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

22 Mar 2016 to 03 Aug 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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

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

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-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-)

EC Number:

285-083-3

EC Name:

Amines, C10-14-branched and linear alkyl, bis[2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-)

Cas Number:

85029-58-9

Molecular formula:

C34H24CrN8O6.C10-14H21-29NH2

IUPAC Name:

reaction mass of: branched and linear(C10-C14)ammonium , bis[2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-)

Details on test material:

The substance was formerly identified as: 73297-13-9 / 615-953-3 / Chromate(1-), bis[2-[2-[4,5-dihydro-3-methyl-5-(oxo-κO)-1-phenyl-1H-pyrazol-4-yl]diazenyl-κN1]benzoato(2-)-κO]-, hydrogen, compd. with 1-tridecanamine (1:1:1)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No. of test material: 003-142715
- CAS: 85029-58-9
- Purity test date: ca. 98 area-% (LC)
- Content: 98.4 g/100 g (titration)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Storage stability of the test material: The stability under storage conditions over the study period was guaranteed by the sponsor
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:
- Homogeneity: The test substance was homogeneous by visual inspection

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)
- Test substance applied minimally moistened with PBS

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

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: EpiDermTM tissues

Cell source:

other: Tissue derived from a single donor

Source strain:

other: Epiderm tissue (24 EPI-200 tissues - reconstructed epidermis)

Details on animal used as source of test system:

- EpiDermTM tissues: human-derived epidermal keratinocytes which have been cultured to form a multi layered, highly differentiated model of the human epidermis

SOURCE
- All cells used to produce EpiDermTM are purchased or derived from tissue obtained by MatTek Corporation from accredited instructions. In all cases consent was obtained by these institutions from the donor or the donor's legal next of kin, for the use of the tissues or derivatives of the tissue for research purposes.
- Tissue lot number: 23328

Vehicle:

unchanged (no vehicle)

Details on test system:

SKIN DISC PREPARATION
- Test system: Three dimensional human epidermis model (EpiDermTM)
- Tissue model: EPI-200
- Tissue Lon Number: 23328
- Origin: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
- Procedure used: Test item tested with EpiDermTM using PBS sterile as negative control and 5% (w/v) sodium dodecyl sulfate (SDS) in water as positive control
- Quality control for skin discs: Time of exposure to reduce tissue viability to 50% (ET50; acceptable range: 4.77-8.72 h): ET50 = 5.81 h (Pass)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature (25°C)
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: The tissues were washed with sterile PBS once (1 hour after start of application)
- Observable damage in the tissue due to washing: No damage observed

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: SunriseTM Absorbance Reader
- Wavelength: 570 nm

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION:
For skin irritation at least 3 tissue replicates should be sufficient for a test. In case of borderline results, a second test should be performed. In case of discordant results between the first 2 tests, a third test should be performed.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- 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 test-substance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability.
- A chemical is considered as "irritant", if the mean relative tissue viability with a test material is ≤ 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

- Amount of test item: 25 µL ( approx. 9 mg)
- Amount of negative control: 30 µL
- Amount of positive control: 30 µL

Duration of treatment / exposure:

The conditioned, treated tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator.

Duration of post-treatment incubation (if applicable):

Tissues were washed 1 h after start of application and then placed into the incubator at 37°C for 24 ± 2 hours. After that 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. Afterwards, the assay mediu was replaced by 0.3 mL MTT solution and tissues were incubated in incubator for 3 hours.

Number of replicates:

3 tissue replicates

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

103.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

101.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

100.2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 4

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

101.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The test substance is not able to reduce MTT directly.
In a pretest it was demonstrated that the color of the test substance did not interfere with the colorimetric test.
Acceptance criteria for negaite control, positive control and variability of the tissues were met.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met