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]-,Sodium bis[1-[(2-hydroxy-5-nitrophenyl)azo]naphthalen-2-olato(2-)]cobaltate(1-)and Sodium bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]cobaltate(1-)

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 February 2017 - 9 June 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:

28 July 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

6 July 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

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]-,Sodium bis[1-[(2-hydroxy-5-nitrophenyl)azo]naphthalen-2-olato(2-)]cobaltate(1-)and Sodium bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]cobaltate(1-)

EC Number:

916-867-1

Cas Number:

12270-00-7

Molecular formula:

C32H18CoN6O8Na - C32H20CoN8O8Na - C32H22CoN10O8Na

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]-,Sodium bis[1-[(2-hydroxy-5-nitrophenyl)azo]naphthalen-2-olato(2-)]cobaltate(1-)and Sodium bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]cobaltate(1-)

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 16/137

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: The stability of the test item under storage conditions over the study period was guaranteed by the sponsor.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Remarks:

test item was applied minimally moistened with PBS

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ 200 kit ( EPI-200 tissues with surface of 0.6 cm²), MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
- Tissue lot number: 23393

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 25 min at room temperature and 35 min in the incubator at 37°C
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Washing: with sterile PBS
- Observable damage in the tissue due to washing: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg / mL
- Incubation time: 3 hours
- Spectrophotometer: SunriseTM Absorbance Reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS
- Viability: 1.52 ± 0.144
- Barrier function: ET-50 = 5.55 hours
- Morphology: functional stratum corneum, a viable basal cell layer, intermediate spinous and granular layers
- Contamination: no, sterile

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- killed tissues
- Procedure used to prepare the killed tissues: EPI-200 tissue that is killed by freezing at –20°C
- N. of replicates : 3

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

PREDICTION MODEL / DECISION CRITERIA
mean tissue viability
(% of negative control) Prediction
< 45 irritant
45 - 55 borderline
> 55 non-irritant
The “borderline“ evaluation (50 ± 5%) was statistically determined by using historic BASF data and hence considers the variance of the test method.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 25 μL sterile PBS was applied first. Thereafter, a bulk volume of ca. 25 μL solid test material was applied with a sharp spoon and homogeneously distributed together with the fluid.

NEGATIVE CONTROL
- Amount(s) applied: 30 µL

POSITIVE CONTROL
- Amount(s) applied: 30 µL
- Concentration: 5% (w/v) sodium dodecyl sulfate (SDS) in water

Duration of treatment / exposure:

1 hour

Duration of post-treatment incubation (if applicable):

ca. 42 hours

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

82.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100 %

Positive controls validity:

valid

Remarks:

2.7 %

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECT
- Direct-MTT reduction: yes
- Colour interference with MTT: no

Brownish discoloration was observed on all test-substance treated tissues after the washing procedure.

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
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.
- Acceptance criteria met for positive control: yes
A viability of ≤ 20% is acceptable.
- Acceptance criteria met for variability between replicate measurements: yes
The inter-tissue variability is considered to be acceptable if the SD of % viability is ≤ 18%.

Historic control data of PC and NC:
Historic Range of NC (Period Jan 2015 - Jan 2017)
Mean OD570: 2.156 ± 0.278

Historic Range of PC (Period Jan 2015 - Jan 2017)
Mean OD570: 0.065 ± 0.012

Viability (%) (Period Jan 2015 - Jan 2017)
Mean % : 3.0 ± 0.5

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met