Sodium bis[4-hydroxy-3-[(2-hydroxy-1-naphthyl)azo]benzenesulphonamidato(2-)]cobaltate(1-)

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation / corrosion

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Sodium bis[4-hydroxy-3-[(2-hydroxy-1-naphthyl)azo]benzenesulphonamidato(2-)]cobaltate(1-)

EC Number:

261-204-5

EC Name:

Sodium bis[4-hydroxy-3-[(2-hydroxy-1-naphthyl)azo]benzenesulphonamidato(2-)]cobaltate(1-)

Cas Number:

58302-43-5

Molecular formula:

C32H22CoN6O8S2.Na

IUPAC Name:

sodium [4-(hydroxy-kappaO)-3-{[2-(hydroxy-kappaO)-1-naphthyl]diazenyl}benzenesulfonamidato(2-)][4-hydroxy-3-{[2-(hydroxy-kappaO)-1-naphthyl]diazenyl}benzenesulfonamidato(2-)]cobaltate(1-)

Details on test material:

- Name of test material (as cited in study report): Eukesolar Rubine EB Liquid - dried
- Physical state: Solid / dark red
- Analytical purity:
HPLC fingerprint:
87.0 area-% (268 nm) sum of all peaks: 99.4 area-%
90.1 area-% (552 nm) sum of all peaks: 100.2 area-%
(For details see analytical report No.: 14L00142)
- Lot/batch No.: Dye powder sample 14/076 from Material no. 52631478; batch no. M-R/G
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility.
- Storage condition of test material: Room temperature

Test animals

Species:

other: in vitro

Strain:

other: not applicable

Details on test animals or test system and environmental conditions:

not applicable

Test system

Type of coverage:

other: not applicable

Preparation of test site:

other: not applicable

Vehicle:

other: not applicable

Controls:

other: not applicable

Amount / concentration applied:

not applicable

Duration of treatment / exposure:

not applicable

Observation period:

not applicable

Number of animals:

not applicable

Details on study design:

OBJECTIVES
The objective was to assess the potential for corrosive activity and skin irritation of the test
material. Using the currently available methods a single in vitro assay may not always be
sufficient to cover the full range of skin irritating/corrosion potential. Therefore, two in vitro
assays were part of this in vitro skin irritation and corrosion test strategy: The Skin Corrosion
Test (SCT) and Skin Irritation Test (SIT).
The present test is based on the experience that corrosive and irritant chemicals produce
cytotoxicity in human reconstructed epidermis after a short term topical exposure. The test is
designed to predict a skin corrosion or 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 EpiDermTM tissue the induced cytotoxicity (= loss of viability)
is measured by a colorimetric 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 density of the extract is determined spectrophotometrically. Optical
density of the extracts of test-substance treated tissues is compared to negative control
values from tissues and expressed as relative tissue viability.

TEST SYSTEM
Three dimensional human epidermis model
The EpiDermTM model consists of normal, human-derived epidermal keratinocytes which
have been 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 EpiDermTM tissues (surface 0.6 cm²) are cultured on specially
prepared cell culture inserts (MILLICELLs, 10 mm ∅) and commercially available as kits
(EpiDerm™ 200), containing 24 tissues on shipping agarose.
Tissue model: Epi-200
Origin: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
Based on the results of ECVAM (European Center for Validation of Alternative Methods)
funded validation studies, it was concluded by the ECVAM Scientific Advisory Committee that
the EpiDerm™ human epidermis model is suitable to be used for distinguishing between
corrosive and non-corrosive chemicals (ECVAM: ESAC statement on the application of the
EpidermTM human skin model for skin corrosivity testing of 14-15 Mar 2000) as well as
between irritant and non-irritant chemicals (ECVAM: ESAC statement on the scientific validity
of in-vitro tests for skin irritation testing of 5 Nov 2008).

ANALYSES
No analysis of test-substance preparation was performed, because the test substance was
applied minimally moistened with de-ionized water or PBS.

EXPERIMENTAL PROCEDURE
Mesh compatibility
For liquid test substances a nylon mesh can be used as a spreading support. However, this
was not necessary for the solid test substance.
Thus no pretest for mesh compatibility was performed.

