Reaction mass of sodium [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(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:

2016-06-22 to 2017-06-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016-06-22 to 2017-06-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015-07-28

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No. of test material: N01-131001

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

OTHER SPECIFICS: Solid / dark brown, pH ca. 5 (undiluted test substance, moistened with de-ionized water; determined in the lab prior to start of the GLP study)

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
- Tissue lot number: 23343
- Date of initiation of testing: 2016-06-28

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 25 min at room temperature, 35 min at 37 °C (Incubator)
- Temperature of post-treatment incubation: 37 °C

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: 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: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues
- Procedure used to prepare the killed tissues: freezing
- N. of replicates : 3
- Method of calculation used:
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 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 three tissues treated in the same way was calculated.
Application of measurements using killed control tissues: In case of direct MTT reduction 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 tissues treated with the test substance (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.
Application of measurements using color control tissues: The OD570 values measured in the color control tissues (CC) was used to correct the mean OD570 of the test-substance treated tissues (mean OD570 CC corrected). The mean net OD570 CC was subtracted from the respective mean OD570 to result in the mean OD570 CC corrected, only when interference of the test substance in the colorimetric test is noticed. The mean OD570 CC corrected represents the formazan production without the absorbance of the colored 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.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritating to skin if the mean relative tissue viability with a test material is less than or equal to 50 %.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

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

NEGATIVE CONTROL
- Amount applied: 30 µL

POSITIVE CONTROL
- Amount applied: 30 µL
- Concentration: 5 % (w/v) in water

Duration of treatment / exposure:

1 h

Duration of post-treatment incubation (if applicable):

42 h ± 4 h

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Value:

95

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: Due to the color of the test substance, it could not be determined whether the test substance can directly reduce MTT. Therefore, an additional MTT reduction control (freeze-killed control tissues (KC)) was introduced.
- Colour interference with MTT: In a pretest, it was demonstrated that the color of the test substance interferes with the colorimetric test. Therefore, color controls (viable tissues CC) and freeze-killed control tissues (KC CC) were performed to differentiate formazan produced by the cells in the MTT test from color residues of the test substance in the colorimetric test.

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

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

			Tissue 1	Tissue 2	Tissue 3	Mean	SD	CV (%)
NC	Viable Tissue	Mean OD570	2.268	2.194	2.033	2.165
		Viability (% of NC)	104.7	101.3	93.9	100.0	5.5	5.5
	KC Tissue	Mean OD570	0.055	0.051	0.066	0.057
		Viability (% of NC)	2.5	2.3	3.0	2.6	0.4	13.7
Test Substance	Viable Tissue	Mean OD570	2.140	2.079	1.988	2.069
		Viability (% of NC)	98.8	96.0	91.8	95.6	3.5	3.7
	CC Tissue	Mean OD570	0.009	0.013	0.014	0.012
		Viability (% of NC)	0.4	0.6	0.6	0.6	0.1	20.4
	Mean viability of Tissues after CC correction (%of NC)					95.0
	KC Tissue	Mean OD570 KC NC corrected	0.018	0.017	0.019	0.018
		Viability (% of NC)	0.8	0.8	0.9	0.8	0.1	7.1
	KC CC Tissue	Mean OD570	0.029	0.034	0.035	0.032
		Viability (% of NC)	1.3	1.5	1.6	1.5	0.1	9.1
	Mean Viability of KC Tissues after KC CC correction (% of NC)*					0.0
	Final Mean Viability of Tissues after KC and CC correction (% of NC)					95.0
PC	Viable Tissues	Mean OD570	0.052	0.053	0.051	0.052
		Viability (% of NC)	2.4	2.4	2.4	2.4	0.0	1.5

* Negative values are set to zero for further calculation.

Brown discoloration of the test-substance treated tissues was noticed after the washing procedure. 

The results of the color control (CC) tissues indicate interference due to the color of the test substance (mean value 0.6% of NC). The results of the KC tissues did not indicate an increased MTT reduction. Thus for the test substance the final mean viability is given after CC correction only.

 

Interpretation of results:

GHS criteria not met

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 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 sodium [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]chromate(1-)

EC Number:

915-758-6

Molecular formula:

C32H18CrN6O8.Na / C32H22CrN10O8.Na / C32H20CrN8O8.Na

IUPAC Name:

Reaction mass of sodium [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and sodium bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(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: N01-131001

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

OTHER SPECIFICS: Solid / dark brown, pH ca. 5 (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: 23343
- Date of initiation of testing: 2016-06-28

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:
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 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 treated in the same way was calculated.
Application of measurements using killed control tissues: In case of direct MTT reduction 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 tissues treated with the test substance (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.
Application of measurements using color control tissues: The OD570 values measured in the color control tissues (CC) was used to correct the mean OD570 of the test-substance treated tissues (mean OD570 CC corrected). The mean net OD570 CC was subtracted from the respective mean OD570 to result in the mean OD570 CC corrected, only when interference of the test substance in the colorimetric test is noticed. The mean OD570 CC corrected represents the formazan production without the absorbance of the colored 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.


