Dimethyl[3-[(1-oxooctadecyl)amino]propyl][2-oxo-2-(tetradecyloxy)ethyl]ammonium chloride

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The experimental start date was 13 Nov 2017, the experimental completion date was 17 Nov 2017.

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 09 October 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyl[3-[(1-oxooctadecyl)amino]propyl][2-oxo-2-(tetradecyloxy)ethyl]ammonium chloride

EC Number:

272-964-2

EC Name:

Dimethyl[3-[(1-oxooctadecyl)amino]propyl][2-oxo-2-(tetradecyloxy)ethyl]ammonium chloride

Cas Number:

68921-83-5

Molecular formula:

C39H79N2O3.Cl

IUPAC Name:

dimethyl(3-octadecanamidopropyl)[2-oxo-2-(tetradecyloxy)ethyl]azanium chloride

Constituent 2

Reference substance name:

Propane-1,2-diol

EC Number:

200-338-0

EC Name:

Propane-1,2-diol

Cas Number:

57-55-6

Molecular formula:

C3H8O2

IUPAC Name:

propylene glycol

Test material form:

semi-solid (amorphous): gel

Details on test material:

Identification Quaternium-70
Appearance Amber gel
Batch 0002000146
Composition UVCB
Test item storage at room temperature
Stable under storage conditions until 02 April 2018 (retest date)

Test animals / tissue source

Details on test animals or tissues and environmental conditions:

EpiOcular™ (OCL-200-EIT MatTek Corporation, Lot: 27406 Kit Q.
Source: MatTek Corporation, Ashland MA, U.S.A.
The EpiOcular tissue construct is a non-keratinized epithelium (0.6 cm2) prepared from normal human keratinocytes (MatTek). It models the cornea epithelium with progressively stratified, but not cornified cells. These cells are not transformed or transfected with genes to induce an extended life span in culture.
The “tissue” is prepared in inserts with a porous membrane through which the nutrients pass to the cells. A cell suspension is seeded into the insert in specialized medium. After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air. This allows the test material to be directly applied to the epithelial surface in a fashion similar to how the corneal epithelium would be exposed in vivo.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Neat, the test item (an excessive amount per tissue) was applied directly on top of the skin tissue and spread to match the size of the tissue. Since the test item induced color interference in aqueous conditions, two tissues were treated with test item and incubated with DMEM instead of MTT solution.

Duration of treatment / exposure:

6 hours ± 15 minutes at 37.0 ± 1.0°C

Duration of post- treatment incubation (in vitro):

Post-Soak: 25 ± 2 minute immersion incubation at room temperature.
After the Post-Soak period cell culture inserts were each dried carefully and transferred to the 6-well plate containing 1.0 ml of warm Assay Medium and were incubated for 18 hours ± 15 minutes at 37°C.
After incubation, cell culture inserts were dried carefully to remove excess medium and subsequently transferred into a 24-wells plate prefilled with 0.3 ml MTT-medium (1.0 mg/ml). The tissues were incubated for 180 ± 10 minutes at 37°C.

Number of animals or in vitro replicates:

2 tissues per test item together with a negative control and positive control

Details on study design:

Quaternium-70 was checked for possible color interference and direct MTT reduction before the study was started.
On the day of receipt the tissues were equilibrated (in its 24-well shipping container) to room temperature. Subsequently, tissues were transferred to 6-well plates and incubated for
20 ± 4 hours at 37°C in 1.0 ml fresh pre-warmed Assay Medium, which was refreshed after approximately 1 hour.
DMEM (Dulbecco’s Modified Eagle’s Medium): Supplemented DMEM medium, serum-free supplied by MatTek Corporation.
MTT medium: MTT concentrate (5 mg/ml) diluted (1:5) with MTT diluent.
The tissues were pre-incubated at standard culture conditions for 30 ± 2 minutes.
After the exposure period, tissues were thoroughly rinsed with Ca2+Mg2+-free D-PBS to remove residual test item.
Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Eye hazard potential of the test item was classified according to remaining cell viability following exposure of the test item.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Remarks:

mean % tissue viability

Run / experiment:

Main

Value:

12

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Milli-Q water

Positive controls validity:

valid

Remarks:

Methyl Acetate

Remarks on result:

positive indication of irritation

Any other information on results incl. tables

The relative mean tissue viability obtained after 6 hours±15 minutes treatment with Quaternium-70 compared to the negative control tissues was 12%. Since the mean relative tissue viability for Quaternium-70 was below 60%, it is considered to be potentially irritant or corrosive to the eye. The positive control had a mean cell viability after 6 hours ±15 minutes exposure of 31%. The absolute mean OD₅₇₀ of the negative control tissues was within the laboratory historical control data range . The difference between the percentage of viability of two tissues treated identically was less than 17%, indicating that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:

other: Category 2 or Category 1

Conclusions:

Quaternium-70 is potentially irritant or corrosive in the EpiOcular™ test under the experimental conditions described in this report. The test item is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1). Assuming a worst case scenario, the ultimate classification is set at cat. 1.