Direct MTT reduction
The direct reduction of MTT by a test substance interferes with the color density produced by
metabolic capacity of the tissue and would falsify the test results.
To assess the ability of the test material to directly reduce MTT a pretest was performed as
described below.
The test substance was added to 0.9 mL of the MTT solution. The mixture was incubated in
the dark at about 37 °C for 55 to 65 minutes. A negative control (de-ionized water) was tested
concurrently.
If the MTT solution color 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.
In case that direct MTT reduction occurred, one freeze-killed control tissue (KC) per exposure
time (corrosion test) was treated with, each, the test article and the negative control, in the
same way as described in section “Experimental procedure” (3.6) additionally.
In the irritation test subsequent testing of killed controls was considered in the case that
direct MTT reduction occurred and visible residues of the test substance remained on the
tissues after washing, only.
Due to the intense color of the test substance it was not possible to evaluate whether or not
the test substance is able to reduce MTT directly, therefore freeze-killed control tissues (KC)
were treated with the test substance in the same way as the viable tissues.

Basic procedure
Several test substances were tested in parallel within the present test (test no. 61) using the
same control tissues (NC and PC).
Corrosion test:
From the day of arrival in the laboratory, tissues were kept in the refrigerator. At least 1 hour
but not more than 1.5 hours before test-substance application, tissues were transferred to 6-
well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. The preincubation
medium was replaced with fresh medium immediately before application.
Two tissues per exposure time (3 minutes at room temperature or 1 hour in the incubator, as
a rule) and test group (test material, negative control and positive control; 12 tissues per test)
were used.
25 μL de-ionized water was applied first. Thereafter, a bulk volume of 25 μL of the solid test
material was applied with a sharp spoon and homogeneously distributed with the water.
Control tissues were concurrently treated with 50 μL of de-ionized water (negative control,
NC) or with 50 μL of 8 N potassium hydroxide (positive control, PC). In addition one killed
tissue per exposure time was treated with the test substance and NC, respectively, in order
to detect direct MTT reduction.
The tissues were washed with PBS to remove residual test material 3 minutes or 1 hour after
start of the application treatment.
Rinsed tissues were kept in 24-well plates (holding plates) at room temperature on assay
medium until all tissues per application time were dosed and rinsed. The assay medium was
then replaced by MTT solution and tissues were incubated 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 6 microtiter wells
filled with isopropanol for each microtiter plate.

Irritation test:
On the day of arrival in the laboratory, 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 preincubation
medium was replaced with fresh medium and preconditioning continued for 18 ± 3
hours.
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.
Control tissues were concurrently treated with 30 μL of sterile PBS (negative control, NC) or
with 30 μL of 5% SDS (positive control, PC). A nylon mesh was placed carefully onto the
tissue surface afterwards.
In addition three freeze-killed control tissues (KC) were treated with the test substance and
one freeze-killed control tissue was treated with the NC, in order to detect direct MTT
reduction.
The tissues were kept under the laminar flow hood at room temperature for 25 minutes
overall and for 35 minutes in the incubator.
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.
Subsequently, the tissues were placed into the incubator at 37°C for 24 ± 2 hours.
After 24 ± 2 hours the tissues were transferred into new 6-well plates pre-filled with 0.9 mL 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 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 6 microtiter wells
filled with isopropanol for each microtiter plate.

Data evaluation
Table(s) and/or figure(s) of measured parameters presented in the report were produced
using PC based tabular calculation software. The mean and individual data were not always
rounded but the significant digits were produced by changing the display format. As a
consequence, calculation of mean values using the individual data presented in the report
will, in some instances, yield minor variations in value.

Principle
The OD570 values determined for the various tissues are
measures of their viability. The quotient of the OD570 of tissues
treated with the test material and the mean OD570 values of
the NC (percent of control) is used for evaluating whether or
not a test material is corrosive or irritant.

Calculation of individual and mean optical densities
The individual tissue OD570 is calculated by subtracting the
mean blank value of the respective microtiter plate from the
respective individual tissue OD570 value. The mean OD570 for a
test group of two tissues (corrosion test) or three tissues
(irritation test) treated in the same way is calculated.

Application of measurements using killed control tissues
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 testsubstance
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 0.1 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.

Tissue viability
The quantification of tissue viability is presented as the
quotient of the mean OD570 (or mean OD570 KC corrected, if
applicable) divided by the respective OD570 NC value in
percent for each exposure time.

ACCEPTANCE CRITERIA
In case one of the below given acceptance criteria is not covered, repetition of the test was
considered.
Assay acceptance criterion for the negative control (NC)
The absolute OD570 of the negative control tissues in the MTTtest
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 ≥ 0.8. The mean OD570 of the NC
should not exceed 2.8.

