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-)

Endpoint summary

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

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

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 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

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.

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 %.

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.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19.04. – 22.04.2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Details on study design:

Details on study design
TISSUES
- The reconstructed human epidermal model EpiDerm (EPI-200 ver. 2.0, MatTek, Bratislava, Slovakia); Lot No. 23329

CHEMICALS AND MEDIA
- PBS (phosphate buffered saline) prepared 25/02/2016, exp. 25/08/2016 and prepared 23/03/2016, exp.23/09/2016
- MTT (3-(4,5-Dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide), Alfa Aesar, Lot No. 10184735 exp.2/2017
- Isopropyl alcohol p.a., Lach-Ner, Lot No. PP/2014/01574, exp. 12/2016
- SDS (sodium dodecyl sulphate) 5 %, MatTek, Lot No. 012616TMB, exp. 26/01/2017 (positive control)
- EPI-100-NMM-SIT/Maintenance Medium, MatTek, Lot No. 041416ZSB, exp. 28/04/2016

TEST METHOD
Principle of the assay
The test consists of a topical exposure of the neat test chemical to a reconstructed human epidermis (RhE) model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT [(3-4,5-dimethyl thiazole 2-yl) 2,5-diphenyltetrazolium-bromide], present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The reduction of the viability of tissues exposed to chemicals in comparison to negative controls (treated with PBS) is used to predict the skin irritation potential.
Evaluation is determined by measuring of optical density (OD) of the formazan extracts using a spectrophotometer at 570 nm. Relative cell viability is calculated for each tissue as % of the mean OD value of the negative control tissues.
Skin irritation potential of the test substance is predicted if the remaining cell viability is below 50%.

Test system
The reconstructed human epidermal model EpiDerm (EPI-200, MatTek, Ashland, USA) consists of normal human-derived epidermal keratinocytes, which have been cultured to form a multilayered highly differentiated model of the human epidermis. The EpiDerm system is manufactured according to defined quality assurance procedures. The EpiDerm tissues (surface 0.63 cm²) are cultured on specially prepared cell culture inserts and shipped as kits, containing tissues on shipping agarose together with the necessary amount of culture media.

Test procedure
a) Application:
The test substance (25 mg of substance/surface ratio 39.7 mg/cm2) is placed directly atop to the tissue moistened with 25 μL of PBS. The material is spread on the tissue surface.
A single testing, composed of three replicate tissues, was run.
b) Procedure:
On the day of receipt, EpiDerm tissues are conditioned by incubation to release transport stress related compounds and debris. After pre-incubations durable for approximately 1 and 18 hours, tissues are topically exposed to the test chemicals for 1 hour (25 minutes at room temperature and the remaining 35 minutes at 37°C, 5%CO2). Three tissues are used per the test substance, for the positive (PC) and negative (NC) controls. Tissues are then thoroughly rinsed with PBS, blotted to remove the test substances, and transferred to fresh medium.
After 24±2 hours post-incubation period, the medium is replaced by fresh one. Tissues are incubated for another 18±2 hours. Afterwards, the MTT assay is performed by transferring the tissues to 24-well plates containing MTT medium (1 mg/mL). After 3 hour MTT incubation, the blue formazan salt formed by cellular mitochondria is extracted with 2.0 ml/tissue of isopropyl alcohol and the optical density of the extracted formazan is determined using a spectrophotometer at 570 nm.
c) OD570 measuring:
OD570 is measured on a spectrophotometer Libra S22. Isopropyl alcohol serves as a blank. Allowed band width is 2-3 nm. No external filter is used.
d) Viability calculation:
Relative cell viability is calculated for each tissue as % of the mean of the negative control tissues. Than the mean relative tissue viability of three individual tissues exposed to the test substance is calculated – this value is used for the comparison with limit.

