Disodium 3(or 5)-[[4-[(7-amino-1-hydroxy-3-sulphonato-2-naphthyl)azo]-1-naphthyl]azo]salicylate

Endpoint summary

Description of key information

As the in vitro eye irritation/corrosion tests did not provide unambiguous results, the in vivo test was carried out to determine the correct classification.

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:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

TISSUESThe reconstructed human epidermal model EpiDerm™ (EPI-200 ver. 2.0, MatTek, Ashland, USA); Lot No. 21675, kit A) has been obtained from MatTek Bratislava

Vehicle:

physiological saline

Details on test system:

PROCEDUREOn the day of receipt, EpiDerm tissues were conditioned by incubation to release transport stress related compounds and debris. After pre-incubations (1 and 18±3hours), tissues were topically exposed to the test chemicals for 1 hour. Three tissues were used per the test substance (C1), for the positive (PC) and negative (NC) controls. Tissues were then thoroughly rinsed, blotted to remove the test substance, and transferred to fresh medium. After 24±2 hours incubation period, the medium was replaced by fresh one. Tissues were incubated for another 18±2 hours. Afterwards, the MTT assay was 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 was extracted with 2.0 mL/tissue of isopropyl alcohol and the optical density of the extracted formazan was determined using a spectrophotometer at 570 nm. Detailed procedure is described in SOP M/48/3 (VUOS-CETA, 2011).OD570 MEASURINGOD570 (optical density at 570 nm) was measured on a spectrophotometer Libra S22. Isopropyl alcohol serves as a blank.NEGATIVE CONTROLThe absolute optical density of the negative control (NC) tissues (treated with sterile phosphate buffered saline) in the MTT test is an indicator of tissue viability obtained in the testing laboratory after shipping and storing procedures and under specific conditions of use. The assay meets the acceptance criterion if the mean OD570 of the NC tissues is ≥ 0.8 and ≤ 2.8.POSITIVE CONTROLA 5% solution sodium dodecyl sulphate in water is used as positive control (PC) and tested concurrently with the test chemicals. Concurrent means here the PC has to be tested in each assay, but not more than one PC is required per testing day. Viability of positive control should be within 95±1 % confidence interval of the historical data. The assay meets the acceptance criterion if the mean viability of PC tissues expressed as % of the negative control tissues is ≤ 20 %.

Control samples:

yes, concurrent vehicle

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL- Amount(s) applied: 25 mg

Duration of treatment / exposure:

1 hour

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Vehicle:

unchanged (no vehicle)

Irritation / corrosion parameter:

% tissue viability

Value:

94.4

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

see Table No. 1

Table No. 1: Viability values

	Treatment	OD(570)			Mean	SD	Relative viability (% NC)
		1	2	3
NC	PBS	2.076	2.058	1.934	2.023	0.063	100.0
	viability (% NC)	102.6	101.7	95.6	100.0	3.12
C1	270/15	1.767	2.065	1.897	1.910	0.122	94.4
	viability (% NC)	87.4	102.1	93.8	94.4	6.031
PC	5% SDS	0.058	0.049	0.062	0.056	0.005	2.8
	viability (% NC)	2.9	2.4	3.1	2.8	0.269

NC - negative control

PC - positive control

C1 - test substance

SD - Standard deviation

Interpretation of results:

GHS criteria not met

Conclusions:

Average viability of tissues treated by the test substance was 94.4 % of negative control average value. i.e. viability was > 50 %. The effect of the test substance was negative in EpiDermTM model. According to the classification criteria given in chapter 4.5. of this method, the test substance, Direct Black 51, is considered to have no category in regard to skin irritation.

Executive summary:

Test substance, Direct Black 51, was assayed for the in vitro skin irritation in human epidermal model EpiDerm™. The test was performed according to the EU Method B.46. In vitro skin irritation: Reconstructed human epidermis model test and Protocol for: In Vitro EpiDerm™ Skin Irritation Test For use with MatTek Corporation’s Reconstructed Human Epidermal Model EPI-200-SIT.

Average viability of tissues treated by the test substance was 94.4 %, i.e.viability was > 50 %.

The effect of the test substance was negative in EpiDerm™ model (the tissue was not damaged).

According to the classification criteria, the test substance is considered to have no category in regard to skin irritation.

