Reaction products of diazotised 2-amino-5-{[2-(sulfooxy)ethyl]sulfonyl}benzenesulfonic acid coupled with 4-amino-5-hydroxynaphthalene-2,7-disulfonic acid under acidic conditions, further coupled with diazotised reaction products of 2,4,6-trifluoro-1,3,5-triazine with 2-[(2-anilinoethyl)sulfonyl]ethyl hydrogen sulfate and 2,4-diaminobenzenesulfonic acid (1:1:1) under alkaline conditions, potassium sodium salts

Endpoint summary

Description of key information

No skin or eye-irritating effects were noted in in-vitro studies.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-10-04 until 2018-10-26

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

Qualifier:

according to guideline

Guideline:

other: • MatTek Corporation Protocol: In Vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200-SIT); Version 07 November 2014

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No. 640/2012, L 193, Part B. 46. “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method” (06 July 2012).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 13-16 July 2015, Date of Signature: 14 September 2015

Test system:

human skin model

Remarks:

human reconstructed epidermis model

Vehicle:

unchanged (no vehicle)

Details on test system:

Epi-200- SIT Kit (Lot No.: 28665):

1 Sealed 24-well plate, Contains 24 inserts with EpiDerm™ tissues on agarose

2 24-well plates, For MTT viability assay

8 6-well plates For storing inserts, or for topically applying test agents
1 bottle Serum-Free Assay Medium, DMEM-based medium
1 bottle DPBS Rinse Solution, for rinsing the inserts in MTT assay
1 vial 5% SDS Solution (TC-SDS-5%), Skin irritant reference chemical –


MTT-100 Assay Kit Components:

1 vial, 2 mL MTT concentrate
1 vial, 8 mL MTT diluent (supplemented DMEM), for diluting MTT concentrate prior to use in the MTT assay
1 bottle Extractant Solution (Isopropanol), for extraction of formazan crystals

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Negative Controls: 30 µL were applied to each of triplicate tissues
Positive Controls: 30 µL were applied to each of triplicate tissues
Test Material: Each approximately 25 mg (~ 39 mg/cm2 according to guideline) of the test item were applied to the tissues

Duration of treatment / exposure:

60 minutes.

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Species:

other:

Type of coverage:

other: topical

Preparation of test site:

other:

Vehicle:

other:

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissues

Value:

71.3

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

viable tissues

Value:

0.33

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

not examined

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Freeze killed tissues

Value:

2.37

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

not examined

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Non specific killed controls

Value:

0.46

Vehicle controls validity:

not examined

Negative controls validity:

not examined

Positive controls validity:

not examined

Other effects / acceptance of results:

Acceptance Criteria:
Criterion 1 (negative control): The absolute OD 570 nm of the negative control tissues in the MTT test is an indicator of tissue viability obtained after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is meeting the acceptance criterion if the mean OD570 of the negative control tissues is  0.8 and ≤ 2.8.
Criterion 2 (positive control): An assay is meeting the acceptance criterion if mean relative tissue viability of the positive control is  20%.
Criterion 3 (standard deviation): The SD of 3 identical replicates should be ≤ 18.
Criterion 4: OD values should not be below historically established boundaries.
Concurrent negative controls (NC) and positive controls (PC) will be used in each run to demonstrate that viability (with the NC), barrier function and resulting tissue sensitivity (with the PC) of the tissues are within a defined historical acceptance range.
Historical data and the quality certificate of the supplier of the test kit demonstrating its robustness are annexed to the report, including quality control data (determined by MatTek Corporation, 82105 Bratislava, Slovakia) of the respective EpiDermTM lot. According to the OECD TG 439, the acceptance limit of the ET50 should be between 4.77 hours and 8.7 hours after treatment with 1% Triton X-100 (QC batch release criteria).

