Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
20 October to 22 October 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The eye irritation potential was determined using the SkinEthic Reconstituted Human Corneal model with a treatment period of 10 minutes. The test is based on the hypothesis that irritant chemicals are able to penetrate the corneal epithelial tissue and are sufficiently cytotoxic to cause cell death.
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of inspection 19th August 2008, Date of signature 4th March 2009

Test material

Constituent 1
Chemical structure
Reference substance name:
undec-10-en-1-yl 2-cyano-3,3-diphenylprop-2-enoate
EC Number:
700-604-0
Cas Number:
947701-81-7
Molecular formula:
C27H31NO2
IUPAC Name:
undec-10-en-1-yl 2-cyano-3,3-diphenylprop-2-enoate
Constituent 2
Reference substance name:
700-824-2
IUPAC Name:
700-824-2
Constituent 3
Reference substance name:
2-Propenoic acid, 2-cyano-3-(4-methoxyphenyl)-3-phenyl, 10-undecen-1-yl ester
IUPAC Name:
2-Propenoic acid, 2-cyano-3-(4-methoxyphenyl)-3-phenyl, 10-undecen-1-yl ester
Constituent 4
Reference substance name:
Undecenyl methoxycrylene
IUPAC Name:
Undecenyl methoxycrylene
Details on test material:
Details on Surrogate material:
Batch number: APB401608;
Date Received: 10 August 2009;
Storage conditions: room temperature in the dark;
Purity =98.6-98.8%.
SMILES Notation: c2cc(OC)ccc2C(c1ccccc1)=C(C(#N))C(=O)OCCCCCCCCCC=C

Test animals / tissue source

Species:
human
Strain:
other: not applicable to study
Details on test animals or tissues and environmental conditions:
Not applicable to study.

Test system

Vehicle:
other: See free text in "details on study design" section.
Controls:
other: not applicable
Amount / concentration applied:
See free text in "details on study design" section.
Duration of treatment / exposure:
10 minutes
Observation period (in vivo):
See free text in "details on study design" section.
Number of animals or in vitro replicates:
not applicable to study.
Details on study design:
The purpose of this study was to determine the eye irritation potential of the test material using the SkinEthic Reconstituted Human Corneal model (HCE, SkinEthic Laboratories Nice, France) after a treatment period of 10 minutes.

The SkinEthic model consists of transformed human keratinocytes of the cell line HCE (LSU EYE Center, New Orleans, USA) that form a corneal epithelial tissue (mucosa), devoid of stratum corneum, resembling, histologically, the mucosa of the human eye. Test materials are applied directly to the culture surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly. The model consists of an airlifted, living, corneal tissue construct, produced in polycarbonate inserts in serum free and chemically defined medium.

The test is based on the hypothesis that irritant chemicals are able to penetrate the stratum corneum of the SkinEthic RHC model and are sufficiently cytotoxic to cause cell death in the underlying cell layers.

Cytotoxicity was determined by the reduction of MTT to formazan by viable cells in the test material treated tissues (quantitative measurement of tissue viability) relative to the negative control.

One tissue for each treatment group was retained for possible tissue histopathology.

Negative and Positive Controls:

The negative control material, Solution A was used as supplied. The composition of 1 liter Solution A in water is as follows:

Na2HPO4 0.142 g/l
HEPES 7.149 g/l
KCl 0.224 g/l
NaCl 7.5.97 g/l

The positive control material, Sodium Dodecyl Sulphate (SDS), was prepared as a 1% solution w/v solution in sterile distilled water.

PRE-TEST

Assessment of Direct Test Material Reduction of MTT:

One limitation of the assay is possible interference of the test material with MTT. A test material may directly reduce MTT to formazan, thus mimicking dehydrogenase activity of the cellular mitochondria of viable cells. This property of the test substance is a problem only if at the time of the MTT test (after the chemical has been rinsed off) there are still sufficient amounts of the test material on or in the tissues. To identify this possible interference, the test material was checked for its ability to reduce MTT directly.

30 microliters of test material was added to a 0.5 mg/ml MTT solution and incubated at room temperature in the dark for 60 minutes. Untreated MTT solution was used as a control. If the MTT solution turned black/purple, the test material was presumed to have reduced the MTT.

