Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 - 13 Dec 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Demonstration of proficiency not in report; no historical control data range reported.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guidelineopen allclose all
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 in 2018
Deviations:
no
Qualifier:
equivalent or similar 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 in 2019
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Medicines & Healthcare products Regulatory Agency, Department of Health of the Government of the United Kingdom

Test material

Constituent 1
Reference substance name:
Reaction product of castor oil with glycerol
EC Number:
949-117-7
Molecular formula:
not applicable, substance is UVCB
IUPAC Name:
Reaction product of castor oil with glycerol
Test material form:
liquid

Test animals / tissue source

Species:
human
Strain:
other: EpiOcular™ (OCL-200)

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 50 µL
Duration of treatment / exposure:
30 ± 2 min
Duration of post- treatment incubation (in vitro):
2 ± 0.25 h
Number of animals or in vitro replicates:
2
Details on study design:
- Details of the test procedure used: The EpiOcular ™ Eye Irritation Test (EIT) consists of a topical exposure of the neat test item to a human reconstructed cornea model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT [(3-4,5-dimethyl thiazole 2-yl) 2,5-diphenyl-tetrazoliumbromide], present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison of untreated negative controls is used to predict eye irritation potential.

- RhCE tissue construct used, including batch number : EpiOcular™ (OCL-200) (MatTek Corporation, Ashland, USA); batch: 27085; date received: 11 Dec 2018

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: 30 ± 2 min (exposure), 12 ± 2 min (post-treatment immersion), 2 ± 0.25 h (post-treatment incubation), temperatures not specified

- Description of any modifications to the test procedure : Only two (2) tissues were used for test substance and controls.

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: The ability of the test substance to directly reduce MTT and to form a blue/purple reaction product was assessed in a pre-experiment. The test confirmed that the test item interfered with MTT but not with the solvent. Therefore, additional freeze killed controls were included for the study that allow determination of the background signal in the absence of viable tissue.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- Spectrophotometer: BMG LabTech FluoStar Optima
- Wavelength: 570 nm
- Filter: no filter used
- Filter bandwidth: not applicable, no filter used
- Spectrophotometer calibration date: 05 Nov 2018

FUNCTIONAL MODEL CONDITIONS (specified by MatTek Corporation)
- Viability: 2.087 ± 0.124 (acceptance range: 1.1 - 3.0)
- Barrier function: 15.82 min (acceptance range: 12.2 - 37.5 min)
- Morphology: 8 layers are present; thickness: 77.4 µm (acceptance range: 40 - 120 µm)
- Contamination: no contamination reported

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model : The test substance is considered to be not irritating to eye if the tissue viability after 30 min exposure, 12 min post-exposure immersion and 120 min post-exposure incubation is > 60% relative to the negative control treated tissue.

- Acceptable variability between tissue replicates for positive and negative controls : The results are acceptable if the negative control optical density (OD) is > 0.8 and < 2.5 and if the mean relative viability of the positive control is below 50% of the negative control viability.

- Acceptable variability between tissue replicates for the test chemical: The results are acceptable if the difference of viability between the two relating tissues of a single test item is < 18% in the same run (for positive and negative control tissues and tissues of test items).

Results and discussion

In vitro

Results
Irritation parameter:
other: % tissue viability
Remarks:
mean value of 2 tissues
Run / experiment:
30 min
Value:
115.188
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean optical density (OD) of the tissue replicates treated with the negative control was ≥ 0.8 and ≤ 2.8 for every exposure time (values of 2.344 and 2.166).
- Acceptance criteria met for positive control: The mean viability of the tissue replicates treated with the positive control was < 50% compared to the negative control (20.783% and 13.215%).
- Acceptance criteria met for variability between replicate measurements: The coefficient of variation (CV) in the range 20 - 100% viability between tissue replicates is ≤ 20% (10.118%).

Any other information on results incl. tables

Table 1: Viability measurements after 30 ± 2 min of application and 2 ± 0.25 h post-incubation of test and reference items. Two tissues per condition were tested, with three measurements taken per tissue.

Condition

Tissue #

Raw Data

Blank Corrected Data

Mean OD

% Viability

1

2

3

1

2

3

NC

Tissue 1

2.509

2.499

2.493

2.352

2.342

2.336

2.344

103.932

Tissue 2

2.304

2.301

2.364

2.147

2.144

2.207

2.166

96.068

PC

Tissue 1

0.623

0.631

0.622

0.466

0.474

0.465

0.469

20.783

Tissue 2

0.457

0.45

0.457

0.300

0.293

0.300

0.298

13.215

TA1

Tissue 1

2.406

2.659

2.64

2.249

2.502

2.483

2.412

106.948

Tissue 2

2.884

2.96

2.976

2.727

2.803

2.819

2.783

123.429

NC: negative control (sterile water),

PC: Positive control (neat methyl acetate),

TA1: Reaction product of castor oil with glycerol.

Table 2: Mean and SD of viability measurements and of viability percentages after 30 ± 2 min of application and 2 ± 0.25 h post-incubation.

Name

Code

Mean of OD

SD of OD

Mean of viability
(%)

SD of viability (%)

CV %

Classification

Sterile water

NC

2.255

0.125

100.000

5.561

5.561

No Category

Methyl acetate

PC

0.383

0.121

16.999

5.352

31.482

No Prediction

Reaction product of castor oil with glycerol

TA1

2.598

0.263

115.188

11.654

10.118

No Category

NC: negative control (sterile water),

PC: Positive control (neat methyl acetate),

TA1: Reaction product of castor oil with glycerol.

Applicant's summary and conclusion

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008
Conclusions:
Under the conditions of the conducted test, the test substance did not possess irritating properties towards human-derived epidermal keratinocytes in the EpiOcular™ model. There is regulatory acceptance in the EU that a substance can be considered non-irritant and non-corrosive based on a negative result in the EpiOcular™ EIT prediction model.
Executive summary:

The available data on eye irritation and serious eye damage are, therefore, conclusive but not sufficient for classification according to the critera of Regulation (EC) No. 1272/2008 (CLP).