Irritation / corrosion parameter:

other: OD570

Value:

8.9

Negative controls validity:

valid

Positive controls validity:

valid

	Treatment	OD570			Avg	SD	Average viability
		1	2	3			(% NC)
NC	PBS	1.790	1.543	1.557	1.630	0.113	100.0
	viability (%)	109.8	94.7	95.5	100.0	6.950
C2	272/15	0.137	0.168	0.132	0.146	0.016	8.9
	viability (%)	8.405	10.307	8.098	8.937	0.977
PC	5% SDS	0.054	0.051	0.050	0.052	0.002	3.2
	viability (%)	3.3	3.1	3.1	3.2	0.104

PBS - phosphate buffered saline

SDS - sodium dodecyl sulphate

Interpretation of results:

other: The test substance is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1). As the test substance was not tested for skin corrosion, it is not possible to decide between these two categories.

Conclusions:

Under the above-described experimental design, average viability of tissues treated by the test substance Reactive Blue 234 was 8.9 % of negative control average value, i.e. viability was < 50 %.
Average viability of treated tissues was 8.9 %, i.e. viability was < 50 %.
The effect of the test item was positive in EpiDermTM model (tissues were damaged).

Executive summary:

The test item, Reactive Blue 234, was assayed for the in vitro skin irritation in human epidermal model EpiDermTM. The test was performed according to the OECD Test Guideline No. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method (2015) and Protocol for: In Vitro EpiDerm^TMSkin Irritation Test For use with MatTek Corporation’s Reconstructed Human Epidermal Model EPI-200-SIT (2014).

After pre-incubation of tissues, 25 mg of the test substance was placed directly atop to the previously moistened tissue and it was spread on the entire tissue surface. Length of exposition was 60 minutes. Three tissues were used for the test substance and every control.

After removal of the test substance, tissues were post-incubated for approximately 42 hours due to leave of damage reparation. Three hours incubation with MTT and two hours extraction period with shaking followed then. Optical density (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.

Average viability of treated tissues was 8.9 %, i.e. viability was < 50 %.

The effect of the test item was positive in EpiDerm^TMmodel (tissues were damaged).

The test substance is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1). As the test substance was not tested for skin corrosion, it is not possible to decide between these two categories.

Reference 3

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Feb - March 1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline available

Principles of method if other than guideline:

- Principle of test: similar to Acute Toxicity: Dermal Irritation / Corrosion
- Short description of test conditions: exposure 4 hours, observation at 24, 48, 72 hours after exposure
- Parameters analysed / observed: hyperemia, edema on intact or scarified skin

GLP compliance:

no

Species:

rabbit

Strain:

New Zealand White

Type of coverage:

not specified

Preparation of test site:

other: scarified skin

Vehicle:

water

Controls:

not required

Amount / concentration applied:

500 mg

Duration of treatment / exposure:

4 hours

Observation period:

24, 48, 72 hours

Number of animals:

1

Irritation parameter:

other: observation of intact skin

Basis:

animal #1

Time point:

24 h

Score:

> 1 - < 2

Remarks on result:

other: yellow-green pigmented spots, hyperemia

Irritation parameter:

other: observation of intact skin

Basis:

animal #1

Time point:

other: 48/72 h

Score:

0

Irritation parameter:

other: observation of scarified skin

Basis:

animal #1

Time point:

24 h

Score:

> 1 - < 2

Remarks on result:

other: yellow-green pigmented spots, weak hyperemia, very weak edema

Irritation parameter:

other: observation of scarified skin

Time point:

other: 48/72 h

Score:

ca. 1 - 2

Remarks on result:

other: very weak edema

Interpretation of results:

study cannot be used for classification

Executive summary:

The tested substance Ostazinová námořnická modř H-5R is not irritating for intact skin and is very weak irritating for scarified skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01.06. – 02. 06. 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

Adopted 26th July 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Breeding service CHOVSERVIS a.s., division TORO® Hlavečník, Hradec Králové, Czech Republic
- Number of animals:
- Characteristics of donor animals (e.g. age, sex, weight): The eyes were enucleated as soon as possible after death. Only healthy animals (12 to 60 months old) considered suitable for entry into the human food chain were used as a source of corneas for use in the BCOP test.

- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions):
The risk of contamination was minimized (e.g., by keeping the container containing the eyes on ice, by adding antibiotics to the HBSS used to store the eyes during transport (e.g., penicillin at 100 IU/mL and streptomycin at 100 μg/mL).

