Reaction product of: stearyl-diethanol-amine with C16-C18 saturated fatty acids

Eye irritation

Administrative data

Endpoint:

eye irritation

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 July 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study according to international guidelines.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for the Testing of Chemicals, adopted 25. Jul. 2014, DRAFT PROPOSAL FOR A NEW TEST GUIDELINE , Reconstructed Human Cornea-like Epithelium (RhCE)

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: “EpiOcular TM Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irri-tation of chemicals” by MatTek Corporation, document no. MK‐24‐007‐0055, dated 10. Dec. 2012

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction product of: stearyl-diethanol-amine with C16-C18 saturated fatty acids

EC Number:

943-080-0

Molecular formula:

Not applicable, UVCB

IUPAC Name:

Reaction product of: stearyl-diethanol-amine with C16-C18 saturated fatty acids

Test material form:

other: pastilles

Details on test material:

Name SABOSTAT A 300
Batch no. 20085596
Appearance whitish pastilles
Composition Reaction product of stearyl-diethanol-amine with C16-C18 saturated fatty acids
CAS No. not applicable
(CAS No. of main compound 2-[(2-hydroxyethyl)octadecylamino]ethyl stearate: 52497-24-2)
EINECS-No. not stated
Molecular formula not applicable
Molecular weight 668 g/mol (calculated based on average UVCB composi-tion)
Purity 100%
Homogeneity homogeneous
Vapour pressure 5.85E-21 Torr Calculated using Advanced Chemistry De-velopment (ACD/Labs) Software V11.02 (© 1994-2015 ACD/Labs) h Pa at 25°C
Stability H2O; EtOH; acetone; CH3CN; DMSO: unknown
Solubility H2O: not soluble DMSO: soluble at 50 °C; EtOH, acetone, CH3CN: unknown
Production date 16. Apr. 2015
Expiry date 10. Apr. 2016
Storage Room temperature (20 ± 5 °C), keep away from light/ hu-midity

Test animals / tissue source

Species:

other: 3D human cornea tissue model

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Commercially available EpiOcularTM kit.
The EpiOcularTM tissue consists of normal, human-derived keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells. These cells are not transformed or transfected with genes to induce an extended life span. The EpiOcularTM tissues are cultured in specially prepared cell culture inserts with a porous membrane through which nutrients can pass to the cells. The tissue surface is 0.6 cm2.
EpiOcularTM tissues were procured from MatTek In Vitro Life Science Laboratories, Mylnské Nivy 73, 82105 Bratislava, Slovakia.
Day of delivery: 14. Jul. 2015
Batch no.: 21562

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: negative and positive control vessels (in vitro study)

Amount / concentration applied:

The following amounts were applied to the tissues:

Tissue 1 51.5 mg
Tissue 2 49.4 mg

Duration of treatment / exposure:

6 hours

Observation period (in vivo):

Not applicable

Number of animals or in vitro replicates:

Not applicable

Results and discussion

In vivo

Results

Irritation parameter:

other: Relative absorbance (%)

Basis:

mean

Score:

107.5

Remarks on result:

other: Absorbance of test item compared to negative control

Any other information on results incl. tables

Findings and Results

Measured Values

As blank, the optical density of isopropanol was measured in eight wells of the 96-well-plate. The measured values and their mean are given in the following table:     

Table 1.Absorbance Values Blank Isopropanol (OD at 570 nm)

Replicate	1	2	3	4	5	6	7	8	Mean
Absorbance	0.035	0.038	0.035	0.035	0.034	0.036	0.036	0.036	0.036

 

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

Table 2. Absorbance Values Negative Control, Positive Control and Test Item (OD at 570 nm)

Designation	Measurement	Negative Control	Positive Control	SABOSTAT A 300
Tissue 1	1	1.289	0.453	1.440
	2	1.368	0.457	1.438
Tissue 2	1	1.358	0.456	1.446
	2	1.360	0.456	1.447

 

From the measured absorbances, the mean of each tissue was calculated, subtracting the mean absorbance of isopropanol as given in table 1 (= corrected values).

Table3 Mean Absorbance Negative Control, Positive Control and Test Item

Designation	Negative Control	Positive Control	SABOSTAT A 300
Mean – blank (Tissue 1)	1.293	0.419	1.403
Mean – blank (Tissue 2)	1.323	0.420	1.411

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 4 % Viability Positive Control and Test Item

Designation	Positive Control	SABOSTAT A 300
% Viability (Tissue 1)	32.0%	107.3%
% Viability (Tissue 2)	32.1%	107.8%
% Viability Mean	32.1%	107.5%

Assessment

Eye irritation is assessed using the criteria given in the following table (source: MatTek Corporation):

Table 5 Assessment of Eye Irritation

% Viability	Assessment	GHS classification
> 60 %	Non eye irritant	No GHS category for eye irritation
≤60 %	Eye irritant	GHS category 1 or 2

 

 Validity

Validity criteria and results are stated in the following table:

Table 6 Validity

Criterion	Demanded	Found
OD of negative control	≥1.0 and≤ 2.6	1.3
% Formazan production of positive control	< 60% of negative control	32.1%
Variation within replicates	< 20%	2.3% (negative control) 0.1% (positive control) 0.6% (test item)

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of the test system, the substance is considered as
not eye irritant in the EpiOcularTM Eye Irritation Test.
After treatment with the test item, the relative absorbance values increased to 107.5 %.
This value is well above the threshold for eye irritation potential (60%).
All validity criteria were met. The criterion for optical density of the negative control was fulfilled: The OD value was 1.3 (> 1.0 and < 2.6).
The positive control induced a decrease in the relative absorbance as compared to the negative control to 32.1% (< 60%). Variation within the replicates was acceptable (<20 %).
For these reasons, the result of the test is considered as valid.

Executive summary:

The substance was applied to a three-dimensional human cornea tissue model in duplicate for a 6 h exposure.

In average, 50.5 mg of the solid test item were applied to each tissue.

After treatment, the respective substance was rinsed from the tissue; then, cell viability of the tissues was evaluated by addition of MTT which can be reduced to a blue formazan. Formazan production was measured by measuring the optical density (OD) of the resulting solution.

Deionised water was used as negative control, Methyl acetate was used as positive control.

The controls showed the expected results. After treatment with the negative control, the absorbance values were within the required acceptability criterion of 1.0 ≤ mean OD ≤ 2.6, OD was 1.3. The positive control showed clear eye irritating effects, the relative absorbance value was reduced to 32.1 %. Variation within tissues was acceptable (< 20%).

 

After treatment with the test item, the relative absorbance values increased to 107.5 %.

This value is well above the threshold for eye irritation potential (60%).

 

Under the conditions of the test system, the substance is considered as

not eye irritant in the EpiOcular^TMEye Irritation Test.