Octadecanoic acid, reaction products with 2-amino-2-methyl-1-propanol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From May 25, 2017 to June 22, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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:

Octadecanoic acid, reaction products with 2-amino-2-methyl-1-propanol

EC Number:

273-159-9

EC Name:

Octadecanoic acid, reaction products with 2-amino-2-methyl-1-propanol

Cas Number:

68951-62-2

Molecular formula:

C20H41NO2 (C16 chain representative) & C22H45NO2 (C18 chain representative)

IUPAC Name:

N-(1-hydroxy-2-methylpropan-2-yl)hexadecanamide; N-(1-hydroxy-2-methylpropan-2-yl)octadecanamide

Test material form:

solid

Details on test material:

Appearance: Off white cream

Test animals / tissue source

Species:

other: EpiOcularTM tissue model (OCL-200-MatTek Corporation)

Strain:

other: Keratinocyte 4F1188

Details on test animals or tissues and environmental conditions:

Test system:
The EpiOcularTM tissue model (OCL-200-MatTek Corporation) is composed of stratified human keratinocytes in a three-dimensional structure, reflecting the morphology and function of the human corneal epithelium found in vivo.

Lot No.: 23787
Keratinocyte Strain: 4F1188

MatTek’s EpiOcularTM system consists of normal, human-derived keratinocytes which have been cultured to form a stratified, squamous epithelium similar to that found in the cornea. Cultured on specially prepared cell culture inserts using serum-free culture medium, the cells differentiate to form a multi-layered structure with progressively stratified, but not cornified cells which closely parallel the corneal epithelium (for information see www.mattek.com). QC results for the specific lot of models received (Lot# 23787) were checked in-house for MatTek acceptance ranges with the following outcome:

- Morphology - TBD
- Tissue viability - TBD
- Skin barrier function (ET50 value for 0.3 % Triton X-100) where ET50 is the time taken for 0.3 % Triton X-100 to reduce the viability of the skin model to 50 % relative to the negative control) - TBD
- Sterility testing showed no contamination during long term antibiotic and antimycotic free culture - TBD

Test system

Vehicle:

other: PBS (Sterile Dulbecco’s Phosphate Buffered Saline)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

20 µl of PBS (Sterile Dulbecco’s Phosphate Buffered Saline) plus 50 mg of test substance.

Duration of treatment / exposure:

6h ± 15 minutes post-treatment immersion, and 18 hours ± 15 minutes post-treatment incubation.

Duration of post- treatment incubation (in vitro):

25 ± 2 minutes

Number of animals or in vitro replicates:

Three tissues per condition (n=3).

Details on study design:

Preliminary test:
The test substance was first checked for its potential for MTT interference and solvent interference (water and isopropanol).

Main test overview:
Day 0: On the day of receipt, EpiOcularTM tissues were pre-incubated overnight at 37 °C, 5 % CO2.
Day 1: Exposure to and removal of test and reference substances (50 mg of test substance or 50 µl of reference substances for 6h ± 15 minutes, followed by a 25 ± 2 minutes post-treatment immersion, and 18 hours ± 15 minutes post-treatment incubation). 
Day 2: End of MTT viability test, readings at 570 nm without reference filter.

Results and discussion

In vitro

Results

Irritation parameter:

other: % viability

Value:

112.829

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

All validity criteria for the test were met:
- Criteria: the mean OD570 of the negative control (treated with sterile water) tissues is ≥ 0.8 and ≤ 2.5.
Result for the test: 1.071
- The mean of the positive control relative percentage viability is below 50% of negative control viability after 6 hours exposure.
Result for the test: 36.696
- The SD between three tissues replicates should not exceed 18 % in the same run (for negative and positive control tissues and tissues of test substances).
Results for the test:
NC: 11.368
PC: 7.373
TA2: 7.528

Applicant's summary and conclusion

Interpretation of results:

other: CLP criteria not met (not classified)

Conclusions:

Under the study conditions, based on the percentage viability of 112.829%, the test substance was concluded to be non-irritant to the human eye

Executive summary:

A study was conducted to determine the eye irritation potential of the test substance, C16-18 AMP using Reconstructed human Cornea-like Epithelium (RhCE) test method, according to OECD 492 Guideline, in compliance with GLP. Three tissues of the EpiOcularTM tissue model were treated with the test substance, positive or negative control. Tissues were pre-wetted with 20 μL of PBS (Sterile Phosphate Buffered Saline) prior to topical application of approximately 50 mg of neat test substance. Sterile water was used as negative control and methyl acetate as positive control. After 6 h exposure on the surface of EpiOcularTM reconstructed ocular epithelium and 18 h post-incubation time, the viability of the tissues was assessed and compared to a negative control. The MTT viability test readings were conducted at 570 nm without reference filter. Under the study conditions, based on the percentage viability of 112.829%, the test substance was concluded to be non-irritant to the human eye (XCellR8, 2017).