Reaction mass of Chromate(1-), [2-(3-chlorophenyl)-2,4-dihydro-4-[[2-hydroxy-5-(methylsulfonyl)phenyl]azo]-5-methyl-3H-pyrazol-3-onato(2-)][methyl [7-hydroxy-8-[[2-hydroxy-5-(methylsulfonyl)phenyl]azo]-1-naphthalenyl]carbamato(2-)]-, sodium and sodium bis[2-(3-chlorophenyl)-2,4-dihydro-4-[[2-hydroxy-5-mesylphenyl]azo]-5-methyl-3H-pyrazol-3-onato(2-)]chromate(1-) and sodium bis[methyl [7-hydroxy-8-[[2-hydroxy-5-mesylphenyl]azo]-1-naphthyl]carbamato(2-)]chromate(1-)

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016-10-06 to 2017-08-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

2015-07-28

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of Chromate(1-), [2-(3-chlorophenyl)-2,4-dihydro-4-[[2-hydroxy-5-(methylsulfonyl)phenyl]azo]-5-methyl-3H-pyrazol-3-onato(2-)][methyl [7-hydroxy-8-[[2-hydroxy-5-(methylsulfonyl)phenyl]azo]-1-naphthalenyl]carbamato(2-)]-, sodium and sodium bis[2-(3-chlorophenyl)-2,4-dihydro-4-[[2-hydroxy-5-mesylphenyl]azo]-5-methyl-3H-pyrazol-3-onato(2-)]chromate(1-) and sodium bis[methyl [7-hydroxy-8-[[2-hydroxy-5-mesylphenyl]azo]-1-naphthyl]carbamato(2-)]chromate(1-)

EC Number:

915-704-1

Molecular formula:

C36H28ClCrN7O10S2.Na / C34H26Cl2CrN8O8S2.Na / C38H30CrN6O12S2.Na

IUPAC Name:

Reaction mass of Chromate(1-), [2-(3-chlorophenyl)-2,4-dihydro-4-[[2-hydroxy-5-(methylsulfonyl)phenyl]azo]-5-methyl-3H-pyrazol-3-onato(2-)][methyl [7-hydroxy-8-[[2-hydroxy-5-(methylsulfonyl)phenyl]azo]-1-naphthalenyl]carbamato(2-)]-, sodium and sodium bis[2-(3-chlorophenyl)-2,4-dihydro-4-[[2-hydroxy-5-mesylphenyl]azo]-5-methyl-3H-pyrazol-3-onato(2-)]chromate(1-) and sodium bis[methyl [7-hydroxy-8-[[2-hydroxy-5-mesylphenyl]azo]-1-naphthyl]carbamato(2-)]chromate(1-)

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No. of test material: 001-140902

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

OTHER SPECIFICS: Solid / brown, pH ca. 6 (undiluted test substance, moistened with de-ionized water; determined in the lab prior to start of the GLP study)

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Demanded by the Guideline.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM model, EPI-200, MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
- Tissue lot number: 23362
- Date of initiation of testing: 2016-10-11

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature, 1 min or 37 °C, 1 h

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: The tissues were washed with PBS to remove residual test material 3 minutes or 1 hour after start of the application treatment.
- Observable damage in the tissue due to washing: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: SunriseTM Absorbance Reader
- Wavelength: 570 nm
- Filter: without reference filter

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
Barrier function and Quality control (QC): The supplier demonstrates that each batch of the model used meets the defined production release criteria. MatTek determines the ET50 value following exposure to Triton X-100 (1 %) for each EpiDermTM batch. The ET50 must fall within a range established based on a historical database of results. The following acceptability range (upper and lower limit) for the ET50 is established by the supplier as described in the cited OECD guidelines. Lower acceptance limit: ET50 = 4.0 hours; Upper acceptance limit: ET50 = 8.7 hours

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues
- Procedure used to prepare the killed tissues: freezing
- No of replicates: 2
- Method of calculation used: see "Any other information on materials and methods"


PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50 %, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 25 µL deionized water + 25 µL test substance (bulk volume)

