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

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From May 17th to 25th, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

July 28th, 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

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

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

- Direct MTT reduction - functional check in tubes: 25 mg of the test item were added to 1 mL MTT medium (red) and incubated in the incubator (37±1°C, 5±1 % CO2, moistened) for 65 minutes. At the end of the exposure time, the presence and intensity of the staining was observed. The test item did not reduce MTT directly.
- Colour interference : 50 mg of the test item were added to 2 mL isopropanol and placed on a shaker for 2 hours 25 minutes at room temperature. Two 200 μL aliquots of isopropanol solutions and of pure isopropanol were transferred to a 96-well plate and the absorbance was measured with a plate reader at the MTT measurement wavelength. Colour of the test item did not interfere with the endpoint.
- MTT test: 25 mg of the test item was dosed directly on tissue (reconstructed human epidermal model EpiDerm™, EPI-200 ver. 2.0, MatTek, Bratislava, Slovakia, Lot No. 28614 kit C) previously moistened with 25 μL DPBS. The item was spread on the entire tissue surface. A single experiment, composed of three replicate tissues, was run. On the day of receipt, EpiDermTM tissues were conditioned by incubation to release transport stress related compounds and debris. Duration of pre-incubations before treatment was 60 minutes and 20 hours, 35 minutes. After pre-incubations, tissues were topically exposed to the test chemicals for 60±1 minutes (25 minutes at room temperature and the remaining 35 minutes at culture conditions). Three tissues were used per the test item, three for the the positive (PC) and three for the negative (NC) controls. Tissues were then thoroughly rinsed with PBS. Inserts were then transferred to fresh medium. After 25 hours and 14 minutes of post-incubation period, the medium was replaced by fresh one. Tissues were incubated for another 18 hours 41 minutes. Afterwards, the MTT assay was performed by transferring the tissues to 24-well plates containing MTT medium (1 mg•mL-1). After 180 minutes of MTT incubation, the blue formazan salt formed by cellular mitochondria was extracted with 2.0 mL/tissue of isopropyl alcohol (for 128 minutes, room temperature, shaking) and the optical density of the extracted formazan was determined using a spectrophotometer at 570 nm. OD570 was measured on a spectrophotometer Libra S22. Isopropyl alcohol served as a blank. Allowed band width is 2-3 nm. No external filter was used.
- Viability calculation: relative cell viability was calculated for each tissue as % of the mean of the negative control tissues. Than the mean relative tissue viability of three individual tissues exposed to the test item was calculated – this value is used for the comparison with limit.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Duration of treatment / exposure:

60±1 minutes

Duration of post-treatment incubation (if applicable):

approximately 42 hours

Number of replicates:

A single experiment, composed of three replicate tissues

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Experiment I

Value:

89.6

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Any other information on results incl. tables

OD₅₇₀values obtained at the MTT test, their averages, standard deviations (%) and relative viabilities

	Treatment	OD570			Avg	SD	Average viability (% NC)
		1	2	3
NC	PBS	1.798	2.132	2.052	1.994	0.142	100
	%	90.2	106.9	102.9	100	7.141
C1	85/18	1.716	1.68	1.964	1.787	0.126	89.6
	%	86.1	84.3	98.5	89.6	6.332
PC	5% SDS	0.048	0.047	0.054	0.05	0.003	2.5
	%	2.4	2.4	2.7	2.5	0.155

 Notes:

NC	negative control
PC	positive control
SDS	sodium dodecyl sulphate
PBS	phosphate-buffered saline
C1, 85/18	test item
avg	arithmetic average
SD	standard deviation calculated from individual % tissue viabilities
Average viability (% NC)	viability of single tissues compared with negative control

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

The average viability of tissues treated by the test item, was 89.6 % of negative control average value i.e. viability was > 50 %.
The effect of the test item was negative in EpiDermTM model.
non skin irritant

Executive summary:

The test item,was assayed forin vitroskin irritation in the human epidermal model EpiDerm^TM.The test was performed according totheOECDTest GuidelineNo.439:In VitroSkin Irritation: Reconstructed HumanEpidermisTest Method(2015) and Protocol for: In Vitro EpiDerm^TMSkin Irritation Test for use with MatTek Corporation’s Reconstructed Human Epidermal Model EPI-200-SIT.

After pre-incubation of tissues,25 mg of the test item was placed directly on tissue and spread on the entire tissue surface.The length of exposure was60 minutes. Three tissues were used for the test item and for positive and negative controls.

After removal of the test item, tissues were post-incubated for approximately 42 hours. Three hours incubation with MTT and two hours extraction period with shaking followed. Optical density (OD₅₇₀) of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

Tests for colour interference and direct reduction did not demonstrate influence of colour or reductive properties of the test substance on study results. Thus, no steps for correction of results were performed.

Under the above-described experimental design,average viability of treated tissues was 89.6%,i.e. viability was >50 %.

The effect of the test item was negative in EpiDerm^TMmodel (tissues were not damaged).

According to the classification criteria,the test item,is considered to have no category with regard to skin irritation.