Fatty acids, tall-oil, reaction products with 2-[(2-aminoethyl)amino]ethanol

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-09-25 to 2013-01-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD 439 and EU method B.46 in a GLP certified testing facility.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test material form:

other: liquid

Details on test material:

Name 400112
Batch no. 0001273679
Appearance yellow-brown, liquid
Composition UVCB substance
Purity 100% (not purified but used as synthesised)
Production date May 2011
Expiry date May 2013
Storage Room Temperature: 20 ± 5°C

In vitro test system

Test system:

human skin model

Source species:

human

Vehicle:

unchanged (no vehicle)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 microliter
- Concentration (if solution): undiluted

VEHICLE
- Amount(s) applied (volume or weight with unit):
- Concentration (if solution):
- Lot/batch no. (if required):
- Purity:

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 microliter
- Concentration (if solution):

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 microliter
- Concentration (if solution):

Duration of treatment / exposure:

35 minutes at 37°C

Number of replicates:

3 for each sample and control

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

formation of formazan from MTT was determined being a measure of cell viability.

Run / experiment:

1

Value:

3.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

formation of formazan from MTT was determined being a measure of cell viability.

Run / experiment:

2

Value:

3.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

4.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: value refers to % formazan production

Any other information on results incl. tables

Blank:

As blank, the optical density of isopropanol was measured in eight wells of the 96-wellplate.

Mean value:0.061

 

Table 1: Absorption values of the negative control, test item and positive control (OD at 570 nm)

Tissue no.	Measurement	Negative Control	400112	Positive Control
1	1	2.132	0.429	0.214
	2	2.151	0.425	0.211
2	1	2.044	0.440	0.215
	2	2.084	0.445	0.216
3	1	2.028	0.452	0.222
	2	2.044	0.469	0.225

 

From the measured absorption values, the mean of each tissue was calculated. From the mean absorption values the mean absorption of isopropanol (0.061) was subtracted. Mean and relative standard deviations (comparison of the three tissues) were also calculated.

Table 2: Mean absorption values subtracted by the mean absorption of the blank

Tissue no.	Negative Control	400112	Positive Control
1	2.081	0.366	0.152
2	2.003	0.382	0.155
3	1.975	0.400	0.163
Mean of the three tissues	2.020	0.383	0.157
Relative standard deviation of the three tissues	2.7%	4.4%	3.6%

 

Table 3: Mean absorption values of freeze killed tissues of negative control and test item (OD at 570 nm)  

Tissue no.	Negative Control	400112
1	0.093	0.380
2	0.087	0.400
3	0.088	0.399
Mean of the three tissues	0.090	0.393

 

The mean OD of the untreated freeze-killed tissue is subtracted from the mean OD of the treated freeze-killed tissue (= 0.304). This difference is then subtracted from the OD of the test item in the test. The calculated value is the “OD corrected”.

Corrected Values Viable Tissues

The absorption values of the negative and positive control and the corrected absorption values of the test item are given in the following table:

Table 4: Mean absorption values

Tissue no.	Negative Control	400112 (corrected)	Positive Control
1	2.081	0.062	0.152
2	2.003	0.078	0.155
3	1.975	0.096	0.163
Mean of the three tissues	2.020	0.079	0.157
Relative standard deviation of the three tissues	2.7%	21.3%	3.6%

 

Comparison of Formazan Production

For the test item and the positive control, the following percentage values of formazan production were calculated in comparison to the negative control:

Table 5: % Formazan production

Tissue no.	400112	Positive Control
1	3.1%	7.5%
2	3.9%	7.7%
3	4.8%	8.1%
Mean of % formazan production	3.9%	7.8%

 

 

Applicant's summary and conclusion

Interpretation of results:

other: irritant according to EU regulation

Conclusions:

400112 is considered irritant in the Human Skin Model Test.

Executive summary:

To determine the Skin Irritation Potential of 400112 the Human Skin Model Test following EU-Method 8.46 resp. OECD 439 was conducted.

Three tissues of the human skin model EpiDerm™ were treated with the test item 400112 for 60 min. 30 µL of the liquid lest item were applied to each tissue (using a nylon mesh) and spread to match the tissue size (0.63 cm²). DPBS-buffer was used as negative control, 5% SDS-solution was used as positive control.

After treatment with the negative control, the absorbance values were within the required acceptability criterion of 1.0 < mean OD < 2.5. The positive control showed clear irritating effects. Variation within tissues was acceptable for the positive and negative control (< 18%).

After the treatment with the test item 400112, the relative absorbance values were reduced to 3.9%. This value is well below the threshold for irritation potential (50%).

Therefore, 400112 is considered as irritant in the Human Skin Model Test.