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

yes, concurrent MTT non-specific colour 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:

84.3

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Value after KC and CC correction

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 h

Value:

108.2

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Value after KC and CC correction

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: Due to the color of the test substance, it could not be determined whether the test substance can directly reduce MTT. Therefore, an additional MTT reduction control (freeze-killed control tissues (KC)) was introduced.
- Colour interference with MTT: In a pretest, it was demonstrated that the color of the test substance interferes with the colorimetric test. Therefore, color controls (viable tissues CC) and freeze-killed control tissues (KC CC) were performed to differentiate formazan produced by the cells in the MTT test from color residues of the test substance in the colorimetric test.

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 mean OD570 values, individual and mean viability values, standard deviations and coefficient of variation

3 min			Tissue 1	Tissue 2	Mean	SD	CV (%)
NC	Viable Tissue	Mean OD570	2.051	1.964	2.007
		Viability (% of NC)	102.2	97.8	100.0	3.1	3.1
	KC Tissue	Mean OD570	0.083	0.095	0.089
		Viability (% of NC)	4.1	4.7	4.4	0.4	9.2
Test Substance	Viable Tissue	Mean OD570	1.802	1.599	1.701
		Viability (% of NC)	89.8	79.7	84.7	7.2	8.4
	CC Tissue	Mean OD570	0.010	0.009	0.010
		Viability (% of NC)	0.5	0.4	0.5	0.0	7.4
	Mean viability of Tissues after CC correction (%of NC)				84.3
	KC Tissue	Mean OD570 KC NC corrected	0.006	0.012	0.009
		Viability (% of NC)	0.3	0.6	0.4	0.2	51.1
	KC CC Tissue	Mean OD570	0.034	0.010	0.022
		Viability (% of NC)	1.7	0.5	1.1	0.8	77.1
	Mean Viability of KC Tissues after KC CC correction (% of NC)*				0.0
	Final Mean Viability of Tissues after KC and CC correction (% of NC)				84.3
PC	Viable Tissues	Mean OD570	0.284	0.188	0.236
		Viability (% of NC)	14.1	9.3	11.7	3.4	28.8

* Negative values were set to zero for further calculation.

 

The results of the color control (CC) tissues indicate interference due to the color of the test substance (mean value 0.5 % of NC). The results of the KC tissues did not indicate an increased MTT reduction. Thus for the test substance the final mean viability is given after CC correction, only.

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

1 h			Tissue 1	Tissue 2	Mean	SD	CV (%)
NC	Viable Tissue	Mean OD570	1.736	1.717	1.726
		Viability (% of NC)	100.6	99.4	100.0	0.8	0.8
	KC Tissue	Mean OD570	0.086	0.099	0.092
		Viability (% of NC)	5.0	5.7	5.3	0.6	10.3
Test Substance	Viable Tissue	Mean OD570	1.898	1.934	1.916
		Viability (% of NC)	109.9	112.0	111.0	1.5	1.3
	CC Tissue	Mean OD570	0.043	0.051	0.047
		Viability (% of NC)	2.5	3.0	2.7	0.3	12.0
	Mean viability of Tissues after CC correction (%of NC)				108.2
	KC Tissue	Mean OD570 KC NC corrected	0.029	0.028	0.029
		Viability (% of NC)	1.7	1.6	1.7	0.0	1.2
	KC CC Tissue	Mean OD570	0.049	0.113	0.081
		Viability (% of NC)	2.8	6.5	4.7	2.6	56.5
	Mean Viability of KC Tissues after KC CC correction (% of NC)*				0.0
	Final Mean Viability of Tissues after KC and CC correction (% of NC)				108.2
PC	Viable Tissues	Mean OD570	0.088	0.111	0.99
		Viability (% of NC)	5.1	6.4	5.7	0.9	16.0

* Negative values are set to zero for further calculation .

Brown discoloration of the test-substance treated tissues was noticed after the washing procedure. 

The results of the color control (CC) tissues indicate interference due to the color of the test substance (mean value 2.7 % of NC). The results of the KC tissues did not indicate an increased MTT reduction. Thus for the test substance the final mean viability is given after CC correction only.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met