Reaction mass of N-(2-hydroxyethyl)alkyl] alkylamide and glycerol

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 24 November 2016 and 09 December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is considered to be reliability 1 as it has been conducted according to OECD Test Guideline 439 using the EPISKIN™ Reconstructed Human Epidermis Model and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test material form:

liquid

Details on test material:

Chemical Name: N-[2-(2-Hydroxyethoxy)ethyl]acetamide
CAS No.: 118974-46-2
Batch: 27191705
Purity: 82% (dose calculation was adjusted to puriy)
Appearance: Pale to yellow liquid
Expiry Date: 16 June 2017
Storage Conditions: At room temperature
Stability in Solvent: Stable in water (not quantified)
Purpose of Use: Industrial chemical

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other:

Source strain:

not specified

Test animals

Species:

other: EPIKSIN in vitro Reconstructed Human Epidermis (RHE) Model

Strain:

other: Not applicable

Details on test animals or test system and environmental conditions:

Epi-200 SIT kits
Supplier : MatTek Corporation, 82105 Bratislava, Slovakia
Date received : Not reported

Epi-200 SIT Kit lot number : 23383
Maintenance Medium lot number :23383
Assay Medium lot number :23383

Test system

Type of coverage:

other:

Preparation of test site:

other:

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

other: Dissiminated (delete when editing): Positive control can be added here.

Amount / concentration applied:

Test material
- Applied volume: 30 ul
- Concentration (if solution): undiluted

Positive and negative control: 30 µL each, used as supplied

Duration of treatment / exposure:

60-Minute exposure period and 43 hours post-exposure incubation period.

Number of animals:

A total of 9 tissues were used: Triplicate tissues were treated with: test substance, positive control or negative control.

Details on study design:

Test for Direct MTT Reduction and Colour Interference
A test item may interfere with the MTT endpoint if: a) it is coloured and/or b) able to directly reduce MTT. The MTT assay is affected only if the test item is present in the tissues when the MTT viability test is performed.
Some non-coloured test items may change into coloured test items in wet or aqueous conditions and thus stain tissues during the 60 min exposure. Therefore, before exposure, a functional check for this possibility has to be performed (step 1).
Step 1
30 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.
Since the test item did not dye water or did not change colour in the presence of water, an additional test (step 2) with viable tissues (but without MTT addition) was not necessary to be performed.
Step 3
All test items (including those already evaluated in step 1 and step 2) were further evaluated for their potential to interfere with MTT assay. To test if a test item directly reduces MTT, 30 µL of the test item were added to 1 mL of the MTT-solution (1mg/mL) and was incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5% CO2) for 60 minutes. Untreated MTT medium was used as control.
Since the MTT solution did not turn blue/purple, the test item was not considered to reduce MTT and an additional test with freeze-killed tissues did not have to be performed.

EXPERIMENTAL PERFORMANCE
Pre-warming of EpiDerm™ Tissues
The plastic bag containing the 24-well plate with epidermal tissues was opened under sterile conditions. Under an airflow using forceps, the gauze was removed and the inserts were taken out. Any remaining agarose that adheres to the outer sides of the inserts was removed by gentle blotting on the sterile filter paper or gauze, and the tissues were placed in the empty, sterile 6-well plate. Prior to the exposure of the test item and of the controls the EpiDerm™ tissues were inspected for quality: If necessary, it was taken care, that
1. air bubbles between agarose and insert were not > 30% of the total surface,
2. liquid on top of the insert was removed with sterile cotton tips,
3. if again moisture is observed on top of the inserts after the pre-incubation or in
case of visible defects the respective skin models were discarded.
0.9 mL of the assay medium (20 – 25 °C) was pipetted into each well of sterile 6-well plates. The inserts with the EpiDerm™ tissues were placed in the upper wells, and were pre-incubated for 60 minutes in the incubator (37 ± 1.5 °C, 5 ± 1% CO2, 95 ± 5% RH). Following, the inserts were transferred from upper wells into the lower wells of the 6-well plates, and, the pre-incubation was continued for further approximately 21 hours (37 ± 1.5 °C, 5 ± 1% CO2, 95 ± 5% RH).

