Reaction mass of Reaction product of 1-chloro-3-{[1-chloro-3-(dodecyloxy)propan-2-yl]oxy}propan-2-ol with methyl diethanolamine and [3-(dodecyloxy)-2-hydroxypropyl]bis(2-hydroxyethyl)methylammonium chloride

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-07-24 to 2017-07-27

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:

adopted 28. Jul. 2015

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of Reaction product of 1-chloro-3-{[1-chloro-3-(dodecyloxy)propan-2-yl]oxy}propan-2-ol with methyl diethanolamine and [3-(dodecyloxy)-2-hydroxypropyl]bis(2-hydroxyethyl)methylammonium chloride

IUPAC Name:

Reaction mass of Reaction product of 1-chloro-3-{[1-chloro-3-(dodecyloxy)propan-2-yl]oxy}propan-2-ol with methyl diethanolamine and [3-(dodecyloxy)-2-hydroxypropyl]bis(2-hydroxyethyl)methylammonium chloride

Test material form:

other: solid, very viscous yellow paste

Test animals / tissue source

Species:

human

Strain:

other: normal, human-derived keratinocytes

Details on test animals or tissues and environmental conditions:

- Source: MatTek In Vitro Life Science Laboratories, Mylnské Nivy 73, 82105 Bratislava, Slovakia.
- Commercially available EpiOcularTM kit: normal, human-derived keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea.
- Surface: 0.6 cm
- Inspection: each tissue was inspected for air bubbles between the agarose gel and insert
- Pre-incubation: 37 ± 1 °C, 5 ± 1 % CO2 and 80–100 % relative humidity for 1 hour
- After the pre-incubation, the medium was replaced with fresh assay medium. All 6-well-plates were incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80–100 % relative humidity for 16 hours
- Exposure: 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 80–100 % relative humidity for 30 minutes.
After that, 50 μL of the controls and a defined amount of the test item were applied in duplicate.
After dosing the last tissue, all plates were transferred into the incubator for 6 hours at 37 ± 1 °C, 5 ± 1 % CO2 and 80–100 % relative humidity.
- Post-Treatment: The inserts were thoroughly rinsed with DPBS.
For post-treatment incubation, the tissues were incubated for 18 hours at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity 1 mL assay medium

Test system

Vehicle:

other: DPBS

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

- Negative control: 50 μL of sterile demineralised water
- Positive control: 50 μL of methyl acetate
- Test substance: 50 ± 5 mg

Duration of treatment / exposure:

6 h

Observation period (in vivo):

not applicable

Duration of post- treatment incubation (in vitro):

18 h

Number of animals or in vitro replicates:

2 replicates per dose group

Details on study design:

- MTT assay: incubation with 0.3 mL of MTT solution for 190 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity. After incubation with MTT the inserts were incubated with isopropanol and shaken for 2 hours at room temperature, protected from light.

- Data evaluation:
- Calculation of mean OD of the blank isopropanol (ODBlk)
- Subtraction of mean ODBlk of each value of the same experiment (corrected values)
- Calculation of mean OD of the two replicates for each tissue
- Calculation of mean OD of the two relating tissues for controls and test item
Note: Corrected OD value of negative control corresponds to 100 % viability

To calculate the relative absorbance, the following equation was used: %viability = (OD corrected of test item or positive control / OD corrected of mean negative control) * 100

- Description of evaluation criteria: If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is non-irritant and if the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is serious eye damage/ eye
irritant.

- Acceptability of the Assay: The results are acceptable if
(1) The negative control OD is > 0.8 and <2.5
(2) The mean relative viability of the positive control is below 50% of the negative control viability
(3) The variation within replicates is < 20%

Results and discussion

In vitro

Results

Irritation parameter:

other: relative tissue viability (%)

Run / experiment:

mean of two replicates

Value:

5.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

The criterion for optical density of the negative control was fulfilled: The OD value was 1.5 (> 0.8 and < 2.5). The positive control induced a decrease in tissue viability as compared to the negative control to 32.4%. The variation within the replicates of negative control, positive control and test item was acceptable (< 20%). All acceptance criteria were fulfilled. For these reasons, the result of the test is considered as valid.

In vivo

Irritant / corrosive response data:

After treatment with the test item, the mean value of relative tissue viability was reduced to 5.2 %. This value is below the threshold for eye irritation potential (≤ 60%).All validity criteria were met.

Any other information on results incl. tables

Mean absorbance values

Designation	Measurement	Negative control	Positive control	Test item
Tissue 1	1	1.589	0.493	0.121
	2	1.666	0.504	0.116
Tissue 2	1	1.540	0.577	0.119
	2	1.541	0.578	0.116

% Viability positive control and test item

Designation	Positive Control	Test item
% viability (Tissue 1)	29.80%	5.30%
% viability (Tissue 2)	34.90%	5.20%
% viability Mean	32.40%	5.20%

Applicant's summary and conclusion

Conclusions:

The test item is considered either eye irritant or inducing serious eye damage in the EpiOcularTM Eye Irritation Test.

Executive summary:

This study was performed in order to evaluate the potential of the test substance to evoke eye irritation in a Reconstructed human Cornea-like Epithelium (RhCE) model in an in vitro study (OECD 492). The study was performed in compliance to GLP.

The pre-tests showed that the test item did not reduce MTT itself and no colour inference with the photometrical measurement could be observed. Therefore no additional tests were necessary.

The test item 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 1.5. The positive control showed clear eye irritating effects, mean value of the relative tissue viability was 32.4 % (< 50%). The variation within tissue replicates of negative control, test item and positive control was acceptable (< 20%).

After treatment with the test item, the mean value of relative tissue viability was 5.2 %. This value is below the threshold for eye irritation potential (≤ 60%). One valid experiment was performed.

In conclusion and under the conditions the test item is considered either eye irritant or inducing serious eye damage in the EpiOcular Eye Irritation Test.