Amines, C16-18 (even numbered)-alkyl , salts with phosphoric acid, mono- and di-C16-18 (even numbered) alkyl esters

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 Sep 2020 - 20 Sep 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

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

1

Reference substance name:

Amines, C16-18 (even numbered)-alkyl , salts with phosphoric acid, mono- and di-C16-18 (even numbered) alkyl esters

EC Number:

952-252-4

Molecular formula:

H4NO4P(C32H66) to H3NO4P(C54H111)

IUPAC Name:

Amines, C16-18 (even numbered)-alkyl , salts with phosphoric acid, mono- and di-C16-18 (even numbered) alkyl esters

Test material form:

solid

Details on test material:

Chemical registery number : 952-252-4
Chemical name : Amines, C16-18 (even numbered)-alkyl , salts with phosphoric acid, mono- and di-C16-18 (even numbered) alkyl esters

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

Non-keratinized epithelium (0.6 cm2) prepared from normal human keratinocytes.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 50.1 to 55.9 mg

Duration of treatment / exposure:

6 hours ± 15 minutes

Duration of post- treatment incubation (in vitro):

18 hours

Number of animals or in vitro replicates:

The test was performed on a total of 2 tissues per test item together with a negative control
and positive control

Details on study design:

- Control substances used:
Negative control: Sterile Milli-Q water
Positive control: Methyl Acetate [CAS Number 79-20-9]

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: After the exposure period with the test item (6 hours ± 15 minutes at 37.0 ± 1.0°C), the tissues were thoroughly rinsed with Ca2+Mg2+-free D-PBS (brought to room temperature) to remove residual test item. After rinsing the cell culture inserts were each dried carefully and immediately transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a pre-labeled 12-well plate for a 25 ± 2 minute immersion incubation at room temperature (Post-Soak). 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.

- Number of tissue replicates used per test chemical and controls: The test was performed on a total of 2 tissues per test item together with a negative control and positive control

- Wavelength and band pass used for quantifying MTT formazan: The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

- Supporting information for the specific RhCE tissue construct used: EpiOcular™ (OCL-200-EIT MatTek Corporation, Lot: 31755, kit A.

Results and discussion

In vitro

Results

Irritation parameter:

percent tissue viability 

Run / experiment:

Mean Tissue Viability

Value:

94

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item is non-irritant in the EpiOcular™ test under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate the eye hazard potential of the test item. For this purpose, the test item was topically applied on the Reconstructed Human EpiOcular™ Model.
The possible eye hazard potential of the test item was tested through topical application for 6 hours.
The study procedures described in this report were based on the most recent OECD guideline.
Batch AEK-20-106 of the test item was a beige solid. The test item (50.1 to 55.9 mg) was applied directly on top of the tissue for 6 hours ± 15 minutes.
After exposure, the cornea epithelial construct was thoroughly rinsed to remove the test item and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 18 hours at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect.
The positive control had a mean cell viability of 9.8% after 6 hours ± 15 minutes exposure.
The absolute mean OD570 (optical density at 570 nm) 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 7%, indicating that the test system functioned properly.
The test item showed color interference in aqueous conditions. In addition to the normal procedure, two tissues were treated with test item. Instead of MTT solution, these tissues were incubated with assay medium. The non-specific colour of the test item was 0.30% of the negative control tissues. The OD of the treated tissues without MTT assay was subtracted from the ODs of the test item treated viable tissues with MTT assay.
Eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 6 hours ± 15 minutes treatment with the test item compared to the negative control tissues was 94%. Since the mean relative tissue viability for the test item was above 60% after 6 hours ± 15 minutes treatment, the test item is considered to be non-irritant.
In conclusion, the test item is non-irritant in the EpiOcular™ test under the experimental conditions described in this report.