Reaction mass of 2-(3-{6-[3-(2-Hydroxyethyl)-3-methylureido]hexyl}-1-methylureido)ethanol and 6-[3-(2-Hydroxyethyl)-3-methylureido]hexylamino 3-(methylamino)propionate

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:

October 2014

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)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N,N''-hexane-1,6-diylbis[N'-(2-hydroxyethyl)-N'-methylurea]

EC Number:

253-281-9

EC Name:

N,N''-hexane-1,6-diylbis[N'-(2-hydroxyethyl)-N'-methylurea]

Cas Number:

36938-15-5

Molecular formula:

C14H30N4O4

IUPAC Name:

2-(3-{6-[3-(2-Hydroxyethyl)-3-methylureido]hexyl}-1-methylureido)ethanol

Constituent 2

Reference substance name:

6-[3-(2-Hydroxyethyl)-3-methylureido]hexylamino 3-(methylamino)propionate

Molecular formula:

C14H30N4O4

IUPAC Name:

6-[3-(2-Hydroxyethyl)-3-methylureido]hexylamino 3-(methylamino)propionate

Test material form:

solid: bulk

Details on test material:

- Batch no.: EGGE 2806-1
- Currenta sample no.: 2187

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING

- Treatment of test material prior to testing: test item was melted in a hot cabinet with 100 °C

FORM AS APPLIED IN THE TEST (if different from that of starting material): liquid (melted and cooled down test item)

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

commercially available test system

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: epiCS® (CellSystems, Troisdorf, Germany).
- Tissue batch number(s): Cat.-No.CS-100

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: RT (room temperature)
- Temperature of post-treatment incubation (if applicable): Incubator temperature: 37 ± 2° C (CO2 gas concentration: 5 %; Humidity: maximum)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1mg/ml
- Incubation time: 3 hours
- Spectrophotometer: EL808, Bio-Tek
- Wavelength: 570 nm

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION:
the optical density of the isopropanol-extracts of 3 insert was determined by duplicate per insert = 6 OD values.

PREDICTION MODEL / DECISION CRITERIA:
- The mean optical density (OD) values obtained with the test item was used to calculate the percentage of viability relative to the negative control, which is set at 100 %.
- According to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post treatment incubation is less than or equal (≤ ) to 50 %.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 30 μl

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μl
- Concentration (if solution): 0.9% NaCl in water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µl
- Concentration (if solution): 5% SDS in physiological saline

Duration of treatment / exposure:

20 min

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

three

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

ca. 81

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
- Reliability of the test was previously confirmed by interlaboratory validation

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: Tabular Summary of the results

Sample No.	Test item	OD mean*	StdDev	% Viability
1 -3	negative control, 0.9% NaCl	2.49	0.05	100
4 -6	positive control, 5% SDS	0.04	0.03	1.53
10 -12	EGGE 2806 -1	2.01	0.36	80.65

*6 values

Applicant's summary and conclusion

Interpretation of results:

other: negative

Executive summary:

A study was performed for the assessment of the skin irritancy of the test item with reconstructed human epidermis (RhE). The experiment was carried out using the commercially available test method epiCS®. The study was conducted in accordance with OECD TG 439 and EU Test Method B.46. The mean value of cell viability was recorded to be 81 %. The test item was thus shown to be not irritating to reconstructed human skin in vitro.