Reaction mass of ethoxylated N-oleyl-1,3-propanediamine, hydrofluorides of and ethoxylated N-palmityl-1,3-propanediamine, hydrofluorides of and ethoxylated N-stearyl-1,3-propanediamine, hydrofluorides of and olaflur

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:

from 2009-08-13 to 2009-09-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 9194M0801
- Expiration date of the lot/batch: 2011-01-04

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:
Commercially available Epi-200-Kit.
The EpiDerm™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi-layered, 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 are cultured on specially prepared cell cultures inserts.
- Tissue batch number: 12243
- Delivery date: 02. September 2009
- Date of initiation of testing: 02. September 2009

PERFORMANCE OF THE STUDY
Description of the Method
Four 6-well-plates were prepared with 0.9 mL assay medium in each well. The inserts containing the tissues were transferred to the wells using sterile forceps and the 6-well-plates were set into the incubator at 37°C and 5% CO2 for one hour (pre-incubation).
For each experiment (“three minutes” and “one hour”), one 24-well-plate was prepared as holding plate. 12 wells of each plate were filled with 300 µL assay medium, the other 12 with 300 µL MTT medium. One additional plate was left empty. The plates were stored in the incubator.
For each experiment (“three minutes” and “one hour”), two 6-well-plates were used. After pre-incubation, the assay medium was replaced by fresh assay medium and the test was started, using two wells as negative control with 50 µL H2O demin., two wells as positive controls with 50 µL potassium hydroxide solution and two other wells for testing the test item.
Approx. 25 mg of the wax-like test item were applied together with 25 µL H2O. At the start of each experiment (application of negative controls), a stop watch was started.
After the respective incubation time, the inserts were removed from the plates using sterile forceps. The inserts were thoroughly rinsed with PBS, blotted with sterile cellulose tissue and set into the respective holding plate, using the wells containing assay medium. After transfer of all inserts, they were immediately moved to the wells containing MTT solution, blotting the bottom with cellulose tissue again before setting the insert into the MTT well. The tissues were incubated with MTT medium for three hours. After this time, the MTT medium was aspirated and replaced by PBS buffer. This was then aspirated, too, and replaced several times. At last, each insert was thoroughly dried and set into the empty, pre-warmed 24-well-plate. Into each well, 2 mL isopropanol were pipetted, taking care to reach the upper rim of the insert. The plate was then covered with Parafilm® and left to stand over night at room temperature.
On the next day, the inserts in which formazan had been produced over night were pierced with an injection needle, taking care that all colour was extracted. The inserts were then discarded and the content of each well was thoroughly mixed in order to achieve homogenisation. From each well, three replicates with 200 µL solution (each) were pipetted into a 96-well-plate which was read in a plate spectral photometer at 570 nm.

NUMBER OF REPLICATE TISSUES: 2

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: Six per tissue

PREDICTION MODEL / DECISION CRITERIA: Please refer to "Any other information on results incl. tables"

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Approx. 25 mg of the wax-like test item were applied together with 25 µL H2O.

Duration of treatment / exposure:

3 min and 1 h respectively

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean (of two tissues) after 3 minutes

Value:

100.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: For details please refer to "Any other information on results incl. tables"

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean (of two tissues) after 1 hour

Value:

90.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: For details please refer to "Any other information on results incl. tables"

Any other information on results incl. tables

Corrosivity was assessed using the criteria given in the following table.

Table 1: Assessment of Corrosivity

% Formazan production after 3 min. incubation time	% Formazan production after 1 h min. incubation time	Assessment
< 50% of negative control	Irrelevant	Corrosive
> 50% negative control	< 15% of negative control	Corrosive
> 50% of negative control	> 15% of negative control	Not corrosive

Table 2: Absorption Values

Negative Control		Test Item		Positive Control		Incubation
Tissue 1	Tissue 2	Tissue 1	Tissue 2	Tissue 1	Tissue 2
1.850	1.855	1.805	1.904	0.508	0.493	3 min
1.838	1.865	1.840	1.906	0.498	0.494
1.894	1.875	1.832	1.919	0.528	0.490
1.748	1.819	1.605	1.646	0.233	0.252	1 hour
1.751	1.803	1.603	1.647	0.253	0.260
1.757	1.812	1.553	1.645	0.251	0.252
Mean		Mean		Mean
1.863		1.868		0.502		3 min
1.782		1.617		0.250		1 hour

Table 3: % Formazan Production

Test Item	Positive Control	Incubation
100.3%	26.9%	3 min
90.7%	14.0%	1 hours

Corrosivity of the Test Item

The relative absorbance value was 100.3% after three minutes treatment. This value is above the threshold for corrosivity (50%). After one hour treatment, the relative absorbance values were reduced to 90.7%, lying well above the threshold for corrosivity (15%). Therefore, the test item is considered as not corrosive.

Validity

The criterion for optical density of the negative control (> 0.8) was fulfilled: optical density was 1.863 (3 minutes) resp. 1.782 (1 hour).

The positive control showed a clear corrosive effect: the value of the three-minute-experiment was 26.9% and the value of the one-hour-experiment was 14.0%.

Values for negative control and for positive control were within the range of historical data of the test facility.

Therefore the experiment is considered valid.

Applicant's summary and conclusion

Interpretation of results:

other: not corrosive

Remarks:

Criteria used for interpretation of results: criteria given in OECD guideline 431

Conclusions:

The test item is considered as not corrosive, based on in vitro testing.

Executive summary:

Two tissues of the human skin model EpiDermTM were treated with Olaflur for three minutes and one hour, respectively, to investigate skin corrosion properties.

25 mg of the wax-like test item were applied to each tissue, wetted with H2O and spread to match the tissue size. Deionised water was used as negative control, 8-m KOH was used as positive control.

After treatment with the negative control, the absorbance values were well above the required acceptability criterion of mean OD > 0.8 for both treatment intervals thus showing the quality of the tissues. The positive control showed clear corrosive effects for both treatment intervals.

After three minutes treatment with the test item, the relative absorbance value was increased to 100.3%. This value is well above the threshold for corrosion potential (50%). After one hour treatment, relative absorbance values were reduced to 90.7%. This value, too, is well above the threshold for corrosion potential (15%).

Therefore, Olaflur is considered as not corrosive in the Human Skin Model Test.