Reaction product of 4,4'-methylenediphenyl diisocyanate (MDI) (CAS: 101-68-8), Octylamine (111-86-4) and Hexamethylenediamine (124-09-4)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02/06/2020 - 02/07/2020

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

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:

2019

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Polyurethane thickener

IUPAC Name:

Polyurethane thickener

Specific details on test material used for the study:

- Direct MTT reduction: The test item was tested for the ability of direct MTT reduction. The MTT solution did not change its colour; therefore, direct MTT reduction had not taken place, and no data correction was necessary.
- Assessment of Coloured or Staining Test Items: After subtraction of the mean OD for isopropanol, the mean OD of the test item solution was 0.00 (≤ 0.08). Therefore, the main test was performed without colourant controls.

Test animals / tissue source

Species:

human

Strain:

not specified

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability:
The EpiOcularTM Eye Irritation Test (EIT) predicts the acute eye hazard potential of chemicals by measurement of tissue damage caused by cytotoxic effects in the reconstructed human cornea-like tissue model.
- Description of the cell system used: The EpiOcularTM tissue consists of normal, human-derived keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells. These cells are not transformed or transfected with genes to induce an extended life span. The EpiOcularTM tissues are cultured in specially prepared cell culture inserts with a porous membrane through which nutrients can pass to the cells. The tissue surface is 0.6 cm2.
- Cell line used, its source, batch number used for testing: EpiOcularTM tissues, MatTek In Vitro Life Science Laboratories, batch 30665

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

- Amount of test material and controls applied: 50 μL

Duration of treatment / exposure:

- Negative and positive controls: 28 minutes
- Test item: 30 minutes

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

Duplicate

Details on study design:

- Details of the test procedure used
: MTT assay
- Doses of test chemical and control substances used
: 50 μL
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: 50 μL of the controls and the test item were applied in duplicate in one- minute- intervals. After dosing the last tissue, the plates were transferred into the incubator for 28 minutes (negative and positive control) and 30 minutes (test item) at 37 ± 1°C, 5 ± 1 % CO2 and ≥ 95% relative humidity. At the end of the exposure time, tissues were rinsed with DPBS then immediately transferred to and immersed in 5 mL of pre-warmed assay medium for 12 minutes post soak at room temperature. For post-treatment incubation, the tissues were incubated in assay medium for 120 minutes at 37 ± 1°C, 5 ± 1 % CO2 and ≥ 95% relative humidity.
- Controls used for direct MTT-reducers and/or colouring test chemicals: Following pre-test checks, no controls for direct MTT-reduction or coloured test item was required.
- Number of tissue replicates used per test chemical and controls: Two
- Wavelength and band pass used for quantifying MTT formazan: Plate spectrophotometer at 570 nm
- Measurements: Optical density (OD)
- Calculation: % viability = (OD corrected of test item or positive control / OD corrected of mean negative control) x 100

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

percent tissue viability 

Run / experiment:

1

Value:

106.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

percent tissue viability 

Run / experiment:

2

Value:

103.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation parameter:

mean percent tissue viability 

Run / experiment:

Mean

Value:

105.2

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:

ACCEPTANCE AND VALIDITY:
- Acceptance criteria met for negative control: Yes - OD value of 2.0 (i.e. >0.8 - < 2.0)
- Acceptance criteria met for positive control: Yes - mean relative viability value of 39.0% compared to negative control (i.e. <50%)
- Variation within replicates: 8.9% for the negative control, 5.3% for the positive control and 3.0% for the test item (i.e. <20%)
- Historical data: The values for negative control and for positive control were within the range of historical data of the test facility.
- Conclusion: The experiment is considered valid.

RESULTS AND CONCLUSION:
- Test results: After treatment with the test item, the mean value of relative tissue viability was increased to 105.2%. This value is above the threshold for eye irritation potential (≤ 60%). Therefore, under the conditions of the test, the test item is considered non-eye irritant in the EpiOcularTM Eye Irritation Test.

Any other information on results incl. tables

Table 1. Mean OD570 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD570 of tissues	Individual tissue viability (%)	Relative mean viability (%)	Variation within replicates (%)
Negative control	2.119	-	-	8.9
	1.939	-
Positive control	0.845	41.6	39.0	5.3
	0.738	36.4
Test item	2.166	106.7	105.2	3.0
	2.105	103.7

OD = Optical density

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the test, the test item is considered non-eye irritant in the EpiOcularTM Eye Irritation Test.

Executive summary:

The in vitro eye irritation potential was tested in the Reconstructed Human EpiOcular™ Model (OECD 492). After treatment with the test item, the mean value of relative tissue viability was increased to 105.2%. This value is above the threshold for eye irritation potential (≤ 60%). Therefore, the test item is considered to be non-irritating and was classified as a non-irritant.