2-[4-[(hexahydro-2,4,6-trioxo-5-pyrimidyl)azo]phenyl]-6-methylbenzothiazole-7-sulphonic acid, compound with 2,2',2''-nitrilotris[ethanol] (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:

2016-02-25 to 2016-04-27

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

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:

Commission Regulation (EU) No 640/2012 of 6 July 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 2-[4-[(Hexahydro-2,4,6-trioxo-5-pyrimidyl)azo]phenyl]-6-methylbenzothiazole-7-sulphonic acid, compound with 2,2',2''-nitrilotris[ethanol] (1:1) and Lithium 2-[4-[(hexahydro-2,4,6-trioxopyrimidin-5-yl)azo]phenyl]-6-methylbenzothiazole-7-sulphonate

IUPAC Name:

Reaction mass of 2-[4-[(Hexahydro-2,4,6-trioxo-5-pyrimidyl)azo]phenyl]-6-methylbenzothiazole-7-sulphonic acid, compound with 2,2',2''-nitrilotris[ethanol] (1:1) and Lithium 2-[4-[(hexahydro-2,4,6-trioxopyrimidin-5-yl)azo]phenyl]-6-methylbenzothiazole-7-sulphonate

Specific details on test material used for the study:

Batch no.: 0013479406

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The EPISKIN model has been validated for irritation testing in an international trial. After a review of scientific reports and peer reviewed publications on the EPISKIN method, it showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purposes of distinguishing between skin irritating and no skin irritating test substances (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPI-200
- Tissue batch number: 23317 (test run 1) and 23328 (test run 2)
- Date of initiation of testing: 2016-03-01

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room termperature for 25 minutes and in the incubator (37 °C) for 35 minutes
- Temperature of post-treatment incubation: 37°C ± 1°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Once, 1 hour after start of application with sterile PBS
- Observable damage in the tissue due to washing: Yes, due to mechanical damage of tissue 1 during the washing procedure, only two tissues of the negative control could be evaluated.
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: SunriseTM Absorbance Reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Positive control: mean 3 %; range: 2.2-3.9

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
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 freeze-killed control tissue (KC), incubated and removed by washing in the same way as in the main experiment. Thereafter extraction in isopropanol was performed and the OD570 of the extract was determined spectrophotometrically.
Based on the result of the pretest it was judged that application of color control tissues is not necessary.

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the mean tissue viability is ≤ 50 %.
- The test substance is considered to be non-irritant to skin if the mean tissue viability is > 50 %

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amounts applied: bulk volume of ca. 25 μL of the solid test material
- Concentration: undiluted

NEGATIVE CONTROL
- Amount applied: 30 μL
- Concentration: no data

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

Duration of treatment / exposure:

1 hour

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3 (per independent experiment, 2 test runs)

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean value of 3 tissues

Value:

92.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100 %

Positive controls validity:

valid

Remarks:

4 %

Remarks on result:

other: Result refers to 2nd run. Due to the high inter-tissue variability of the test substance (1st run), a 2nd test run was performed to clarify the result.

Other effects / acceptance of results:

Yellowish discoloration of the test-substance treated tissues was observed after the washing procedure.

Values for single tissues
1st test run: 101.1%, 95.3% and 7.6%. Due to the high inter-tissue variability of the test substance, a 2nd test run was performed to clarify the result.
2nd test run: 91.6%, 94.7% and 90.5%.

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, for the 2nd test run.
Due to the non-concordant replicate measurements of the test-substance treated tissues after the 1st test run, the study was repeated.

- Range of historical values:

Historic Range of NC: OD570
Period: Jan 2014 - Jan 2016;
Mean OD: 2.373; SD: 0.263; Mean + 2 SD: 2.899; Mean - 2 SD: 1.847

Historic Range of PC: OD570
Period: Jan 2014 - Jan 2016; Mean OD: 0.071; SD: 0.011; Mean + 2 SD: 0.093 Mean - 2 SD: 0.050

Viability (%)
Period: Jan 2014 - Jan 2016; Mean %: 3.0; SD: 0.4; Mean + 2 SD: 3.9; Mean - 2 SD: 2.2

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met