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:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Details on test animals or tissues and environmental conditions:

TEST SYSTEM:Bovine eyes source: Breeding service CHOVSERVIS a.s., division TORO® Hlavečník, Hradec Králové, Czech RepublicEyes were collected by slaughterhouse employees. 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. 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).The time interval between collection of the eyes and use of corneas in the BCOP was minimized (typically collected and used on the same day). The results were based on the selection criteria for the eyes, as well as the positive and negative control responses. All eyes used in the assay were from the same group of eyes collected on a specific day.SELECTION CRITERIA FOR EYES USED IN BCOP:The eyes, once they arrive at the laboratory, were carefully examined for defects including scratched, and neovascularisation. Only corneas from eyes free of such defects 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. From 22 eyes the 6 eyes were eliminated after inductive incubation, because the baseline opacity values were >7. Nine corneas were used for the study, 4 eyes was superfluous and remaining 3 were used for the testing of another substance.NUMBER OF CORNEAS PER GROUP:Exposed group (test substance) - 3 corneasPositive control group (20% Imidazole) – 3 corneasNegative control group (0.9% NaCl) – 3 corneas

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 the solution in physiological saline solution- Concentration (if solution): 2g of the test substance was dissolved in 10 mL of 0.9% sodium chloride solution.VEHICLE- Amount(s) applied (volume or weight with unit): 2g of the test substance was dissolved in 10 mL of 0.9% sodium chloride solution- Concentration (if solution): 0.9% sodium chloride in water- Lot/batch no. (if required): 145068131

Number of animals or in vitro replicates:

NUMBER OF CORNEAS PER GROUP:Exposed group (test substance) - 3 corneasPositive control group (20% Imidazole) – 3 corneasNegative control group (0.9% NaCl) – 3 corneas

Details on study design:

PROCEDURE SCHEME:Selection of corneas, mounting in holders → incubation with EMEM 1 hour (32 ± 1°C) → removed EMEM, measurement of baseline opacity → treatment by positive and negative control substances and test substance (incubation 4 hour) → washing epithelium, measurement of opacity after application → application of sodium fluorescein (5 mg/ml), incubation 1.5 hour (32 ± 1°C) → measurement of absorbance (490 nm).PREPARATION OF THE EYES: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 and 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.CONTROL SUBSTANCES: - Positive control: physiological saline solution (0.9% NaCl solution in water)- Negative control: imidazole (CAS: 288-32-4) 20% solutionPOST-EXPOSURE: After the exposure period, the test substance, the negative control, or 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 test substance was removed multiplicity by reason of colouring of the test substance (black). 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. ENDPOINTS MEASURED:- Opacity: the amount of light transmission through the cornea. Corneal opacity was measured quantitatively with the aid of an opacitometer (Opacitometer, MC2 - Le spécialiste du laboratoire – France) resulting in opacity values measured on a continuous scale. - Permeability: the amount of sodium fluorescein dye that penetrates all corneal cell layers (i.e., the epithelium on the outer cornea surface through the endothelium on the inner cornea surface) measured indirectly using visible light spectrophotometry. 1 mL sodium fluorescein solution (5 mg/mL) was added to the anterior chamber of the corneal holder, which interfaced with the epithelial side of the cornea, while the posterior chamber, which interfaced with the endothelial side of the cornea, is filled with fresh EMEM. The holder was incubated in horizontal position for 1.5 hours at 32 ± 1 ºC. 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.EVALUATION OF RESULTS- Mean opacity:Opacity values of treated corneas were corrected by subtracting individual background opacity values and the mean opacity is calculated.- Mean permeability:Mean optical density value of treated corneas was corrected by subtracting the mean optical density value of negative control and the mean opacity is calculated. - IVIS calculation:Resulting mean opacity and OD490 values for each treatment group was combined in an empirically-derived formula to calculate an in vitro irritancy score (IVIS) for each treatment group as follows:IVIS = mean opacity value + (15 x mean permeability OD490 value)- Decision criteria:A substance that induces an IVIS ≥ 55.1 is defined as a corrosive or severe irritant. - Study acceptance criteria: A test is considered acceptable if the positive control gives IVIS that falls within one standard deviations of the current historical mean, which is to be updated at least every three months, or each time an acceptable test is conducted in laboratories where tests are conducted infrequently. The negative or solvent/vehicle control responses should result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneas treated with the respective negative or solvent/vehicle control.

Irritation parameter:

in vitro irritation score

Value:

48.61

Negative controls validity:

valid

Positive controls validity:

valid

Tab. No. 1: Permeability and opacity:

Group	Opacity					Permeability
	Baseline	Opacity after treatment	Difference	Mean difference	Mean Opacity (corrected)	OD490^a	Mean Permeability	Mean Permeability(corrected)
negative control (0.9% NaCl)	7	7	0	0.67	-	0.012	0.008	-
	6	7	1			0.011
	7	8	1			0.000
positive control (20% Imidazole)	7	62	55	54.67	54.00 (54.67 – 0.67)	2.068	1.893	1.885 (1.893 – 0.008)
	6	64	58			1.814
	7	58	51			1.796
Direct Black 51	5	40	35	41.00	40.33 (41.00 – 0.67)	0.417	0.560	0.552 (0.560 – 0.008)
	5	48	43			0.551
	6	51	45			0.710

^a - Values of permeability (Optical density at 490nm)

Tab. No. 2: IVIS values

Group	IVIS
	Calculation	Result
negative control (0.9% NaCl)	0.67 + 15 x 0.008	0.79
positive control (20% Imidazole)	54.00 + 15 x 1.885	82.28
Direct Black 51	40.33 + 15 x 0.552	48.61

Interpretation of results:

other: not corrosive (Category 1)

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

The In Vitro Irritancy Score (IVIS) for Direct Black 51 was 48.61. This is less than the limit value of IVIS (55.1), which means that the test substance was not identified as a corrosive or severe irritant.