Results after treatment with Reactive Blue F07 -0195 and the controls

					Mean	Mean OD	Mean	Relative		Mean
					OD	of 3 wells	OD of	Viability		Relative
		OD	OD	OD	of	blank	3 tissues	[%] Tissue		Viability
Treatment Group	Tissue No.	570 nm	570 nm	570 nm	3 Wells	corrected	blank corrected	1, 2 + 3*	Standard Deviation	[%]**
		Well 1	Well 2	Well 3
Blank		0.036	0.037	0.038	0.037
	1	2.028	2.108	2.148	2.095	2.058		105.412
Negative Control	2	1.906	1.917	1.886	1.903	1.866	1.952	95.587	5.0	100.0
	3	1.985	1.969	1.955	1.970	1.933		99.001
	1	0.090	0.095	0.095	0.093	0.057		2.894
Positive Control	2	0.103	0.099	0.097	0.100	0.063	0.060	3.224	0.2	3.08
	3	0.099	0.098	0.098	0.098	0.061		3.131
	1	1.504	1.611	1.669	1.595	1.558		79.811
Test Item	2	1.321	1.383	1.378	1.360	1.323	1.381	67.789	8.0	71.30***
	3	1.281	1.298	1.320	1.300	1.263		64.690
Blank		0.038	0.037	0.037	0.037
Negative Control	1	0.045	0.045	0.046	0.045	0.008	0.009	0.410	0.1	0.470
Viable Tissues	2	0.049	0.047	0.047	0.048	0.010		0.529
Test Item	1	0.043	0.043	0.044	0.043	0.006	0.006	0.299	0.0	0.33
Viable Tissues	2	0.044	0.044	0.045	0.044	0.007		0.352
Negative Control	1	0.091	0.093	0.092	0.092	0.055	0.054	2.804	0.0	2.78
Freeze killed Tissues	2	0.091	0.089	0.093	0.091	0.054		2.747
Test Item	1	0.085	0.084	0.085	0.085	0.047	0.046	2.421	0.1	2.37
Freeze Killed Tissues	2	0.083	0.082	0.083	0.082	0.045		2.312
Test Item NSKC	1	0.045	0.046	0.045	0.045	0.008	0.009	0.417	0.1	0.460
	2	0.043	0.046	0.052	0.047	0.010		0.497

*                      Relative viability [rounded values]:            

**                    Mean relative viability [rounded values]:   

***                  corrected value

The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water resulted in blue colour due to the intrinsic colour of the test item itself.

Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did showed blue/purple colour.

The mean relative viability of the test item, corresponding to cell viability, decreased to71.3% (threshold for irritancy:≤50%), consequently the test item was declared as non-irritant to skin. 

Interpretation of results:

GHS criteria not met

Remarks:

not irritant to skin according to UN GHS and EU CLP regulation

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, Reactive Blue F07-0195 is not irritant to skin according to UN GHS and EU CLP regulation.

Executive summary:

This in vitro study was performed to assess the irritation potential of Reactive Blue F07-0195 by means of the Human Skin Model Test.

The test item reduced MTT (test for direct MTT reduction), and its intrinsic colour was intensive (test for colour interference). Consequently, additional tests with freeze-killed and viable tissues were necessary.

Each three tissues of the human skin model EpiDerm^™were treated with the test item, the negative control (DPBS) or the positive control (5% SDS) for 60 minutes.

After treatment with the negative control the absorbance values were well within the required range of the acceptability criterion of mean OD³0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues.

Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 60 minutes treatment interval, thus assuring the validity of the test system.

After treatment with the test item Reactive Blue F07-0195 the mean relative viability value decreased to 71.3% compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

In conclusion, it can be stated that in this study and under the experimental conditions reported, Reactive Blue F07-0195 is non-irritant to skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Experimental start date 26 November 2018 Experimental completion date 26 November 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

chicken

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Collection and Transport of Eyes to the Laboratory
Multiple chicken heads (Spring chickens (Gallus Gallus e.g. Ross 308 Broiler)), were supplied by Baileys Turkeys Ltd., Cheshire, UK. The chickens weighed approximately 3 kg and were approximately 56 days old prior to being humanely killed for human consumption.

Heads were removed immediately after the chickens had been humanely killed at the source, for use on the same day. The time interval between collection of chicken heads and placing the eyes in the superfusion chamber following enucleation was minimized although all eyes had to fall within the acceptance criteria identified in the test guideline.