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL
- Concentration: 8 N

Duration of treatment / exposure:

3 min or 1 h

Number of replicates:

2

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min

Value:

113.3

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 h

Value:

95.6

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: Mechanical damage of KC tissue No. 2 (1 h incubation) occurred during the washing procedure
- Direct-MTT reduction: Due to the intense color of the test substance, it was not possible to evaluate whether the test substance is able to directly reduce MTT. Therefore, freeze-killed control tissues (KC) were treated with the test article and the negative control in the same way as described above.
- Colour interference with MTT: Due to the color of the test substance, a pretest (experimental conduct in accordance with GLP, but without a GLP status) was performed as follows: the test substance was applied to a KC tissue, incubated for 1 hour and removed by washing. Thereafter, extraction in isopropanol was performed and the OD570 of the extract was spectrophotometrically determined. Based on the result of the pretest, it was judged that application of color control tissues is not necessary.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Any other information on results incl. tables

Table 3: Individual and ean OD570 values, individual and mean viability values, standard deviations and coefficient of variation

Exposue Time: 3 min			Tissue 1	Tissue 2	Mean	SD	CV (%)
NC	Viable Tissue	Mean OD570	1.971	1.899	1.935
		Viability (% of NC)	101.8	98.2	100.0	2.6	2.6
	KC Tissue	Mean OD570	0.120	0.102	0.111
		Viability (% of NC)	6.2	5.3	5.7	0.7	11.4
Test Substance	Viable Tissue	Mean OD570	2.141	2.273	2.207
		Viability (% of NC)	110.6	117.5	114.0	4.8	4.2
	KC Tissue *	Mean OD570	0.000	0.029	0.015
		Viability (% of NC)	0.0	1.5	0.7	1.1	141.4
	Final mean viability of tissues after KC correction (% of NC)				113.3
PC	Viable Tissue	Mean OD570	0.370	0.462	0.416
		Viability (% of NC)	19.1	23.9	21.5	3.3	15.6

* Negative values are set to zero for further calculation.

Brownish discoloration and minimal compound residues were observed on all test-substance treated tissues after the washing procedure.

Due to the intense color of the test substance, the ability of the test substance to directly reduce MTT could not be determined in a pretest. Therefore, KC tissues were applied in parallel. The results of the KC tissues indicate an increased MTT reduction (mean viability 0.7 % of NC). Thus for the test substance, the final mean viability is given after KC correction.

Table 4: Individual and ean OD570 values, individual and mean viability values, standard deviations and coefficient of variation

Exposue Time: 1 h			Tissue 1	Tissue 2	Mean	SD	CV (%)
NC	Viable Tissue	Mean OD570	1.814	1.984	1.899
		Viability (% of NC)	95.5	104.5	100.0	6.3	6.3
	KC Tissue	Mean OD570	0.0889	0.089	0.089
		Viability (% of NC)	4.7	4.7	4.7	0.0	0.0
Test Substance	Viable Tissue	Mean OD570	1.832	1.838	1.835
		Viability (% of NC)	96.5	96.8	96.6	0.2	0.3
	KC Tissue**	Mean OD570	0.020	0.233	0.020
		Viability (% of NC)	1.1	12.3	1.1
	**Final mean viability of tissues after KC correction (% of NC)				95.6
PC	Viable Tissue	Mean OD570	0.078	0.087	0.082
		Viability (% of NC)	4.1	4.6	4.3	0.3	7.3

** Mechanical damage of KC tissue No. 2 occurred during the washing procedure, thus, the result of KC tissue 2 is excluded from calculation of the mean value. Due to the unambiguous results, this deviation did not adversely affect the evaluation of the study.

Brownish discoloration and minimal compound residues were observed on all test-substance treated tissues after the washing procedure.

Due to the intense color of the test substance, the ability of the test substance to directly reduce MTT could not be determined in a pretest. Therefore, KC tissues were applied in parallel. The result of KC tissue 1 indicate an increased MTT reduction (viability 1.1 % of NC). Thus for the test substance, the final mean viability is given after KC correction.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met