Amines, C10-14-branched and linear alkyl, bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]chromate(1-) (1:1)

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-04-07 to 2016-07-25

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

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)

Test material

Test material information

Constituent 1

Reference substance name:

Amines, C10-14-branched and linear alkyl, bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]chromate(1-) (1:1)

EC Number:

284-628-2

EC Name:

Amines, C10-14-branched and linear alkyl, bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]chromate(1-) (1:1)

Cas Number:

84961-40-0

Molecular formula:

C32H22CrN10O8.C10-14H21-29NH2

IUPAC Name:

Amines, C10-14-branched and linear alkyl, bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]chromate(1-) (1:1)

Test material form:

solid

Details on test material:

- State of aggregation: solid (powdeQ

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No. of test material: 002-141407, Test-substance No.: 16/0046-1

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

OTHER SPECIFICS: Solid / orange, pH ~ 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

Cell source:

other: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPI-200
- Tissue lot number: 23328
- Date of initiation of testing: 2016-04-12

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature, 37 °C, incubator
- Temperature of post-treatment incubation: 37 °C, incubator

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: The tissues were washed with sterile PBS to remove residual test substance.
- Observable damage in the tissue due to washing: Due to mechanical damage of tissue 1 during the washing procedure, only two tissues of the negative control could be evaluated. Since the OD570 values of tissue 2 and 3 are well within the historical control data, this deviation is not considered to adversely affect the result of this study.

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, without reference filter

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability:
MTT QC assay, 4 hours.
Acceptance criteria OD (540 - 570 nm) [1.0 - 3.0]
Result:1.744 ± 0.201
- Barrier function:
ET-50 assay, 100 µL 1 % Triton X-100. 4 timepoints, n = 3, MTT assay
Acceptance criteria: ET-50 (4.77 - 8.72 h)
Result: 5.81 h
- Contamination: Longterm antibiotic and antimycotic free culture, no contamination observed

NUMBER OF REPLICATE TISSUES: 3

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the mean relative tissue viability with a test substance is less than or equal to 45 %.
- The test substance is considered to be non-irritant to skin if the mean relative tissue viability with a test substance is higher than 55 %.
- The test substance is considered to be borderline, if the mean relative tissue viability with a test substance is lesser than or equal to 55 % and higher then 45 %.

The „borderline"-evaluation (50 ± 5 %) was determined statistically using historic BASF data and hence considers the variance of the test method. This evaluation is confirming the borderline range provided in OECD Guideline 439.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 25 µL bulk volume of the solid test substance


NEGATIVE CONTROL
- Amount applied: 30 µL

POSITIVE CONTROL
- Amount applied: 30 µL
- Concentration: 5 %

Duration of treatment / exposure:

1 h

Duration of post-treatment incubation (if applicable):

42 ± 4 h

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

109.6

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: Yes, tissue 1
- Direct-MTT reduction: No
- Colour interference with MTT: No

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

Test substance identification		tissue 1	tissue 2	tissue 3	mean	SD	CV [%]
NC	mean OD570	*	1.953	2.198	2.075
	viability [% of NC]	*	94.1	105.9	100.0	8.3	8.3
16/0046-1	mean OD570	2.311	2.104	2.412	2.275
	viability [% of NC]	111.3	101.4	116.2	109.6	7.6	6.9
PC	mean OD570	0.087	0.088	0.075	0.084
	viability [% of NC]	4.2	4.3	3.6	4.0	0.3	8.7

 

* Due to mechanical damage of tissue 1 during the washing procedure, only two tissues of the negative control could be evaluated. Since the OD570 values of tissue 2 and 3 are well within the historical control data, this deviation is not considered to adversely affect the result of this study.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met