Following slaughter, the intact chicken heads were placed into individual plastic compartments within a plastic box in order to minimize any damage to the eyes. The base of each compartment was lined with a paper towel moistened with isotonic saline. The heads were transported to the test facility at ambient temperature.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

0.03g of test item

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

Treated corneas were evaluated prior to treatment and at 30, 75, 120, 180 and 240 minutes (±5 minutes) after the eyes had been decontaminated with the isotonic saline.

Number of animals or in vitro replicates:

The test item and positive control item groups consisted of three eyes and the negative control item group consisted of two eyes.

Details on study design:

Eye Preparation
Eyes that had a high baseline fluorescein staining (>0.5) or corneal opacity score (>0.5) after the enucleation process were rejected.

Eyelids were carefully excised whilst taking care not to damage the cornea. The integrity of the cornea was measured with a drop of 2% (w/v) sodium fluorescein to the surface of the cornea and then rinsed with isotonic saline after a few seconds. The treated eyes were examined with the use of the Haag-Streit BQ 900 (Switzerland) microscope, to examine for damage to the cornea. An acceptable eye for the ICE test was one where the fluorescein retention and corneal opacity scores are ≤ 0.5.

Acceptable eyes were dissected from the skull and pulled from the orbit by holding the nictitating membrane firmly with surgical forceps. The tissue behind the eye was carefully removed with bent, blunt tipped scissors. Once the eye was removed from the orbit a portion of the optic nerve remained. Other connective tissue was removed from the eye on an absorbent tray liner.

Enucleated eyes were transferred to an appropriate clamp keeping the cornea vertical. They were then transferred to chambers within the superfusion apparatus ensuring the corneas received sufficient isotonic saline from the saline drip. The temperature of the chambers was at 32 ±1.5 °C.

Once all eyes were placed in the superfusion apparatus, the eyes were examined again with the Haag-Streit BQ 900 to ensure the eyes had not been damaged by the dissection procedure. Corneal thickness measurements are taken with a depth measuring device no. 1 on the Haag-Streit BQ 900 slit lamp microscope at the center of each cornea.

Eyes were replaced when: (i) the fluorescein score was > 0.5; (ii) the corneal opacity score was > 0.5; or (iii) there was any additional signs of damage, (iv) the corneal thickness measurements for individual eyes deviated more than 10% from the mean value for all eyes.

After the approval process the eyes were incubated for approximately 45 minutes for equilibrium purposes. Time zero measurements for corneal thickness and opacity were taken to serve as a baseline. The baseline for the fluorescein measurements were taken at dissection.

Application of Test Item, Negative and Positive Control Items
The test item and positive control item groups consisted of three eyes and the negative control item group consisted of two eyes.

Immediately following the zero reference measurements, each test eye (including clamp) was removed from the superfusion apparatus and placed horizontally (cornea facing upwards) into a petri dish. 0.03 g of test item was applied to the cornea. The entire surface of the cornea was evenly covered. The test item remained in place for 10 seconds and was then rinsed from the eye using 20 mL of isotonic saline. The treated eye (including clamp) was subsequently returned to the superfusion apparatus in the original upright position.

0.03 g of the positive control item, Imidazole, was similarly applied to the cornea of each positive control eye and 0.03 mL of the negative control item was applied to the cornea of each negative control eye.

Irritation parameter:

cornea opacity score

Run / experiment:

Test item

Value:

0.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class II

Irritation parameter:

fluorescein retention score

Run / experiment:

Test item

Value:

0.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class II

Irritation parameter:

other: Corneal swelling compared to time zero

Run / experiment:

Test item

Value:

5.07

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class II

Other effects / acceptance of results:

Corneal Opacity Scores
Very faint opacity was noted in the negative control treated eyes.

‘Complete corneal opacity; iris invisible’ was noted in all positive control treated eyes.

Very faint opacity was noted in one test item treated eye.

‘Scattered or diffused areas; details of the iris are clearly visible’ of opacity were noted in two test item treated eyes.

No morphological effects were noted in the test item, positive or negative control item treated eyes.

Fluorescein Retention Scores
No fluorescein retention was noted in one negative control treated eye during the study period.

Very minor single cell staining was noted in one negative control treated eye.

‘Confluent large areas of the cornea retaining fluorescein’ was noted in all positive control treated eyes.

Very minor single cell staining was noted in two test item treated eyes.

