Tripotassium [5-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-4-hydroxy-3-[(2-hydroxy-4-sulpho-1-naphthyl)azo]naphthalene-2,7-disulphonato(5-)]cuprate(3-)

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28.06. – 07.07.2016

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted: July 28th, 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Tripotassium [5-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-4-hydroxy-3-[(2-hydroxy-4-sulpho-1-naphthyl)azo]naphthalene-2,7-disulphonato(5-)]cuprate(3-)

EC Number:

304-149-5

EC Name:

Tripotassium [5-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-4-hydroxy-3-[(2-hydroxy-4-sulpho-1-naphthyl)azo]naphthalene-2,7-disulphonato(5-)]cuprate(3-)

Cas Number:

94246-75-0

Molecular formula:

C23H11ClCuN7O11S3.3K

IUPAC Name:

tripotassium 18-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-13λ³,15λ³-dioxa-1λ⁴,2-diaza-14-cuprahexacyclo[12.11.0.0³,¹².0⁴,⁹.0¹⁶,²⁵.0¹⁷,²²]pentacosa-1,4(9),5,7,10,16,18,20,22,24-decaene-14,14-bis(ylium)-13,15-diide-10,20,24-trisulfonate

impurity 1

Reference substance name:

Potassium chloride

EC Number:

231-211-8

EC Name:

Potassium chloride

Cas Number:

7447-40-7

Molecular formula:

ClK

IUPAC Name:

potassium chloride

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): Reactive Blue 234
- Other name: Ostazin Blue H-5R
- Lot/batch No.: 8002
- Expiration date of the lot/batch: unlisted

In vitro test system

Test system:

human skin model

Remarks:

a reconstructed human epidermal model EpiDerm™ (EPI-200, MatTek)

Source species:

human

Cell type:

other: normal human-derived epidermal keratinocytes

Cell source:

other: Keranocyte strain: 00267

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: The reconstructed human epidermal model EpiDerm™ (EPI-200, MatTek, Ashland, USA)
- Tissue batch number(s): Lot No. 23343, kit F

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37±1°C
- Temperature of post-treatment incubation (if applicable): 37±1°C

REMOVAL OF TEST MATERIAL AND CONTROLS
tissues were thoroughly rinsed and blotted to remove the test substance (controls)
Detailed procedure is described in internal SOP M/46/2.
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration:
tissues were transferred to 24-well plates containing MTT medium (1 mg·mL-1)
- Incubation time: 3 hr
- Spectrophotometer: Libra S22. Isopropyl alcohol serves as a blank.
- Wavelength: 570±30 nm

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues
- Procedure used to prepare the killed tissues (if applicable): n.a.
- N. of replicates : 2
- Method of calculation used:
Data correction for colour interference
True viability = %Viability of treated tissue – %NSCliving

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
1. Direct MTT reduction - functional check in tubes (was a part of another study No. 272/15/4AI: Reactive Blue 234 - In vitro Skin Irritation Test (EpiDermTM Model); VUOS-CETA Report No. 16-394, 2016)
2. Direct MTT reduction – test in frozen tissues
3. MTT test

DECISION CRITERIA
According to the OECD TG 431 as well as to the EU Method B.40, the test substance is considered to be corrosive to skin:
i) if the viability after 3 minutes exposure is less than 50 %, or
ii) 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 %.
The test substance is considered to be non-corrosive to skin:
i) if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is greater than or equal to 15 %

Control samples:

yes, concurrent vehicle

yes, concurrent positive control

Amount/concentration applied:

test substance C2: 25 mg, no vehicle
NC: water
PC: KOH 8N

Duration of treatment / exposure:

3 and 60 minutes

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

73

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Direct MTT reduction - functional check in tubes:
Decision if the test substance is directly-reducing or not could not be made.

- Direct MTT reduction - test in frozen tissues:
OD570 were lower in tissues treated with the test substance than in these treated with water. Consequently, the test substance remained in tissues had no contribution to OD570 in the MTT test.


ACCEPTANCE OF RESULTS: All assay acceptance criteria have been met.

