Reaction mass of: N,N'-[1,3-Phenylenebis(methylene)bis[12-hydroxyoctadecanamide], N,N'-hexane-1,6-diylbis(12-hydroxyoctadecanamide) and N,N'-ethane-1,2-diylbis(12-hydroxyoctadecan-1-amide)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 . Jan. 2019 - 21. Feb. 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-ethane-1,2-diylbis(12-hydroxyoctadecan-1-amide)

EC Number:

204-613-6

EC Name:

N,N'-ethane-1,2-diylbis(12-hydroxyoctadecan-1-amide)

Cas Number:

123-26-2

Molecular formula:

C38H76N2O4

IUPAC Name:

12-hydroxy-N-[2-(12-hydroxyoctadecanamido)ethyl]octadecanamide

Test material form:

solid

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Tissue 1: 52.8 mg
Tissue 2: 50.0 mg

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

18 hours and 15 minutes

Number of animals or in vitro replicates:

2 replicates

Details on study design:

On the day of the start of the experiment, the MTT concentrate was thawed. The MTT concentrate was diluted with assay medium directly before use. The assay medium was warmed in the water bath to 37 ± 1°C. 6-well-plates were labelled with test item, negative control and positive control and filled with 1 mL assay medium in the appropriate wells. All inserts were inspected for viability and the presence of air bubbles between agarose gel and insert. Viable tissues were transferred in the prepared 6-well-plate and incubated at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity for 1 hour. After the pre-incubation, the medium was replaced and the wells were filled with 1 mL fresh assay medium. All 6-well-plates were incubated at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity for 16 hours and 29 minutes. 7.2.2 Exposure and Post-Treatment After overnight incubation, the tissues were pre-wetted with 20 μL DPBS buffer and the tissues were incubated at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity for 29 minutes. After that, 50 μL of the controls and a defined amount of the test item were applied in duplicate in one- minute- intervals. At the beginning of each experiment (application of negative controls), a stop watch was started. After dosing the last tissue, all plates were transferred into the incubator for 6 hours at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity. At the end of exposure time, the inserts were removed from the plates in one-minute-intervals using sterile forceps and rinsed immediately. The inserts were thoroughly rinsed with DPBS. Then, the tissues were immediately transferred into 5 mL of assay medium in pre-labelled 12-well plate for 24 minutes post soak at room temperature. After that, each insert was blotted on absorbent material and transferred into a pre-labelled 6-well plate, containing 1 mL assay medium. For post-treatment incubation, the tissues were incubated for 18 hours and 15 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity. After the post-treatment incubation, the MTT assay was performed.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

other: Tissue Viability

Remarks:

in % of the negative control

Run / experiment:

1

Value:

104.9

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

other: Tissue Viability

Remarks:

in % of the negative control

Run / experiment:

2

Value:

105

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

All validity criteria are met. Positive and negative controls are within the range of historical data of the test facility. Therefore, the experiment is considered valid.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

All validity criteria are fulfilled. The study can be considered valid. Under the conditions of the test, Bisamid is considered non-eye irritant in the EpiOcularTM Eye Irritation Test.

Executive summary:

One valid experiment was performed. The test item Bisamid was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 6 hours. 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 formazan. The formazan production was evaluated by measuring the optical density (OD) of the resulting solution. Demineralised water was used as negative control and methyl acetate was used as positive control. The controls showed the following results: After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD > 0.8 and < 2.5, OD was 1.7. The positive control showed clear eye irritating effects, the mean value of the relative tissue viability was 30.1% (< 50%). The variation within tissue replicates of the controls and the test item was acceptable (< 20%). After treatment with the test item, the mean value of relative tissue viability was 104.9 %. This value is well above the threshold for eye irritation potential (≤ 60%). Test items that induce values above the threshold are considered non-eye irritant. Under the conditions of the test, Bisamid is considered non-eye irritant in the Epi-OcularTM Eye Irritation Test.