Fatty acids, C18-unsatd., dimers, hydrogenated, reaction products with N1,N1-dimethyl-1,3-propanediamine

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

between 14 July 2009 and 16 July 2009.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

The assay has undergone inter-laboratory validation and has been shown to reliably detect test materials that are negligible or moderate to severe ocular irritants. The strain of rabbit used in these laboratories has been shown to produce satisfactory responses using known ocular-irritants and non-ocular irritants during in house validation. Study conducted to GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2010

Materials and methods

Test guideline

Qualifier:

equivalent or similar 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)

Principles of method if other than guideline:

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 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)

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-bis[3-(dimethylamino)propyl]-9-nonyl-10-octylnonadecanediamide; N-[3-(dimethylamino)propyl]-9-[2-(7-{[3-(dimethylamino)propyl]carbamoyl}heptyl)-3-[(2E)-oct-2-en-1-yl]-4-pentylcyclohexyl]nonanamide; N-[3-(dimethylamino)propyl]-9-[2-(7-{[3-(dimethylamino)propyl]carbamoyl}heptyl)-3-octyl-4-pentylcyclohexyl]nonanamide

EC Number:

937-688-5

Cas Number:

1391530-05-4

Molecular formula:

C46H82N4O2 to C46H94N4O2

IUPAC Name:

N,N'-bis[3-(dimethylamino)propyl]-9-nonyl-10-octylnonadecanediamide; N-[3-(dimethylamino)propyl]-9-[2-(7-{[3-(dimethylamino)propyl]carbamoyl}heptyl)-3-[(2E)-oct-2-en-1-yl]-4-pentylcyclohexyl]nonanamide; N-[3-(dimethylamino)propyl]-9-[2-(7-{[3-(dimethylamino)propyl]carbamoyl}heptyl)-3-octyl-4-pentylcyclohexyl]nonanamide

Details on test material:

- Substance type: UVCB
- Physical state: amber coloured viscous liquid
- Analytical purity: > 97.5%
- Lot/batch No.: CM71117612
- Expiration date of the lot/batch: 14 April 201 0
- Storage condition of test material: room temperature in the dark

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Date Recieved: 14 April 2009
- Purity test date: 13 October 2008

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Stability under storage conditions: Stable

Test animals / tissue source

Species:

human

Strain:

not specified

Details on test animals or tissues and environmental conditions:

The SkinEthic RHC 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.
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 μl 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°C, 5% CO2 in air.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30ul
- Concentration (if solution): Undiluted


VEHICLE
- Amount(s) applied (volume or weight with unit): N/A
- Concentration (if solution): N/A
- Lot/batch no. (if required): N/A
- Purity: N/A

Duration of treatment / exposure:

10 min

Observation period (in vivo):

All the tissues were rinsed after 10 min treatment, and each tissue was placed into a pre-labelled 24-well plate containing 300 μl of maintenance medium (at room temperature for 3 h) .

Number of animals or in vitro replicates:

Triplicate tissues for negative control group;
Triplicate tissues for test material treated group;
Triplicate tissues for positive control group

Details on study design:

See information presented in the below section "Any other information on materials and methods incl. tables"

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

other: Mean Tissue Viability (%)

Run / experiment:

1

Value:

0.626

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

other: Mean Tissue Viability (%)

Run / experiment:

2

Value:

0.616

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The relative mean viability of the test material treated tissues after a 10 minute exposure was 50.9%. According to the protocol followed the test material was considered to be an irritant (I).
The test material was not able to directly reduce MTT.
Control Tissue: The positive control result of 48.9% fell within the historical range of 12.4 to 75.0% for the positive control for this study type in this testing facility.

Any other information on results incl. tables

The test material was classified according to the following criteria:

_i)            If the % relative mean tissue viability was 160% the test material was considered to be non-irritant.

_ii)           If the % relative mean tissue viability was < 60% the test material was considered to be an irritant.

Results.The relative mean viability of the test material treated tissues after a 10 minute exposure was 50.9%.

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 Irritant.

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

According to the protocol followed the test material was considered to be Irritant.

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 (HCE, SkinEthic Laboratories,,) 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-diphen-tetrazoum bromide) by the test material followed by the main test.

For the main test, triplicate SkinEthic tissues were treated with 30 HI of the test material for 10 minutes. Triplicate tissues treated with 30 p1 of Solution A served as the negative control and triplicate tissues treated with 30 p1 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 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 (OD5&)). Data are presented in the form of % viability (MTT conversion relative to negative controls).

The test material was classified according to the following criteria:

_i)            If the % relative mean tissue viability was 160% the test material was considered to be non-irritant.

_ii)           If the % relative mean tissue viability was < 60% the test material was considered to be an irritant.

Results.The relative mean viability of the test material treated tissues after a 10 minute exposure was 50.9%.

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 Irritant.