‘Single cell staining scattered throughout the treated area of the cornea’ was noted in one test item treated eye.

  Ocular Reactions

The ocular reactions observed in treated eyes were as follows:

Test Item
Maximal mean score for corneal opacity	:	0.8	ICE Class II
Mean score of Fluorescein Retention	:	0.7	ICE Class II
Maximal mean corneal swelling compared to time zero	:	5.07%	ICE Class II
Positive Control Item
Maximal mean score for corneal opacity	:	4.0	ICE Class IV
Mean score of Fluorescein Retention	:	3.0	ICE Class IV
Maximal mean corneal swelling compared to time zero	:	26.36%	ICE Class III
Negative Control Item
Maximal mean score for corneal opacity	:	0.5	ICE Class I
Mean score of Fluorescein Retention	:	0.3	ICE Class I
Maximal mean corneal swelling compared to time zero	:	-3.42%	ICE Class I

Interpretation of results:

GHS criteria not met

Conclusions:

No prediction can be made following assessment of the data for all endpoints.

Executive summary:

Introduction

The study was performed to evaluate the possible corrosivity or severe irritancy potential of the test item as measured by its ability to induce toxicity in an enucleated chicken eye.

Method

0.03 g of thetest item was applied onto the cornea of each of three enucleated eyes. A further three enucleated eyes were treated with positive control item. A further two enucleated eyes were treated with negative control item. 

Results

Maximal ocular irritation observations recorded for the test item treated eyes were as follows:

Mean Corneal Opacity (ICE class)	Mean Fluorescein Retention (ICE class)	Mean Corneal Thickness compared to time zero % (ICE class)					Combination of the 3 Endpoints
		30 mins	75 mins	120 mins	180 mins	240 mins
0.8 (II)	0.7 (II)	1.38 (I)	-1.38 (I)	1.38 (I)	5.07 (II)	2.76 (I)
		(II)					3 x II
Classification:		No prediction can be made

 

Conclusion

No prediction can be madefollowing assessment of the data for all endpoints.

 

mins=Minutes following treatment

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

An in vitro study was performed to assess the irritation potential of Reactive Blue F07-0195 by means of the Human Skin Model Test. The test item reduced MTT (test for direct MTT reduction), and its intrinsic colour was intensive, consequently, additional tests with freeze-killed and viable tissues were conducted.

Each three tissues of the human skin model EpiDerm^™were treated with the test item, the negative control (DPBS) or the positive control (5% SDS) for 60 minutes.

After treatment with the negative control the absorbance values were well within the required range of the acceptability criterion of mean OD³0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues.

Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 60 minutes treatment interval, thus assuring the validity of the test system.

After treatment with the test item Reactive Blue F07-0195 the mean relative viability value decreased to 71.3% compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

In conclusion, it can be stated that in this study and under the experimental conditions reported, Reactive Blue F07-0195 is non-irritant to skin.

An in-vitro eye irritation study was performed to evaluate the possible corrosivity or severe irritancy potential of the test item as measured by its ability to induce toxicity in an enucleated chicken eye. 0.03 g of thetest item was applied onto the cornea of each of three enucleated eyes. A further three enucleated eyes were treated with positive control item. A further two enucleated eyes were treated with negative control item. 

Maximal ocular irritation observations recorded for the test item treated eyes were as follows:

Mean Corneal Opacity (ICE class)	Mean Fluorescein Retention (ICE class)	Mean Corneal Thickness compared to time zero % (ICE class)					Combination of the 3 Endpoints
		30 mins	75 mins	120 mins	180 mins	240 mins
0.8 (II)	0.7 (II)	1.38 (I)	-1.38 (I)	1.38 (I)	5.07 (II)	2.76 (I)
		(II)					3 x II
Classification:		No prediction can be made

  mins=Minutes following treatment

Based on the test results, no severe eye irritating effects were observed, however, according to the scoring dsystem of the test, no prediction can be made following assessment of the data for all endpoints. As structurally similar compounds as the chromophores of this substance were not irrtitating to the eye and a QSAR prediction using the main constituent of the test subtsance was negative, the information gained was considered sufficient to consider the test substance not an eye-irritant.