Reaction mass of 2-hexadecyl-N-(2-hydroxyethyl)-3-oxoicosanamide, 2-hexadecyl-N-(2-hydroxyethyl)-3-oxo-, N-(2-hydroxyethyl)-3-oxo-2-tetradecylicosanamide and N-(2-hydroxyethyl)-3-oxo-2-tetradecyloctadecanamide.

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 08 November 2011 and 10 November 2011.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: SkinEthic Reconstructed Human Corneal model (HCE, SkinEthic Laboratories, Nice, France)

Deviations:

no

Principles of method if other than guideline:

The purpose of this study was to determine the eye irritation potential of the test item using the SkinEthic Reconstructed Human Corneal model (HCE, SkinEthic Laboratories, Nice, France) after a treatment period of 10 minutes.

The test is based on the hypothesis that irritant chemicals are able to penetrate the stratum corneum of the SkinEthic HCE 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 item treated tissues (quantitative measurement of tissue viability) relative to the negative control.

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

PC-9S

IUPAC Name:

PC-9S

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Sponsor's identification: PC-9S
Description: off white powder
Lot number: PS1 011 03
Purity: not supplied
Date received: 11 July 2011
Expiry date: 18 November 2012
Storage conditions: room temperature in the dark

Test animals / tissue source

Species:

other: SkinEthic HCE model

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

Not applicable as an invitro method was used.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 30 mg

Duration of treatment / exposure:

10 minutes

Observation period (in vivo):

not applicable as an in vitro method was conducted

Number of animals or in vitro replicates:

not applicable as an in vitro study was conducted

Details on study design:

Pre-Test:
Assessment of Direct Test Item Reduction of MTT:

The MTT assay, a colourimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) to a purple formazan dye by mitochondrial succinate dehydrogenase in viable cells.

One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT to formazan, thus mimicking dehydrogenase activity of the cellular mitochondria of viable cells. This property of the test item is only a problem, if at the time of the MTT test (after the chemical has been rinsed off) there are still sufficient amounts of the test item on or in the tissues. To identify this possible interference, the test item was checked for its ability to reduce MTT directly. 30 mg of test item was added to 1 ml of a 0.5 mg/ml MTT solution and incubated at 37°C, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control. If the MTT solution turned blue, the test item was presumed to have reduced the MTT.

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 labelled with details of the treatment and the appropriate exposure time. Separate treatment plates were used for the test item, 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' into the wells of the 'treatment plates' containing the maintenance medium.

Results and discussion

In vivo

Results

Irritation parameter:

other: in vitro study

Basis:

mean

Time point:

other: 10 minute exposure period

Score:

102

Max. score:

102

Reversibility:

other: no effects seen

Remarks on result:

other: According to the study plan followed the test item was considered to be a Non-Irritant (NI).

Irritant / corrosive response data:

The relative mean viability of the test item treated tissues after a 10-Minute exposure period was 102.0%.

Other effects:

The MTT solution containing the test item turned yellow which indicated that the test item did not directly reduce MTT.

Any other information on results incl. tables

Assessment of Direct Test Item Reduction of MTT:

The MTT solution containing the test item turned yellow which indicated that the test item did not directly reduce MTT.

The relative mean viability of the test item treated tissues after a 10-Minute exposure period was 102.0%. It was considered unnecessary to proceed with tissue histopathology.

Conclusion:

According to the study plan followed the test item was considered to be a Non-Irritant (NI).

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

According to the study plan followed the test item 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 item using the SkinEthic Reconstructed Human Corneal model (HCE, SkinEthic Laboratories, Nice, France) after 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.

Methods.

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 item followed by the main test. For the main test, triplicate SkinEthic tissues were treated with 30 mg of the test item for 10 minutes. Triplicate tissues treated with 30 µl of Solution A served as the negative control and triplicate tissues treated with 30 µl of 2% w/v Sodium Oodecyl 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 remaining 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 (00540). Data are presented in the form of percentage viability (MTT conversion relative to negative controls).

The test item was classified according to the following criteria:

If the percentage relative mean tissue viability was > 60% the test item was considered to be non-irritant (NI).

If the percentage relative mean tissue viability was < 60% the test item was considered to be an irritant (I).

Results.

The relative mean viability of the test item treated tissues after a 10-Minute exposure period was 102 % (not an irritant). It was considered unnecessary to proceed with tissue histopathology.

Quality criterion. The quality criterion required for acceptance of results in the test was satisfied.

Conclusion: According to the study plan followed, the test item was considered to be a Non-Irritant (NI).