Aluminum, 2-(2-quinolinyl)-1H-indene-1,3(2H)-dione sulfo derivs. complexes

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From November 13th to December 6th, 2018

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Aluminum, 2-(2-quinolinyl)-1H-indene-1,3(2H)-dione sulfo derivs. complexes

EC Number:

309-264-4

EC Name:

Aluminum, 2-(2-quinolinyl)-1H-indene-1,3(2H)-dione sulfo derivs. complexes

Cas Number:

100208-62-6

Molecular formula:

Not applicable

IUPAC Name:

Aluminum, 2-(2-quinolinyl)-1H-indene-1,3(2H)-dione sulfo derivs. complexes

Test material form:

solid: particulate/powder

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

The reconstructed human cornea-like epithelial model EpiOcular™ (OCL-200 ver. 2.0) were used.
The RhCE tissues are reconstructed from primary human cells, which have been cultured for several days to form a stratified, highly differentiated squamous epithelium, morphologically similar to that found in the human cornea. The EpiOcular™ RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinized surface, showing a cornea-like structure analogous to that found in vivo.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The test item (50 mg of item/surface ratio 39.7 mg/cm2) was placed directly atop to the tissue moistened with 20 µL of PBS.

Duration of treatment / exposure:

6 ± 0.25 hours

Duration of post- treatment incubation (in vitro):

18 ± 0.25 hours

Number of animals or in vitro replicates:

Two tissues were used for the test item and every control

Details on study design:

- Tissues preparation and treatment: on the day of receipt, EpiOcularTM tissues were conditioned to release transport stress related compounds and debris by incubation in assay medium for test performance for 1 hour at standard culture conditions and, after media replacement, overnight at culture conditions. After pre-incubations, tissues were wetted with 20 μL of PBS. After 30 minutes incubation, tissues were topically exposed to the test item (50 mg per tissue) for 6 ± 0.25 hours. Two tissues were used per test item, two for the positive control and two for the negative control. At the end of the treatment time, the test item was removed by extensively rinsing the tissues with PBS brought to room temperature. After rinsing, tissues were immediately transferred to and immersed in 5 ml of previously warmed assay medium (room temperature) in a pre-labelled 12-well plate for a 25 ±2 minutes immersion incubation (post-soak) at room temperature. This incubation in assay medium was intended to remove any test item absorbed into the tissue. At the end of the post-soak immersion, each insert was removed from the assay medium, the medium was decanted off the tissue, and the insert was blotted on absorbent material and transferred to the appropriate well of the pre-labelled 6-well plate containing 1 mL of warm assay medium. The tissues were incubated for 18 ± 0.25 hours at standard culture conditions (post-treatment incubation).
MTT assay: at the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent material. Tissues were placed into the 24-well plate containing 0.3 ml of MTT solution and incubated for 180±10 minutes at standard culture conditions. Afterwards, the bottom of all inserts was blotted on absorbent material, inserts were then transferred to a pre-labelled 24-well plate and immersed in 2 mL of isopropyl alcohol. The plates were sealed with parafilm, and were let in refrigerator overnight. The second day, the plates were placed on an orbital plate shaker and shaken for 2-3 hours at room temperature.
- OD570 measuring: extracts were collected, mixed and two 200 μL aliquots from every well were transferred to the appropriate wells of a pre-labelled 96-well plate for OD570 reading. As average OD570 of negative control was higher than 1.999, the same measurement was performed with 100 μL aliquots. OD570 was measured on a plate reader Biotek Epoch. Isopropyl alcohol serves as a blank. No external filter was used.

Results and discussion

In vitro

Results

Irritation parameter:

other: Mean tissue viability (%)

Value:

83.8

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The test item was hardly washable as it adhered to tissue. The experiment had to be repeated because of damage of tissue at stripping of the test item off tissues. The test item got stuck on tissues and it was present on them even after rinsing, stripping with spatula, post-soaking and post-incubation also in the repeated experiment. Average NC OD570 of neat extracts was 2.099 which is higher than 1.999. for this reason, 0.1 mL of extracts and controls were pipetted into other wells.
The mean negative control (neat extract) OD570 was 2.099 which is > 0.8 and < 2.5. This criterion was fulfilled.
The mean relative viability of the positive control was 34.7 % which is below 50% of negative control viability. This criterion was fulfilled.
The difference of viability between the two relating tissues of the negative control was 1.8 %. The difference of viability between the two relating tissues of the test item was 9.9 %. The difference of viability between the positive control tissues was 12.0 % what is < 20%. This criterion was fulfilled.
Under the above-described experimental design, average viability of tissues treated by the test item Quinoline Yellow Lake was 83.8 % of negative control average value. i.e. viability was > to 60 %. The effect of the test item was negative in EpiOcularTM model (tissues were not damaged).

Any other information on results incl. tables

MTT test results

Code	Treatment	OD570		avg	DT	Viability %NC
		Tissue 1	Tissue 2	%NC	%NC
NC	water	0.967	0.985	0.976	1.8	100.0
	% NC	99.1	100.9	100.0
C1	85/18	0.769	0.866	0.818	9.9	83.8
	% NC	78.8	88.8	83.8
PC	99% MA	0.397	0.280	0.339	12.0	34.7

Notes:

NC	negative control
PC	positive control
DT	difference between tissues
C1, 85/18	the test item
avg	arithmetic average
SD	standard deviation calculated from individual % tissue viabilities
viability (%)	viability of single tissues compared with negative control
NT	not tested

Applicant's summary and conclusion

Interpretation of results:

other: No classification required according to CLP Regulation (EC) No 1272/2008

Conclusions:

Average viability of treated tissues was 83.8 % i.e. viability was > 60 %.
The effect of the test item was negative in EpiOcularTM model (tissues were not damaged).
Non-eye irritant

Executive summary:

Results of the repeated experiment were used for evaluation. Experimental design average viability of treated tissues in the second experiment was 83.8 %i.e. viability was >60 %. The effect of the test item was negative in EpiOcular^TMmodel (tissues were not damaged). According to the classification criteria, the test item is identified as not requiring classification and labelling according to UN GHS (No Category).