Negative control: The assay meets the acceptance criterion - OD570 of the NC tissues was 1.770 (3 min) and 1.759 (60 min) what is ≥ 0.8 and ≤ 2.8.
Positive control: Viability of tissues treated with 8N KOH after 60 minutes treatment was 4.9 % what is ≤ 15%.

Coefficient of variance: CV values in all triplets of tissues were ≤ 0.3.

Any other information on results incl. tables

Direct MTT reduction: test in frozen tissues

Treatment 60 min	tissue 1	tissue 2	mean	SD	CV	%NC
water (NC)	0.064	0.065	0.065	0.001	0.008
272/15 (C2)	0.046	0.048	0.047	0.001	0.021	72.9

OD₅₇₀were lower in tissues treated with the test substance than in these treated with water. Consequently, the test substance remained in tissues had no contribution to OD₅₇₀in the MTT test.

MTT test: viable tissues

OD₅₇₀values obtained at the MTT test, their averages, standard deviations (%), coefficients of variance and relative viabilities

Treatment 3 min	OD570			mean	SD	CV	%NC
water (NC)	1.807	1.735	1.769	1.770	0.029	0.017	100.0
272/15 (C2)	1.128	1.488	1.499	1.372	0.172	0.126	77.5
8N KOH (PC)	0.116	0.114	0.111	0.114	0.002	0.018	6.4
Treatment 60 min	OD570			mean	SD	CV	%NC
water (NC)	1.877	1.775	1.624	1.759	0.104	0.059	100.0
272/15 (C2)	1.345	1.297	1.252	1.298	0.038	0.029	73.8
272/15 no MTT (C2)	0.013	0.014	NT	0.014	0.001	0.037	0.8
8N KOH (PC)	0.075	0.091	0.091	0.086	0.008	0.088	4.9

In addition, two viable tissue replicates, which underwent the entire testing procedure (treatment for 60 minutes) but were incubated with medium instead of MTT solution during the MTT incubation step to generate a non-specific colour (NSC_living) control. OD₅₇₀average value is 0.014.

Data correction for colour interference:

True viability = %Viability of treated tissue – %NSC_living

3 minutes experiment: no correction was done, because 3 minutes experiment without MTT was not performed. In this case the expected contribution of the test substance is < 1.0%, what could not influence study outcome.

60 minutes experiment: true relative viability 73.8 % - 0.8 % = 73.0 %.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the above-described experimental design, the test substance Reactive Blue 234 was non-corrosive in In vitro Skin Corrosion Test on EpiDermTM tissues.

Executive summary:

Test substance Reactive Blue 234 was assayed for the in vitro skin corrosion in human epidermal model EpiDermTM. The test was performed according to OECD Test Guideline 431, In Vitro Skin Corrosion: Human Skin Model Test, Adopted: July 28th, 2015.

Direct reduction test in the test tubes was performed before MTT test. As the test substance is coloured violet, the test gave no information about direct MTT reduction. Potential direct reduction caused by rests of the test substance in tissues was excluded after doing the test in frozen tissues.

In MTT test, the test substance (25 mg) was placed atop the previously moistened tissue. Length of exposition was 3 and 60 minutes. Nine tissues were used for the experiment in each time, three per test substance (C2), three for positive control (PC) and three for negative control (NC).

After rinsing, tissues were incubated with MTT for three hours and then extracted with isopropyl alcohol for two hours at room temperature with shaking. OD570 of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

Besides two living tissues underwent the same procedure (60 minutes) with exception of staining with MTT in the last step to detect influence of violet colour which could interfere with evaluation. Influence of the colour was 1% of relative cell viability. This value was subtracted from measured relative cell viability in 60 minutes experiment.

Under the above-described experimental design, average viability of tissues treated by the test substance Reactive Blue 234 was 77.5 % of negative control average value after 3 minutes treatment and 73.0 % (corrected) after 60 minutes of treatment.

The test substance is considered to be non-corrosive to skin:

i) if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is greater than or equal to 15 %.

In the experiment arrangement given above, the test substance Reactive Blue 234 was non-corrosive in EpiDermTM model.