Receipt of Tissues:
On arrival, the SkinEthic HCE tissues (Day 6 cultures), were stored at room temperature prior to transferring into 24-well plates designated “arrival plates” containing 300 microliters of maintenance medium. It was important to ensure that there were no air bubbles present under the tissue inserts. The tissues were incubated overnight at 37 deg C, 5% CO2 in air.

Preparation of Tissues:
Using sterile techniques, 1 ml of maintenance medium at room temperature, was dispensed into the appropriate number of wells of 6-well plates designated “treatment plates.” Each well was labeled with details of the treatment and the appropriate exposure time. Separate treatment plates were used for the test material and negative and positive controls to avoid the possibility of cross contamination occurring. Before treatment the 7 day old tissues were transferred from the “arrival plates” to the wells of the “treatment plates” containing the maintenance medium.

MAIN TEST:

Triplicate tissues were treated with 30 microliters of the test material for 10 minutes. The tissues were dosed at regular timed intervals to allow for the period taken to rinse each insert following exposure and to ensure each tissue received an equal exposure time. Triplicate tissues were treated with 30 microliters of Solution A to serve as negative controls and triplicate tissues were treated with 30 microliters of 1% w/v SDS to serve as positive controls. The plates were incubated at 37 deg C, 5% CO2 in air during the exposure time.

At the end of the relevant exposure period each tissue insert was removed from the well using forceps and rinsed using a wash bottle containing Dulbecco’s Phosphate Buffered Saline (DPBS). Rinsing was achieved by filling and emptying each tissue insert using a constant soft stream of DPBS to gently remove any residual test material. Excess DPBS was removed by blotting the bottom of the insert with absorbent paper. Each tissue was placed into a pre-labelled 24-well plate designated “holding plate” containing 300 microliters of maintenance medium (at room temperature) until all the tissues were rinsed. Following rinsing the tissues (two per group) were transferred to a pre-labeled 24-well plate designated ”MTT Loading plate” containing 300 microliters of a 0.5 mg/ml MTT solution freshly prepared in a maintenance medium. The MTT loading plate was placed into an incubator for approx. three hours at 37 deg C, 5% CO2 in air.

At the end of the incubation period the tissues were visually examined and the degree of MTT staining evaluated (qualitative evaluation of tissue viability). The inserts were blotted on absorbent paper to remove residual MTT solution and transferred to a pre-labeled 24-well plate designated “MTT extraction plate” containing 0.75 ml of isopropanol in each of a sufficient number of wells. An extra 0.75 ml of isopropanol was added onto each tissue and the plate sealed to prevent isopropanol evaporation. The plate was wrapped in aluminium foil (to protect from light) and allowed to stand overnight at room temperature to extract the formazan crystals out of the tissue.

At the end of the extraction period, each tissue insert was pierced with a pipette fitted with a 1000 microliter tip and the extraction solution forced vigorously up and down through the tissue insert until a homogeneous solution was obtained. The empty inserts were discarded. For each tissue triplicate 200 microliter samples were transferred to the appropriate wells of a pre-labeled 96-well plate. 200 microliters of isopropanol alone was added to three wells designated as “blanks.” The optical density was measured (quantitative measurement of tissue viability) at 540 nm (OD540) using the Anthos 2001 microplate reader.

Tissue Histology:

One tissue for each treatment group was retained for possible tissue histopathology.

The tissues were carefully cut out of the polycarbonate inserts with a sharp scalpel. The tissues were carefully cut in half. Both halves were placed into a pre-labelled 1.5 ml Eppendorf tube containing 1 ml of 10% Formalin and stored at room temperature.

Interpretation of Results:

The mean OD540 values of the duplicate tissues were calculated. Each of these OD540 values had already been corrected for blanks by the microplate reader.

The relative mean tissue viabilities (% of the negative control) were calculated as follows:

Relative mean = (mean OD540 of test material/mean OD540 of negative control) x 100
viability (%)

The mean tissue viabilities for the test material was compared to the respective untreated negative control and classified according to the table in the “Any other information on materials and methods including tables” section.

