Amides, adipic acid

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 February 2019 to 22 February 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

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)

GLP compliance:

yes

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

solid

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

EpiOcular™ tissues, Lot No. 27466 Kit B, were received from MatTek and refrigerated at 2-8°C. Before use, the tissues were incubated (37±1°C, 5 ±1 % CO2) with assay medium (MatTek) for a one-hour equilibration. Equilibration medium was replaced with fresh medium for an additional overnight equilibration of 16 to 24 .After the overnight incubation, the tissues were moistened with 20 μl of phosphate-buffered saline (PBS) and incubated at 37±1°C, 5±1% CO2 for 30±2 min.

Test system

Vehicle:

not specified

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The test substance was ground to a fine powder and an aliquot of 50 mg of the test substance were mixed with 1 mL of MTT solution (1 mg/mL methyl thiazole tetrazolium diluted in Dulbecco's Modified Eagle's Medium.

Duration of treatment / exposure:

6 h ± 15 min

Duration of post- treatment incubation (in vitro):

18 h

Number of animals or in vitro replicates:

duplicate

Results and discussion

In vitro

Results

Irritation parameter:

other: tissue viability

Remarks:

The tissue viability of tissue substace was found to be 98.1%

Negative controls validity:

valid

Remarks:

The mean OD570 of the negative control tissues was 1.972, which met the acceptance criteria of greater than 0.8 and less than 2.5

Positive controls validity:

valid

Remarks:

The mean relative viability of the positive control tissues was 30.2%, which met the acceptance criterion of less than 50%.

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The differences in viability between identically treated tissues were 1.24% to 11.76%, which met the acceptance criterion of less than 20%.

Any other information on results incl. tables

The R-squared value calculated for the plate reader linearity check was 0.9997,which met the acceptance criterion of greater than 0.999. All other quality controls passed the acceptance criteria.

The experimental data and summarized results and irritation classifications are as follows

Test and Control Substance Identity	Tissue No.	Mean of Aliquots	% Viability	OD		Viabilities (%)		Irritancy classification
				Mean	S.D.	Mean	S.D.
TR063A L#252-PE	1	1.818	92.2	1.934	0.232	98.1	11.76	Non-irritant
	2	2.050	104.0
TCH2O (Negative Control)	1	1.859	94.3	1.972	0.226	100.0	11.46	Non-irritant
	2	2.085	105.7
Methyl Acetate (Positive Control)	1	0.583	29.6	0.596	0.025	30.2	1.24	Irritant
	2	0.608	30.8

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the study conditions, the test substance was not irritating to eye.

Executive summary:

A study was conducted to determine the eye irritation potential of the test substance according to OECD Guideline 492 in compliance with GLP. Eye irritation potential was examined using reconstructed Human Cornea-like Epithelium (RhCE) following a treatment period of 6 h and a post-exposure incubation period of 18 h. The principle of the assay is based on the measurement of cytotoxicity in reconstructed Human Cornea-like Epithelium cultures following topical exposure to the test substance by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt to a blue formazan salt in the substance treated tissues relative to the negative controls. To identify possible interference of the test substance with the MTT endpoint, the test substance was checked for the ability to directly reduce MTT prior to the main assay. On the day of dosing, duplicate tissues were treated with the test substance for an exposure period of 6 h at 37 ±1°C, 5% ±1 CO2 in air. At the end of the exposure period, each tissue was rinsed and soaked tissue were then incubated in fresh assay medium at incubated at 37 ±1°C, 5 ±1°% CO2 for 18 h. At the end of the incubation period, each EpiOcular™ tissue was transferred to a 24-well plate containing 300 μL of MTT solution. The tissues were then returned to the incubator for a 3 h MTT incubation period. The relative mean viability of the test substance treated tissues was 98.1 %. Under the study conditions, the test substance was not irritating to eye (Troese, 2019).