs., monosodium salts

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

accordign to guideline

Vehicle:

unchanged (no vehicle)

Details on test system:

EpiSkin™ kits are purchased from SkinEthic Laboratories (69007 Lyon, France). The EpiSkin™ tissue 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 EpiSkin™ tissues (surface 0.38 cm²) are cultured on specially prepared cell culture inserts. EpiSkin™ tissues were shipped at ambient temperature on medium-supplemented agarose gels in a 12-well plate and reached Envigo CRS GmbH on 20 October 2015. On the same day of experiment EpiSkin™ tissues were transferred to 12-well plates with maintenance medium and the pre-incubation phase of the EpiSkin™ tissues started.

TREATMENT:
Three tissues of the human skin model EpiSkin™ were treated each with 10 µL of the test item, the negative control (deionised water) or the positive control (5% Sodium lauryl sulfate) for 15 minutes.
After the end of the treatment interval the inserts were removed immediately from the 12-well plate. Using a wash bottle the tissues were gently rinsed with PBS to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper. The inserts were placed in the plates with 2 mL maintenance medium. The tissues were incubated for nearly 43 hours at 37 ± 1.5 °C, 5 ± 0.5% CO2 .

MTT ASSAY:
The MTT concentrate was prepared freshly and diluted with the MTT diluent. A 12-well plate was filled with 2 mL assay medium containing 0.3 mg/mL MTT per well.
After the treatment procedure was completed for all tissues the cell culture inserts were transferred from the holding plates to the MTT-plates. After a 3 hour incubation period (37 ± 1.5 °C, 5 ± 0.5% CO2) MTT solution was aspirated from the wells and the wells were rinsed three times with PBS. Tissue samples were cut out of the inserts with a biopsy punch and transferred into plastic vials. The tissue samples were immersed into extractant solution by gently pipetting 0.5 mL extractant solution (isopropanol containing 0.04 N HCl) into each vial. The tissue samples were completely covered by isopropanol. The vials were sealed to inhibit isopropanol evaporation. The formazan salt was extracted for about 68 hours without shaking in the refrigerator.
Per each tissue sample 2 × 200 µL aliquots of the formazan blue solution were transferred into a 96-well flat bottom microtiter plate. OD was read in a microplate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, version 4.7.1) with 570 ± 1 nm filter. Mean values were calculated from the 2 wells per tissue sample.

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL (26.3 µL/cm²)
- Concentration (if solution): 40% a.i.

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

43 h

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

102.1

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

In vivo

Irritant / corrosive response data:

The mean relative absorbance value of the test item, corresponding to the cell viability, did not decrease (102.1%; threshold for irritancy ≤ 50%), consequently the test item was not irritant to skin.

Any other information on results incl. tables

Evaluation of Results:

The mean OD of the three negative control tissues was calculated. This value corresponds to 100% tissue viability in the current test.

For the test item and the positive control the mean relative viability ± rel. standard deviation of the three individual tissues are calculated and used for classification according to the following prediction model:

For the current test, an irritation potential of a test item according to EU classification H315 (according to regulation (EC) 1272/2008), and GHS category 2 according to UN GHS (published 2003, last (3rd) revision 2009) is recommended if the mean relative tissue viability of three individual tissues is reduced ≤ 50% of the negative control.

Results after treatment with test item and controls

Dose Group	Treatment Interval	Absorbance 570 nm Tissue 1	Absorbance 570 nm Tissue 2	Absorbance 570 nm Tissue 3	Mean Absorbance of 3 Tissues	Relative Absorbance [%] Tissue 1,2,3	Relative Standard Deviation [%]	Rel. Absorbance [% negative control]
Negative control	15 min	0.991	0.915	0.870	0.925	107.1	6.6	100.0
						98.9
						94.1
Positive control	15 min	0.113	0.134	0.139	0.129	12.2	10.6	13.9
						14.5
						15.0
Test Item	15 min	0.917	0.973	0.943	0.944	99.1	3.0	102.1
						105.1
						102.0

The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not led to a change in colour. Optical evaluation of the MTT-reducing capacity of the test item after 3 hours incubation with MTT-reagent did not show blue colour.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this in vitro skin irritation test Amphopropionates C12-18 (40% a.i.) was not irritating.

Executive summary:

In an in vitro dermal irritation study according to OECD guideline 439, adopted July 28, 2015, Amphopropionates C12-18 (40% a.i.) was applied in triplicate (10 µL) to human skin model EpiSkin™ tissues for 15 min. Deionised water was used as negative control, 5% SLS as positive control. After exposure the skin tissues were thoroughly rinsed to remove the test substance and transferred to fresh medium.

After a 43-hour post-treatment incubation the cell viability measured by dehydrogenase conversion of MTT into a blue formazan salt was assessed as predictor for skin irritancy potential. 

After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD ≥ 0.6 till ≤ 1.5 for the 15 minutes treatment interval thus showing the quality of the tissues. Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 15 minutes treatment interval thus ensuring the validity of the test system.

The relative mean tissue viability obtained after 15 minutes treatment with the test substance compared to the negative control tissues was 102.1%. Since the mean relative tissue viability was above 50%, the test substance is considered to be non-irritant.

The positive control had a mean cell viability of 13.9% after 15 minutes exposure.

In this in vitro skin irritation test Amphopropionates C12-18 (40% a.i.) was not irritating.