Fatty acids, C18-(unsaturated) dimers and their monoesters and diesters with 2-ethylhexanol

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-08-31 to 2011-10-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD guideline No. 439 and EU method B.46 in a GLP certified testing facility.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test material form:

liquid: viscous

Details on test material:

Name: 400160
Batch no.: KL-11-038
Appearance: yellow, clear viscous liquid
Composition: UVCB, 2-Ethylhexyl esters of dimerised fatty acids, C18-unsaturated
Purity: 100% (GPC)
Production date: 24 March 2011
Expiry date: 24 March 2016
Storage: room temperature (20 ± 5 °C)

Test system

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

30 µL test item were applied on the tissue surface

Duration of treatment / exposure:

60 min

Observation period:

not applicable (cell viability was determined after the end of treatment period)

Number of animals:

not applicable (this is an in vitro test)

Details on study design:

The human skin model test was performed with commercially available Epi-200-SIT-Kit. The EpiDermTM tissues were produced from MatTek Corporation in Ashland, USA. The day of delivery was 01 July 2010 batch: 13657

Negative control: Dulbecco’s Phosphate Buffered Saline (DPBS) without CaCl2 and MgCl2
One plate (three tissues) was used as negative control; each tissue was treated with 30 µL DPBS buffer
Positive control: Sodium dodecyl sulphate (SDS), CAS No. 151-21-3, solution in deionised H2O, 5%
One plate (three tissues) was used as positive control; each tissue was treated with 30 µL SDS-solution
Test item: BYK-400160
One plate (three tissues) was used for treatment with the test item

Experimental performance:
After dosing the tissues with negative control, positive control and test item, they were incubated at 37°C for 35 min. After 60 min a rinsing step and two incubation periods at 37°C over a total period of 42 h followed.
After incubation, a MTT assay was performed and formazan production was measured by a spectral photometer at 570 nm.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: other: formazan production in comparison to the negative control

Value:

100.9

Remarks on result:

other:

Remarks:

Time point: 60 min. Max. score: 102.5. Remarks: Result: not irritant. (migrated information)

In vivo

Irritant / corrosive response data:

After the treatment with the test item, the mean relative absorbance values were increased to 100.9 % in comparison to the negative control. This value is well above the threshold for irritation potential (50%).

Other effects:

no other effects observed

Any other information on results incl. tables

Evaluation and calculation:

The photometric absorption of the negative controls was considered as 100%. For each replicates of the test item and positive control, formazan production was calculated as % photometric absorption compared with the negative control.

% Formazan production = (OD test item resp. positive control / OD mean negative control) x 100

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

Designation	Measurement	Negative Control	400160	Positive Control
Tissue 1	1	1.734	1.595	0.145
	2	1.713	1.582	0.149
Tissue 2	1	1.504	1.654	0.143
	2	1.513	1.626	0.144
Tissue 3	1	1.562	1.602	0.147
	2	1.577	1.629	0.149

 

From the measured absorption values, the mean of each tissue was calculated and the mean absorption of isopropanol (0.046) was subtracted.

Table 2      Mean Absorption Values

Designation	Negative Control	400160	Positive Control
Mean – blank (Tissue 1)	1.678	1.543	0.101
Mean – blank (Tissue 2)	1.463	1.594	0.098
Mean – blank (Tissue 3)	1.524	1.570	0.102
Mean of the three Tissues	1.555	1.569	0.100
Relative Standard Deviation of the three tissues	7.1%	1.6%	2.1%

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 3     % Formazan Production

Designation	400160	Positive Control
% Formazan production (Tissue 1)	99.2%	6.5%
% Formazan production (Tissue 2)	102.5%	6.3%
% Formazan production (Tissue 3)	101.0%	6.6%
% Formazan production Mean	100.9%	6.5%

All validity criteria were fulfilled.

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the experimental conditions reported, 400160 did not exhibit any skin irritating potential.

Executive summary:

This in-vitro human skin model test (EU method B.46 resp. OECD 439) was performed in order to evaluate the potential of 400160 to evoke skin irritation in a human-skin-model.The study was performed under GLP compliance.

400160 was spread on the surface of three sections of an EpiDerm^TM tissue and incubated for 60 min. The negative control consisting of Dulbecco’s phosphate buffered saline was applied to another three tissue sections. As positive control a SDS solution (50 g/L) was used for three other tissue sections. After rinsing and two incubation steps of altogether 42 h an enzyme assay to determine cell viability was performed. The tissues were incubated with MTT-reagent for 3 h. The tissues were washed with PBS, dried and transferred to a 24 -well-plate. After incubating with isopropanol at room temperature over night, the developed formazan concentration was measured in a spectral photometer at 570 nm.

The mean percentage values of formazan production were calculated in comparison to the negative control. The relative absorbance values were increased to 100.9% after the treatment with 400160. This value is well above the threshold for irritation (50%). The positive control showed a reduction of formazan production of 6.5%. All validity criteria were fulfilled

In conclusion, it can be stated that the test item 400160 is considered as non skin irritant under the experimental conditions used in this study.