Reaction mass of 5,12-dihydroquino[2,3-b]acridine-7,14-dione and aluminium tris(5,7,12,14-tetrahydro-7,14-dioxoquino[2,3-b]acridine-2-sulphonate) and dialuminium tris(5,7,12,14-tetrahydro-7,14-dioxoquino[2,3-b]acridine-2,9-disulphonate)

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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 22, 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: B.46: In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test; Official Journal of the European Union, No. L 193

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 5,12-dihydroquino[2,3-b]acridine-7,14-dione and aluminium tris(5,7,12,14-tetrahydro-7,14-dioxoquino[2,3-b]acridine-2-sulphonate) and dialuminium tris(5,7,12,14-tetrahydro-7,14-dioxoquino[2,3-b]acridine-2,9-disulphonate)

EC Number:

909-092-5

Molecular formula:

Unspecified

IUPAC Name:

Reaction mass of 5,12-dihydroquino[2,3-b]acridine-7,14-dione and aluminium tris(5,7,12,14-tetrahydro-7,14-dioxoquino[2,3-b]acridine-2-sulphonate) and dialuminium tris(5,7,12,14-tetrahydro-7,14-dioxoquino[2,3-b]acridine-2,9-disulphonate)

Test material form:

solid: particulate/powder

Details on test material:

Batch 22426
100% UVCB
storage at room temperature
storage stability until 10 Dec 2025

Specific details on test material used for the study:

Batch no 22426
pH-value: Ca. 5 (undiluted test substance, moistened with de-ionized water; determined in the lab prior to start of the GLP study
Physical state / color: Solid / violet to brown
Storage conditions: Room temperature
Purity 100% UVCB

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from multiple donors

Source strain:

not specified

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM model
- Tissue batch number(s): 23377
- Date of initiation of testing: 8 Nov 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator.(37°C)
After removal of the test material, the tissues were incubated in the incubator at 37°C for 24 ± 2 hours.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: The tissues were washed with sterile PBS to remove residual test material 1 hour after start of
application. Rinsed tissues were blotted on sterile absorbent paper and transferred into new 6-well plates,
pre-filled with 0.9 mL fresh medium. When all tissues were rinsed, the surface of each tissue was carefully dried with a sterile cotton swab.
- Observable damage in the tissue due to washing: None




FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA

Historic Range negative control (OD570)
Mean OD SD Mean + 2 SD Mean - 2 SD
2.289 0.285 2.860 1.719

Historic Range of positive control (OD570)
Mean OD SD Mean + 2 SD Mean - 2 SD
0.070 0.011 0.092 0.047

Viability (%)
Mean % SD Mean + 2 SD Mean - 2 SD
3.1 0.5 4.0 2.1


NUMBER OF REPLICATE TISSUES: Three per test group

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues and killed tissues
- Procedure used to prepare the killed tissues: freeze-kill
- N. of replicates : 3
In case of direct reduction of MTT by the test substance, the OD570 values measured in the freeze-killed control tissues (KC) will be used to correct the mean OD570 of the test-substance treated tissues (mean OD570 KC corrected). Since killed tissue might still have a residual enzyme activity that is able to produce some formazan net OD570 KC is calculated by subtracting the OD570 KC of the NC from the OD570 KC of the test substance. In case the net OD570 KC is greater than zero it is subtracted from the respective mean OD570 to result in the mean OD570 KC corrected. The mean OD570 KC corrected represents the formazan production linked to the tissue viability and therefore indicates the cytotoxic potency of the test substance.


NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
A chemical is considered as "irritant", if the mean relative tissue viability with a test material is less than or equal to 50% after a 60min exposure.

A single test composed of at least three tissue replicates should be sufficient for a test chemical when the result is unequivocal. However, in cases of borderline results, such as nonconcordant
replicate measurements and/or mean percent tissue viability equal to ±5% of the cited cut-off value, a second test should be considered, as well as a third one in case of discordant results between the first two tests.

- Observable damage in the tissue due to washing: None

MTT-Treatment:
After the postincubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours.
After incubation, the tissues were washed with PBS to stop the MTT-incubation. The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in
isopropanol. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 4 microtiter wells filled with isopropanol for each microtiter plate.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

Bulk volume of 25 microliter

Duration of treatment / exposure:

60 min

Duration of post-treatment incubation (if applicable):

24h

Number of replicates:

3

Test system

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): xx µL bulk volume (about xxx mg)

Duration of treatment / exposure:

1 hour followed by a 42-hours post-incubation period

Observation period:

not applicable (in vitro test)

Number of animals:

not applicable (in vitro test)

Details on study design:

Test system:
- In vitro test system on three dimensional human epidermis models. The EpiDermTM model consists of normal, human-derived epidermal keratinocytes cultured to form a multi layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo.
- The test system is based on the experience that irritant chemicals produce cytotoxicity in human reconstructed epidermis after a 1-hour topical exposure and about 42 hours postincubation.

Material and technical equipment:
- EpiDerm™ 200 kit: MatTek Corp., Bratislava, Slovakia containing 24 Epi-200 tissues (reconstructed epidermis): surface 0.6 cm² cultured in Millicells® diameter 1 cm.

Controls:
- Negative control (NC): 30 μL PBS, sterile
- Positive control (PC): 30 µL 5% (w/v) sodium dodecyl sulfate (SDS, Sigma, Germany) in deionized water, sterile

Experimental procedure:
- Direct MTT reduction:
To assess the ability of the test material to directly reduce MTT a pretest was performed. Thereby, the test substance was added to 0.9 mL of the MTT solution. A negative control (de-ionized water) was tested concurrently. If the MTT solution colour or, in case of water-insoluble test substances the border to the water-phase, turned blue / purple, the test substance was presumed to directly reduce MTT. The direct reduction of MTT by a test substance interferes with the colour density produced by metabolic capacity of the tissue and would falsify the test results.
- Basic procedure:
Three tissues were treated with the test substance, the PC and NC, respectively.
• 25 μL sterile PBS was applied first. Thereafter, a bulk volume of 25 μL of the solid test material was applied with a sharp spoon and homogeneously distributed together with the fluid.
• 1 hour after start of application of the test material to the stratum corneum surface of the EpiDermTM tissue, residual test material was removed with sterile PBS and the surface of each tissue was dried. Subsequently, the tissues were incubated at 37°C for 24 ± 2 hours, transferred into new 6-well plates pre-filled with 0.9 mL of fresh medium and thereafter placed into the incubator for additional 18 ± 2 hours post-incubation period.
• After the postincubation period induced tissue destruction (cytotoxicity = loss of viability) was determined by measuring the metabolic activity of the tissue using a colourimetric assay. Thereby, cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity. The mitochondrial dehydrogenase reduces the yellow coloured water-soluble 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to the insoluble blue coloured formazan. After isopropanol extraction of the formazan from the tissues, the optical densitiy of the extract is determined spectrophotometrically. Optical density of the extracts of test substance treated tissues is compared to negative control values and expressed as relative tissue viability.

Acceptance criteria:
In case one of the below given acceptance criteria is not covered, repetition of the test is considered.
- Assay acceptance criterion for the negative control (NC): The absolute OD570 of the negative control tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is acceptable if the mean OD570 of the NC is ≥ 1.0. The mean OD570 of the NC should not exceed 2.5.
- Assay acceptance criterion for the positive control (PC): 5% SDS is used as PC and reflects the sensitivity of the tissues used in the test conditions. A viability of ≤ 20% is acceptable.
- Assay acceptance criterion for tissue variability: For every treatment, 3 tissues are treated in parallel. The inter-tissue variability is considered to be acceptable if the SD of %-viability is ≤ 20.

Evaluation criteria:
- Irritant potential of the test materials is predicted from the mean relative tissue viabilities compared to the negative control tissues concurrently treated with sterile PBS. A chemical is considered as "irritant", if the mean relative tissue viability with a test material is less than or equal to 50%

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

99.6

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The test substance is able to reduce MTT directly. Therefore an additional MTT reduction control KC (freeze-killed control tissues) was introduced.
In a pretest it was demonstrated that the color of the test substance did not interfere with the colorimetric test.

Any other information on results incl. tables

			tissue 1	tissue 2	tissue 3	mean	SD	CV(%)
negative control	viable tissues	mean OD570	1.802	1.693	1.695	1.730
	viable tissues	viability [% of NC]	104.2	97.9	98.0	100.0	3.6	3.6
	killed tissues	mean OD570	0.051	0.053	0.047	0.050
	killed tissues	viability [% of NC]	2.9	3.1	2.7	2.9	0.2	6.6
test substance	viable tissues	mean OD570	1.541	1.746	1.895	1.727
	viable tissues	viability [% of NC]	89.0	100.9	109.5	99.8	10.3	10.3
	killed tissues	mean OD570	0.004	0.007	0.003	0.005
	killed tissues	viability [% of NC]	0.2	0.4	0.2	0.3	0.1	45.1
			Final mean viability of tissues after KC correction [% of NC]:			99.60%
positive control	viable tissues	mean OD570	0.041	0.055	0.051	0.049
	viable tissues	viability [% of NC]	2.4	3.2	2.9	2.8	0.4	14.3

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In-vitro testing showed that the substance is not irritating to skin (OECD 439, GLP).