Castor oil, hydrogenated, ethoxylated

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 July 2017 to 31 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

MatTek Corporation Protocol: EpiOcular™ Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; for use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model; 29 June 2015.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Castor oil, hydrogenated, ethoxylated

EC Number:

500-147-5

EC Name:

Castor oil, hydrogenated, ethoxylated

Cas Number:

61788-85-0

Molecular formula:

C57H110O9 (C2H4O)n where n = 1-6.5

IUPAC Name:

Castor oil, hydrogenated, ethoxylated

Test material form:

solid

Remarks:

pasty wax

Test animals / tissue source

Species:

other: human keratinocytes

Details on test animals or tissues and environmental conditions:

EpiOcular™ kits and MTT-100 kits are purchased from MatTek Corporation (82105 Bratislava, Slovakia).

Lot No.: 27002
Sealed 24-well plate Contains 12/24 inserts with EpiOcular™ tissues on agarose
Serum-free test medium DMEM-Medium
Positive control Methyl Acetate (CAS#79-20-9)
12-well plate Holding plate
24-well plates For MTT viability assay
6-well plates For storing inserts, or for topically applying test agents
Ca++Mg++-Free D-PBS Dulbecco´s Phosphate Buffered Saline

MTT-100 Assay Kit Components
1 vial, 2 mL MTT concentrate
1 vial, 8 mL MTT diluent (supplemented DMEM) For diluting MTT concentrate prior to use in the MTT assay
1 bottle, 60 mL Extractant solution (Isopropanol) For extraction of formazan crystals

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 uL

Duration of treatment / exposure:

30 minutes

Duration of post- treatment incubation (in vitro):

12 minutes in Assay Medium (to remove any test item absorbed into the tissue) and 120 minutes in Assay Mediumat 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 (post-treatment incubation).

Number of animals or in vitro replicates:

2 replicates per treatment (except for blank)

Details on study design:

At the end of the 30 minutes treatment time, the test item was removed by extensively rinsing the tissues (3 times) with Ca++Mg++-free DPBS (brought to room temperature).
After rinsing, the tissues were immediately transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a pre-labelled 12-well plate for 12 minute immersion incubation (post-soak) at room temperature. This incubation in Assay Medium was intended to remove any test item absorbed into the tissue.
At the end of the post-soak immersion, each insert was removed from the Assay Medium, the medium was decanted off the tissue, and the inserts were blotted on absorbent material, and transferred to the appropriate well of the pre-labelled glass tube containing 1 mL of warm Assay Medium. The tissues are incubated for 120 minutes at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 (post-treatment incubation).
At the end of the post-treatment incubation, each insert was removed and gently blotted on absorbent material. The tissues were placed into the 24-well plate containing 0.3 mL of MTT solution. Once all the tissues are placed into the 24-well plate, the plate was incubated for 180 minutes at standard culture conditions.
Each insert was removed from the 24-well plate after 180 minutes, the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labelled 24-well plate containing 2.0 mL of isopropanol in each designated well so that no isopropanol was flowing into the insert on the tissue surface. The plates were sealed with a standard plate sealer, and were stored overnight at 2-8 °C in the dark and then shaken for 2-3 hours at room temperature. At the end of the extraction period, the tissue was pierced and the liquid within each insert was decanted into the well from which it was taken.
The extract solution was mixed and two 200 µL aliquots were transferred to the appropriate wells of a pre-labelled 96-well plate.
The absorbance at 570 nm (OD570) of each well was measured with a plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro Enterprise, version 4.7.1). No reference wavelength measurement was used.

Results and discussion

In vitro

Results

Irritation parameter:

other: viability %

Value:

92.1

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Dose Group	Ab-sorbance Well 1 (Tissue 1/2)	Ab-sorbance Well 2 (Tissue 1/2)	Mean Absor-bance (Tissue 1/2)	Mean Absorbance* Tissue 1 and 2	Mean Absorbance of 2 Tissues*	Rel. Absorbance [%] Tissue 1 and 2**	Absolute Value of the Difference of the Rel. Absorbances [%] Tissue 1 and 2	Mean Rel. Absorbance [%]***
Blank	0.038	0.038	0.038	0.000		99.1
Negative Control	1.674	1.753	1.714	1.676	1.691	100.9	1.8	100.0
	1.735	1.753	1.744	1.706		36.8
Positive Control	0.626	0.693	0.660	0.622	0.617	36.2	0.6	36.5
	0.647	0.652	0.649	0.611		92.6
Test Item	1.555	1.651	1.603	1.565	1.558	91.7	0.9	92.1
	1.581	1.595	1.588	1.550		99.1

*                            Mean of two replicate wells after blank correction
**                          Relative absorbance [rounded values]: 100 * (absorbace test item/positive control/negative control)/absorbance negative control

***                        Mean relative absorbance [rounded values]: 100 * (mean absorbace test item/positive control/negative control)/mean absorbance negative control

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The substance is not irritating to the eyes

Executive summary:

The eye irritation potential of the substance was assessed by means of the Human Cornea Model Test. The substance did not prove to be an MTT reducer and color interference was excluded.

Each 50 µL of the test item, the negative control (deionised water) or the positive control (methyl acetate) were applied to each of duplicate tissue for 30 minutes.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of OD > 0.8 and < 2.5 thus showing the quality of the tissues.

Treatment with the positive control induced a decrease below 50% compared with the negative control value in the relative absorbance thus ensuring the validity of the test system.

The difference of viability between the two relating tissues was < 20% in the same run (for test item tissues, positive and negative control tissues).

Irritating effects were not observed following incubation with the substance. Compared with the value of the negative control the relative mean absorption value corresponding to the viability of the tissues did not decrease below 60% (92.1%).

In conclusion, it can be stated that in this study and under the experimental conditions reported, the substance does not possess any eye irritating potential.