Assay acceptance criterion for the positive control (PC)
Corrosion test: Potassium hydroxide as 8.0 normal ready
made solution is used as positive reference. A 3-minute
treatment with 8.0 N KOH usually reveals a mean relative
tissue viability of ~20%. An assay is acceptable if mean
relative tissue viability of the 3 min positive control is ≤ 30%.
Irritation test: 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, except the killed controls, 2 tissues
(corrosion test) or 3 tissues (irritation test) are treated in
parallel. The inter-tissue variability is considered to be
acceptable if the difference of the OD570 values of the two
tissues is ≤ 0.3 (corrosion test) or if the SD of %-viability is
≤ 20 (irritation test).

Assay acceptance criterion for killed controls (KC)
The OD570 of the killed control tissues treated as negative
control should be ≤ 0.35.

EVALUATION OF RESULTS
The evaluation of the in vitro skin irritation potential of the test substance is based on the
results of the Skin Corrosion Test (SCT) and the Skin Irritation Test (SIT).
If a test substance is not tested in both systems or an inconclusive result is obtained in one of
the studies, the test strategy may still lead to an overall evaluation, when the result of a single
study gives a clear prediction. However, if both studies are inconclusive or contradictory
results are obtained a test evaluation may not be possible.
Corrosive potential of the test materials is predicted from the mean relative tissue viabilities
obtained after 3 min treatment compared to the negative control tissues concurrently treated
with de-ionized water. A chemical is considered as "corrosive", if the mean relative tissue
viability after 3 min treatment with a test material is decreased below 50%. In addition, those
materials with a viability of ≥ 50% after 3 min treatment are considered as "corrosive" if the
mean relative tissue viability after 1 hour treatment with a test material is decreased below
15%.

Mean tissue viability (% of negative control)
3 min: < 50
Prediction: Corrosive (Optional Sub-category 1A)
3 min: ≥ 50 and 1 hour: < 15
Prediction: Corrosive (Optional Sub-category 1B and 1C)
3 min: ≥ 50 and 1 hour: ≥ 15
Prediction: Non-corrosive

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%.

Mean tissue viability (% of negative control)
≤ 50 Prediction: Irritant
> 50 Prediction: Non-irritant

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

other: Corrosion Test (viability [% of NC])

Basis:

mean

Time point:

other: 3 min

Score:

117

Remarks on result:

other: Corrosion Test: The mean viability of the test-substance treated tissues determined after an exposure period of 3 minutes was 117%.

Results 2

Irritation parameter:

other: Corrosion Test (viability [% of NC])

Basis:

mean

Time point:

other: 1 h

Score:

101

Remarks on result:

other: Corrosion Test: The mean viability of the test-substance treated tissues determined after an exposure period of 1 hour was 101%.

Results 3

Irritation parameter:

other: Irritation Test (viability [% of NC])

Basis:

mean

Time point:

other: 1 h

Score:

105

Remarks on result:

other: The mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 105%.

Any other information on results incl. tables

The objective was to assess the potential for corrosive activity and skin irritation of Eukesolar Rubine EB Liquid; dried. Using the currently available methods a single in vitro assay may not always be sufficient to cover the full range of skin irritating/corrosion potential. Therefore, two in vitro assays were part of this in vitro skin irritation and corrosion test strategy: The Skin Corrosion Test (SCT) and Skin Irritation Test (SIT).

The potential of Eukesolar Rubine EB Liquid; dried to cause dermal corrosion/irritation was assessed by a single topical application of 25 μL bulk volume (about 12 mg) of the undiluted test substance to a reconstructed three dimensional human epidermis model (EpiDerm™).

For the corrosion test two EpiDerm™ tissue samples were incubated with the test substance for 3 minutes and 1 hour, respectively. The irritation test was performed with three EpiDerm™ tissue samples, which 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 of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the testsubstance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability.

The EpiDerm™ skin corrosion/irritation test showed the following results:

Due to the intense color of the test substance it could not be determined whether the test substance is able to reduce MTT directly. Therefore an additional MTT reduction control was introduced. However, the result of the control tissues inactivated by freezing (KC) did not indicate an increased MTT reduction.

In both tests minimal compound residues remained on the tissues after the washing procedure. However, this did not interfere with the colorimetric test as was demonstrated in a pretest.

Corrosion test:

The mean viability of the test-substance treated tissues determined after an exposure period of 3 minutes was 117%, and it was 101% after an exposure period of 1 hour.

Irritation 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 105%.

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU