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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
according to OECD and GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Principles of method if other than guideline:
Based on a “Statement on the Scientific Validity of In Vitro Tests for Skin Irritation” of the European Commission (November 2008), official acceptance of the test method in the EU was achieved and implemented in EU, 2008a, Council Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to EC Regulation No 1907/2006 of the European Parliament and of the Council on REACH; 1st ATP 2009: EC Regulation No 761/2009 of 23 July 2009 amending, for the purpose of its ATP, EC Regulation No 440/2008 laying down test methods pursuant to EC Regulation No 1907/2006 of the European Parliament and of the Council on REACH, section B46.
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Aluminium tris(5,7,12,14-tetrahydro-7,14-dioxoquino[2,3-b]acridine-2-sulphonate)
EC Number:
278-207-2
EC Name:
Aluminium tris(5,7,12,14-tetrahydro-7,14-dioxoquino[2,3-b]acridine-2-sulphonate)
Cas Number:
75431-69-5
Molecular formula:
C20H12N2O5S.1/3Al
IUPAC Name:
aluminum tris(7,14-dioxo-5,7,12,14-tetrahydroquino[2,3-b]acridine-2-sulfonate)
Constituent 2
Chemical structure
Reference substance name:
5,12-dihydroquino[2,3-b]acridine-7,14-dione
EC Number:
213-879-2
EC Name:
5,12-dihydroquino[2,3-b]acridine-7,14-dione
Cas Number:
1047-16-1
Molecular formula:
C20H12N2O2
IUPAC Name:
5,12-dihydroquino[2,3-b]acridine-7,14-dione
Constituent 3
Chemical structure
Reference substance name:
Dialuminium tris(5,7,12,14-tetrahydro-7,14-dioxoquino[2,3-b]acridine-2,9-disulphonate)
EC Number:
243-319-2
EC Name:
Dialuminium tris(5,7,12,14-tetrahydro-7,14-dioxoquino[2,3-b]acridine-2,9-disulphonate)
Cas Number:
19795-24-5
Molecular formula:
C20H12N2O8S2.2/3Al
IUPAC Name:
dialuminium tris(5,7,12,14-tetrahydro-7,14-dioxoquino[2,3-b]acridine-2,9-disulphonate)

In vitro test system

Test system:
human skin model
Vehicle:
other: DPBS (MatTek)
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
25 µL bulk volume (~ 17 mg)
Duration of treatment / exposure:
Three EpiDerm tissues were incubated with the test substance for 1 hour followed by a 42 hours post-incubation period
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3

Test animals

Species:
other: reconstituted human epidermis model

Test system

Type of coverage:
other: Topical
Preparation of test site:
other: Not applicable
Vehicle:
other: DPBS (MatTek)
Controls:
yes
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
Each approximately 25 µL bulk volume (~ 17 mg) of the test item were applied to the tissues, wetted with 25 µL DPBS, and spread to match the surface of the tissue for a complete treatment time of 60 minutes.


NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL DPBS (MatTek) were used as negative control and negative control KC per tissue.


POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL of a 5 % SLS solution in deionised water (MatTek) were used as positive control per tissue.
Duration of treatment / exposure:
1 hour followed by a 42 hours post-incubation period
Observation period:
Not applicable
Number of animals:
Not applicable
Details on study design:
The test is based on the experience that irritant chemicals produce cytotoxicity in human reconstructed epidermis after a 1 hour topical exposure and about 42 hours post-incubation. The test is designed to predict skin irritation potential of a chemical by using the three dimensional human epidermis model EpiDermTM. After application of the test material to the stratum corneum surface of the tissue the induced cytotoxicity (= loss of viability) is measured in a colometric assay. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity. The mitochondrial dehydrogenase reduces the yellow colored water-soluble 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to the insoluble blue colored 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 values from negative control (NC) tissues and expressed as relative tissue viability.

The EpiDermTM model (EPI-200) consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi layered, hight differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers arranged in patterns analogous to those found in vivo. used to model the human corneal epithelium. The EpiDermTM tissues (surface 0.6 cm2) are cultured on cell culture inserts (MILLICELLsR, diameter 10 mm) and are commercially available as kits (EpiDermTM 200), containing 24 tissues on shipping agarose.