Treatment
After pre-incubation of EpiDerm™ tissues was completed, medium was replaced by 0.9 mL of fresh medium per well. The negative and positive control, and the test item were added into the insert atop the corresponding EpiDerm™ triplicate tissues. The treatment time was 60 minutes in total. Within this period the 6-well plates were put into the incubator for 35 minutes at 37 ± 1.5 °C, 5 ± 0.5 % CO2. In the remaining period the plates were placed in a sterile bench at room temperature until the end of treatment.
After the end of the treatment interval the inserts were removed immediately from the 6-well plate and tissues were gently rinsed with DPBS at least 15 times in order to remove any residual test material. After the rinsing the inserts were submerged in DPBS at least three times. Afterwards the inserts were once again rinsed with sterile DPBS from the inside and the outside. Excess DPBS was removed by gently shaking the inserts and blotting the bottom with sterile blotting paper. The tissues were then transferred into new 6-well plates with 0.9 mL of fresh assay medium in the upper row. The inserts were placed in the prepared holding plate. Tissues were incubated for nearly 25 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2. After incubation the inserts were transferred into new 6-wells plates containing fresh medium. Thereafter tissues were incubated for another 18 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2. The complete incubation period was approximately 43 hours.

MTT Assay
On the day of testing the MTT concentrate was diluted with the MTT diluent (1 mg/mL). The 24-well plates were prepared before the end of the tissue pre-warming period. A volume of 300 µL of the MTT solution was added to each well and the plates were kept in an incubator (37 ± 1 °C, 5 ± 0.5 % CO2) until further use.
After the 42-hours incubation period was completed for all tissues and exposure groups, culture inserts were transferred from the holding plates to the MTT-plates. After a 3-hour incubation period (37 ± 1 °C, 5 ± 0.5 % CO2), the tissues were rinsed three times with DPBS, and carefully dried with blotting paper. The tissues were transferred into new 24-well plates containing 2 mL of extractant solution (isopropanol) in each well ensuring that the tissues are completely covered. The 24-well plate was sealed to inhibit the isopropanol evaporation.
The formazan salt was extracted for about 2 hours while shaking (~120 rpm) at room temperature.
After the extraction period was completed, the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken. Afterwards the insert was discarded. The 24-well plates were placed on a shaker for 15 minutes until the solution was homogeneous in colour.
Per each tissue, 3  200 µL aliquots of the blue formazan solution were transferred into a 96-well flat bottom microtiter plate from the 15 minutes exposure. OD was read in a microplate reader (Versamax® Molecular Devices, Softmax Pro, version 4.7.1) with a 570 nm filter. Mean values were calculated from the 3 wells per tissue

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: other: Mean relative Absorbance (% Negative Control)

Value:

> 80.6

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Any other information on results incl. tables

Results after treatment with the test item and the controls

Treatment	Tissue No.	Mean Absorbance in 3 wells after Blank correction	Mean Absorbance of 3 tissues after blank correction *	Rel. Absorbance [%] Tissue 1, 2 + 3**	Relative Standard Deviation [%]	Mean Rel. Absorbance [% of Negative Control]***
Blank		0.000		-	-	-
Negative Contol	1	1.469	1.336	1.110	8.7	100.0
	2	1.254		93.9
	3	1.283		96.1
Test Item	1	1.121	1.076	83.9	7.4	80.6
	2	0.984		73.6
	3	1.124		84.1
Positive Control	1	0.079	0.072	5.9	14.8	5.4
	2	0.076		5.7
	3	0.059		4.5

*       Mean of three replicate wells after blank correction

**     relative absorbance per tissue [rounded values]

***   relative absorbance per treatment group [rounded values]

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 1 hour incubation with MTT-reagent did not show blue colour.

The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 80.6% (threshold for irritancy: ≤ 50%), consequently the test item was not irritant to skin.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used for interpretation of results: UN GHS and EU CLP regulation

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, ElfaMoist AC is not irritant to skin according to UN GHS and EU CLP regulation.

Executive summary:

This in vitro study was performed to assess the irritation potential of Reaction mass of N-[2-(2-hydroxyethoxy)ethyl]acetamide and glycerol by means of the Human Skin Model Test.

The test was performed under monochromatic yellow light.

The test item did not reduce MTT (test for direct MTT reduction), and it did not change colour when mixed with deionised water (test for colour interference). Also its intrinsic colour was not intensive. Consequently, additional tests with freeze-killed or viable tissues to determine correction factors for calculating the true viability in the main experiment were not necessary.

Each three tissues of the human skin model EpiDerm™ were treated with the test item, the negative control (DPBS), or the positive control (5% SLS) for 60 minutes.

After treatment with the negative control the absorbance values were well within the required range of the acceptability criterion of mean OD  0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring 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 60 minutes treatment interval, and thus assuring the validity of the test system.

After treatment with the test item ElfaMoist AC the mean relative absorbance value decreased to 80.6% compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

In conclusion, it can be stated that in this study and under the experimental conditions reported, ElfaMoist AC is not irritant to skin.