- Time interval prior to initiating testing:
The time interval between collection of the eyes and use of corneas in the BCOP was minimized, so the collected eyes were processed on the same day.
All eyes used in the assay were from the same group of eyes collected on a specific day.

- indication of any existing defects or lesions in ocular tissue samples:
Only corneas from eyes free of defects including scratched, and neovascularisation were used.
The isolated corneas, after achieve normal metabolic activity (inductive incubation at 32 ± 1°C for one hour), were examined again. The corneas that show macroscopic tissue damage (e.g., scratches, pigmentation, neovascularization) or an opacity >7 opacity units were discarded.

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 μl of suspension
2g of the test substance was suspended in 10 mL of 0.9% sodium chloride solution.

Duration of treatment / exposure:

4 hrs

Duration of post- treatment incubation (in vitro):

1.5 hr

Number of animals or in vitro replicates:

The results were based on the selection criteria for the eyes, as well as the positive and negative control responses.
Number of corneas per group:
Exposed group (test substance) - 3 corneas (No. 1, 4, 6)
Positive control group (20% Imidazole) – 3 corneas (No. 12, 13, 14)
Negative control group (0.9% NaCl) – 3 corneas (No. 2, 3, 9)

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Selection criteria for eyes used in BCOP:
Only corneas from eyes free of defects including scratched, and neovascularisation were used.
The isolated corneas, after achieve normal metabolic activity (inductive incubation at 32 ± 1°C for one hour), were examined again. The corneas that show macroscopic tissue damage (e.g., scratches, pigmentation, neovascularization) or an opacity >7 opacity units were discarded.

Preparation:
Corneas free of defects were dissected with a 2 to 3 mm rim of sclera remaining to assist in subsequent handling, with care taken to avoid damage to the corneal epithelium an endothelium. Isolated corneas were mounted in specially designed corneal holders that consisted of anterior and posterior compartments, which interfaced with the epithelial and endothelial sides of the cornea, respectively. Both chambers were filled to excess with pre-warmed Eagle's Minimum Essential Medium (EMEM). The device was then equilibrated at 32 ± 1°C for at least one hour in water bath to allow the corneas to equilibrate with the medium and to achieve normal metabolic activity, to the extent possible. Following the equilibration period, fresh pre-warmed EMEM was added to both chambers and baseline opacity readings were taken for each cornea. Any corneas that showed macroscopic tissue damage (e.g., scratches, pigmentation, neovascularization) or an opacity >7 opacity units were discarded.
Each test group (test substance, concurrent negative and positive controls) consisted of the three eyes. The three corneas with opacity values close to the median value for all corneas were selected as negative control corneas. The remaining corneas were then distributed into treatment and positive control groups.

QUALITY CHECK OF THE ISOLATED CORNEAS
From 22 eyes the 3 eyes were eliminated after inductive incubation, because the baseline opacity values were >7. Nine corneas were used for the study (the corneas No. 1, 2, 3, 4, 6, 9, 12, 13 and 14), 7 eyes was superfluous and remaining 3 were used for the testing of another substance.

NUMBER OF REPLICATES
Number of corneas per group:
Exposed group (test substance) - 3 corneas (No. 1, 4, 6)
Positive control group (20% Imidazole) – 3 corneas (No. 12, 13, 14)
Negative control group (0.9% NaCl) – 3 corneas (No. 2, 3, 9)

NEGATIVE CONTROL USED
0.9% NaCl
SOLVENT CONTROL USED (if applicable)
0.9% NaCl
POSITIVE CONTROL USED
20% Imidazole

APPLICATION DOSE AND EXPOSURE TIME
750 µL of application form (2g of the test substance was suspended in 10 mL of 0.9% sodium chloride solution).
4 hrs

TREATMENT METHOD: closed-chamber method

POST-INCUBATION PERIOD: yes/no. If YES please specify duration


REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period:
After the exposure period, the negative control and the positive control substance was removed from the anterior chamber with EMEM (containing phenol red - the effectiveness of rinsing acidic or alkaline materials). The corneas were given a final rinse with EMEM (without phenol red). The EMEM (without phenol red) was used as a final rinse to ensure removal of the phenol red from the anterior chamber prior to the opacity measurement. The anterior chamber was then refilled with fresh EMEM without phenol red. The opacity and permeability of each cornea were recorded.