Tissue model: EPI-200
Lot number: 23377
Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia


On the day of arrival the tissues were transferred to sterile 6-well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour the pre-incubation medium was replaced with fresh medium and preconditioning continued for 18 ± 3 hours.
Three tissues were treated with the test substance, the positive and negative control respectively. In addition three killed tissues were used for the test substance and the negative control respectively in order to detect direct MTT reduction.
25 µL sterile DPBS was applied first. Therafter a bulk volume of 25 µL of the test substance was applied and distributed together with the fluid.
Control tissues were concurrently applied with 30 µL of sterile DPBS (NC, NC KC) or with 30 µL of 5 % SLS (PC) or test substance (KC). A nylon mesh was placed carefully onteh the tissue surface afterwards.
The tissues were washed with sterile DPBS to remove residual test material 1 hour after start of application.
Rinsed tissues were blotted on sterile absorbent paper and transferred iinto 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.
Subsequently the tissues were incubated in the incubated at 37°C for 24 ± 2 hours. Then the tissues were transferred into new 6-well plates pre-filled with 0.9 nL of fresh medium and placed into the incubator for additional 18 ± 2 hours post-incubation period. After the post-incubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated for 3 hours. Thereafter the tissues were wahsed with DPBS 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 densitiy at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 4 microtiter wells filled with isopropanol fro each microtiter plate.
The OD570 values determined for the various tissues are measures of their viability. The quotient of the OD570 of tissues treated with the test substance and the mean OD570 values of the NC is used for evaluating whether a test substance is irritant or not irritant.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean viability of tissues after KC correction
Value:
99.6
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Any other information on results incl. tables

Findings:

substance

 

 

tissue 1

tissue 2

tissue 3

mean

standard deviation

coefficient of variation [%]

negative control

viable tissues

mean OD570

1.802

1.693

1.695

1.730

 

 

viability

[% of NC]

104.2

97.9

98.0

100.0

3.6

3.6

KC tissues

mean OD570

0.051

0.053

0.047

0.050

 

 

viability

[% of NC]

2.9

3.1

2.7

2.9

0.2

6.6

positive control

viable tissues

mean OD570

0.041

0.055

0.051

0.049

 

 

viability

[% of NC]

2.4

3.2

2.9

2.8

0.4

14.3

test substance

viable tissues

mean OD570

1.541

1.746

1.895

1.727

 

 

 

viability

[% of NC]

89.0

100.9

109.5

99.8

10.3

10.3

 

KC tissues

mean OD570

KC NC corrected

0.004

0.007

0.003

0.005

 

 

 

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]

99.6

 

 

Due to the ability of the test substance to reduce MTT directly, KC tissues were applied in parallel. The results of the KC tissues indicate an increased MTT reduction (mean viability 0.3 % NC). thus for the test substance the final mean viability is given after KC correction.

The data show, that a treatment with the test item did not significantly affect the viability of tissues (relative viabilty = 99.6 %)

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
Regulation (EC) 1272/2008
Conclusions:
In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is not irritant to skin according to Regulation (EC) 1272/2008.
Executive summary:

The potential of the test substance to cause dermal irritaion was assessed by single topical application of ca. 25 µL bulk volume (about 17 mg) of the undiluted test substance to a reconstructed three dimensional human epidermis model (EpiDermTM).

Three EpiDermTM tissues were incubated with the test substance for 1 hour followed by a 42 -hours post-incubation period.

Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction ot mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the test substance treated epidermal tissues was compared to that of negative control tissues. The quotient of both values indicated the realtive tissue viability.

The following test results were obtained:

The test substance was able to reduce MTT directly. Therefore an additional MTT reduction control KC (freeze-killed control tissues) was introduced.

In a pre-test it was demonstraed that the color of the test substance did not interfere with the colorimetric test.

The mean viability of the test substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 99.6 %. All acceptance criteria were met.

Thus, the test substance does not show a skin irritation potential in the EpiDermTM in vitro skin irritation test.