Hydrogen [3-[[1-[anilinocarbonyl]-2-oxopropyl]azo]-2-hydroxy-5-nitrobenzene-1-sulphonato(3-)]hydroxychromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1)

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:

2016-12-09 to 2017-03-22

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 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

2016-07-29

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Hydrogen [3-[[1-[anilinocarbonyl]-2-oxopropyl]azo]-2-hydroxy-5-nitrobenzene-1-sulphonato(3-)]hydroxychromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1)

EC Number:

304-519-6

EC Name:

Hydrogen [3-[[1-[anilinocarbonyl]-2-oxopropyl]azo]-2-hydroxy-5-nitrobenzene-1-sulphonato(3-)]hydroxychromate(1-) , compound with 3-[(2-ethylhexyl)oxy]propylamine (1:1)

Cas Number:

94276-33-2

Molecular formula:

C16 H12 Cr N4 O9 S . C11 H25 N O . H

IUPAC Name:

hydrogen 3-[(2-ethylhexyl)oxy]propan-1-amine 11-methyl-4-nitro-12-(phenylcarbamoyl)-6-sulfonato-8λ³-oxa-10λ³-oxa-1λ⁴,13-diaza-9-chromatricyclo[7.4.0.0²,⁷]trideca-1(13),2(7),3,5,10-pentaene-9,9,9-tris(ylium)-8,12-diid-9-olate

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No of test material: 002-152503

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

OTHER SPECIFICS: Solid / orange to 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 corrosponding OECD Guideline.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPI-200
- Tissue lot number: 23385
- Date of initiation of testing: 2016-12-13

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 min at room temperature or 1 h at 37 °C.
- Temperature of post-treatment incubation: 37 °C

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
- Modifications to validated SOP: 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 EpiDerm 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

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

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 25 µL (bulk volume)

NEGATIVE CONTROL
- Amount applied: 50 µL


POSITIVE CONTROL
- Amount applied: 50 µL

Duration of treatment / exposure:

3 min or 1 h

Duration of post-treatment incubation (if applicable):

3 h

Number of replicates:

2

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min

Value:

90.9

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 h

Value:

96.3

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: Minimal yellowish discoloration of the test-substance treated tissues was observed after the washing procedure.
- Direct-MTT reduction: No
- Colour interference with MTT: Based on the result of the pretest it was judged that application of color control tissues is not necessary.

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: Exposure period 3 min: Individual and mean OD570 values, individual and mean viability values, standard deviations and coefficient of variation

		Tissue 1	Tissue 2	Mean	SD	CV [%]
NC	Mean OD570	1.914	1.662	1.788
	Viability [% of NC]	107.1	92.9	100.0	10.0	10.0
Test Substance	Mean OD570	1.662	1.589	1.626
	Viability [% of NC]	93.0	88.9	90.9	2.9	3.2
PC	Mean OD570	0.358	0.235	0.297
	Viability [% of NC]	20.0	13.2	16.6	4.8	29.2

 

Table 4: Exposure period 1 h: Individual and mean OD570 values, individual and mean viability values, standard deviations and coefficient of variation

		Tissue 1	Tissue 2	Mean	SD	CV [%]
NC	Mean OD570	1.649	1.711	1.680
	Viability [% of NC]	98.1	101.9	100.0	2.6	2.6
Test Substance	Mean OD570	1.658	1.576	1.617
	Viability [% of NC]	98.7	93.8	96.3	3.5	3.6
PC	Mean OD570	0.085	0.081	0.083
	Viability [% of NC]	5.1	4.8	5.0	0.2	3.4

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met