The test substance was removed from the anterior chamber with EMEM (containing phenol red) and sequentially was removed mechanically using a cotton swab (the test substance was coloured and had tendency to stick on the cornea surface) ; also the test substance was removed from the anterior chamber with EMEM (containing phenol red) once more. The corneas (applied the test substance) were also repeatedly rinsed with EMEM (without phenol red). Rinsing was finalized after complete removal of the test substance.
The anterior chamber was then refilled with fresh EMEM without phenol red. The opacity and permeability of each cornea were recorded.
- POST-EXPOSURE INCUBATION: application of sodium fluorescein (5 mg/ml), incubation 1.5 hour (32 ± 1°C)


METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:
measured quantitatively with the aid of an opacitometer (Opacitometer, MC2 - Le spécialiste du laboratoire – France) resulting in opacity values measured on a continuous scale

- Corneal permeability:
The amount of sodium fluorescein that crosses into the posterior chamber was quantitatively measured with the aid of UV/VIS spectrophotometry (Spectrophotometer GENESYSTM 10 UV/VIS Scanning). The values of absorbance measured at 490 nm were recorded as optical density (OD490) values. This term was used because the measuring is performed with visible light spectrophotometer using a standard 1 cm path length.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
IVIS = mean opacity value + (15 x mean permeability OD490 value)

DECISION CRITERIA:
IVIS UN GHS
≤ 3 No Category
> 3; ≤ 55 No prediction can be made
≥ 55 Category 1

Irritation parameter:

in vitro irritation score

Value:

9.3

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

other: no prediction can be made

Conclusions:

The In Vitro Irritancy Score (IVIS) for Reactive Blue 234 was 9.30.
This value of IVIS is > 3 and simultaneously ≤ 55 therefore the classification according to UN GHS criteria for eye irritation or serious eye damage is: no prediction can be made.

Executive summary:

The test substance, Reactive Blue 234, was tested for the evaluation the potential ocular corrosivity or severe irritancy as measured by its ability to induce opacity and increased permeability in an isolated bovine cornea.

The test was performed according to the OECD Test Guideline No. 437, Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage, Adopted 26th July 2013.

 

The test was performed using nine isolated bovine corneas. The testing was performed on three groups of corneas: test substance treatment group, positive control group and negative control group. Three corneas per group were used.

Closed-chamber method was used, because the test substance was applicable by micropipette. The opacity and permeability of each cornea were measured. The In Vitro Irritancy Score (IVIS) was calculated from the values of opacity and permeability.

 

The In Vitro Irritancy Score (IVIS) for Reactive Blue 234 was 9.30.

This value of IVIS is > 3 and simultaneously ≤ 55 therefore the classification according to UN GHS criteria for eye irritation or serious eye damage is: no prediction can be made.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Feb - March 1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline available

Principles of method if other than guideline:

- Principle of test: not specified
- Short description of test conditions: not specified
- Parameters analysed / observed: damage of cornea and mucosa (hyperemia, edema)

GLP compliance:

no

Species:

rabbit

Strain:

New Zealand White

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

0.1 g of sample

Duration of treatment / exposure:

not specified

Observation period (in vivo):

24, 72 h

Number of animals or in vitro replicates:

1

Irritation parameter:

overall irritation score

Basis:

animal #1

Time point:

24 h

Score:

ca. 1

Reversibility:

fully reversible within: max. 48 h

Remarks on result:

other: very weak hyperemia

Irritation parameter:

overall irritation score

Basis:

animal #1

Time point:

72 h

Score:

0

Remarks on result:

no indication of irritation

Interpretation of results:

other: non-irritating to eyes

Conclusions:

non-irritating

Executive summary:

The tested substance Ostazinová námořnická modř H-5R is not irritating for rabbit eye.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The tested substance Reactive Blue 234 was non corrosive based on in vitro test and not irritating for rabbit intact skin or rabbit eye during in vivo tests therefore overall conclusion is non-irritating.