Assay Acceptance Criterion: Positive Control:

The assay meets the acceptance criterion for an acceptable test if a decrease in relative tissue viability is observed following the 10 minute exposure period.

Results and discussion

In vivo

Results
Irritation parameter:
other: Qualitative evaluation of tissue viability
Basis:
other: Qualitative evaluation of tissue viability
Time point:
other: 10 min
Score:
0
Max. score:
0
Reversibility:
not specified
Remarks on result:
other: Non irritating (NI)
Irritant / corrosive response data:
Assessment of Direct Test Material Reduction of MTT: The test material was not able to directly reduce MTT.

Assessment of Eye Irritation Potential: The mean OD540 values and mean viabilities for each treatment group were determined and tabulated.

The relative mean viability of the test material treated tissues after a 10 minute exposure was 95.2%. Therefore it was considered unnecessary to proceed with tissue histopathology.

Qualitative Evaluation of Tissue Viability (MTT Uptake Visual Assessment):

The qualitative evaluation of tissue viability is presented in the table shown in the “Remarks on results including tables and figures” section below.

The test material and negative control material treated tissues appeared blue, which was considered to be indicative of viable tissue. The positive control material treated tissues appeared blue/white, which was considered to be indicative of semi-viable tissue.

Assay Acceptance Criterion: The quality criterion required for the acceptance of results in the test was satisfied.

Conclusion: According to the protocol followed the test material was considered to be a Non-Irritant (NI).

Any other information on results incl. tables

Assessment of Eye Irritation Potential – Viability of RHC Tissues  

Material

Mean Tissue Viability

MeanOD540

Viability (%)

Negative Control

1.000

0.972

100*

0.944

Positive Control

0.703

0.730

75.1

0.757

Test Material

0.875

0.925

95.2

0.975

  • The mean viability of the negative control tissues is set at 100%

Qualitative Evaluation of Tissue Viability

 (MTT uptake visual evaluation)

Material

Score

Tissue 1

Tissue 2

Negative Control

-

-

Positive Control

+

+

Test Material

-

-

 

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: other: Qualitative Evaluation of Tissue Viability (visual evaluation))
Conclusions:
Conclusion: According to the protocol followed the test material was considered to be a Non-Irritant (NI).

Executive summary:

Introduction: The purpose of this study was to determine the eye irritation potential of the test material using the SkinEthic Reconstituted Human Corneal model (HSE, Skin Ethic Laboratories, Nice, France) after a pretreatment period of about 10 minutes. The test is based on the hypothesis that irritant chemicals are able to penetrate the corneal epithelial tissue and are sufficiently cytotoxic to cause death.

Method: The experimental design of the study consists of a test for direct reduction of MTT (3 -[4,5 -dimethylthiazol-2 -yl]-2,5 -diphenyl tetrazolium bromide) by the test material followed by the main test.

For the main test, triplicate skinetich tissues were treated with 30µl of the test material for 10 minutes. Triplicate tissues treated with 3 0µl of Solution A served as the negative control and triplicate tissues treated with 30µl of 1% w/v Sodium Dodecyl Sulphate served as the positive control.

At the end of the exposure period each skinEthic tissue was rinsed. The rinsed tissues (two per group) were taken for MTT loading. The remianing tissues were retained for possible histopathology. Following MTT loading the reduced MTT was extracted from the tissues.

After extraction the absorbency of triplicate aliquots of the extracted MTT solution for each SkinEthic tissue was measured. The optical density was measured at 540 nm (OD540). Data are presented in the form of percentage viability (MTT conversion relative to negative controls).

The test material was classified according to the following data:

If the percentage relative mean tissue viability was > 60% the test material was considered to be a non irritant

If the percentage relative mean tissue viability was < 60% the test material was considered an irritant.

Results: The relative mean viability of the test material treated tissues after a 10 minute exposure was 95.2%. It was considered unnecessary to proceed with tissue histopathology.

Quality criteria: The quality criteria required for acceptance of results in the test were satisfied.

Conclusion: According to the protocol followed the test material was considered to be a non-irritant (NI).