Executive summary:

The test substance, Direct Black 51, 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 Method B.47 Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants, Council Regulation (EC) No.1152/2010, published in O.J. L 324, 2010.

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 Direct Black 51 was 48.61. This is less than the limit value of IVIS (55.1), which means that the test substance was not identified as a corrosive or severe irritant.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

MatTek 06/29/2015

Deviations:

yes

Remarks:

see Any other information on material and methods

GLP compliance:

yes (incl. QA statement)

Details on test animals or tissues and environmental conditions:

The reconstructed human cornea-like epithelial model EpiOcular (OCL-200 ver. 2.0) comes from MatTek, Bratislava, SK. The Reconstructed human Cornea-like Epithelium (RhCE) tissues are reconstructed from primary human cells, which have been cultured for several days to form a stratified, highly differentiated squamous epithelium, morphologically similar to that found in the human cornea. The EpiOcular RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinized surface, showing a cornea-like structure analogous to that found in vivo.Lots No. of tissues used for this test: 23744 (1st experiment) and 23754 (2nd experiment).

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 mg per tissueTEST MATERIAL - Amount(s) applied (volume or weight with unit): 50 mg per tissue

Duration of treatment / exposure:

6 ± 0.25 hours

Duration of post- treatment incubation (in vitro):

18 ± 0.25 hours

Number of animals or in vitro replicates:

4 tissues

Details on study design:

TEST PROCEDUREDirect MTT reduction - functional check in tubes Some test substances may interfere with the MTT endpoint if they are able to directly reduce MTT. Therefore, before exposure, functional check for this possibility is performed as follows: the test substance is added to 1 mL MTT medium (red) and incubated at the culture conditions for 1 hour. At the end of the exposure time, the presence and intensity of the staining (if any) is observed. If the solution changes colour from red to blue, other steps to correction have to be done.Colour interference Each interfering coloured test chemical is applied on at least two viable tissue replicates (colorant control), which undergo the entire testing procedure but are incubated with medium instead of MTT solution during the MTT incubation step to generate a non-specific colour (NSCliving) control. The colorant control needs to be performed concurrently to the testing of the coloured test chemical due to the inherent biological variability of living tissues. The true tissue viability is then calculated as the percent tissue viability obtained with living tissues exposed to the interfering test chemical and incubated with MTT solution minus the percent non-specific colour obtained with living tissues exposed to the interfering test chemical and incubated with medium without MTT, run concurrently to the test being corrected (%NSCliving).MTT testApplication: The test substance (50 mg of substance/surface ratio 39.7 mg/cm2) is placed directly atop to the tissue moistened with 20 µL of phosphate buffered saline (PBS). The material should cover the entire tissue surface. A single testing, composed of two replicate tissues, is run. Besides, two more tissues are used as colorant control.ProcedureOn the day of receipt, EpiOcular tissues are conditioned to release transport stress related compounds and debris by incubation in assay medium delivered by MatTek for test performance for 1 hour at standard culture conditions and, after media replacement, overnight (following 16-24 hours) also standard at culture conditions. After pre-incubations, tissues are wetted with 20 μl of PBS spread across entire tissue surface. After 30 minutes incubation tissues are topically exposed to the test chemical (50 mg per tissue) for 6 ± 0.25 hours. Four tissues (2 for MTT test 2 for colorant control) are used per test substance (TS) and two for the positive control (PC) and negative control (NC). At the end of the 6 ± 0.25 hours treatment time, the test articles should be removed by extensively rinsing the tissues with PBS brought to room temperature. After rinsing, tissues are immediately transferred to and immersed in 5 ml of previously warmed assay medium (room temperature) in a pre-labelled 12-well plate for a 25 ± 2 minute immersion incubation (post-soak) at room temperature. This incubation in assay medium is intended to remove any test article absorbed into the tissue. At the end of the post-soak immersion, each insert is removed from the assay medium, the medium is decanted off the tissue, and the insert is blotted on absorbent material and transferred to the appropriate well of the pre-labelled 6-well plate containing 1 ml of warm assay medium. The tissues should be incubated for 18±0.25 hours at standard culture conditions (post-treatment incubation). At the end of the post-treatment incubation, each insert is removed from the 6-well plate and gently blotted on absorbent material. Two tissues are placed into the 24-well plate containing 0.3 ml of MTT solution and incubated for 180±10 minutes at standard culture conditions. Two tissues undergo the same procedure with medium instead of MTT solution (colorant control).In the end of staining (incubation with medium) the bottom of all inserts is blotted on absorbent material, and inserts are then transferred to a pre-labelled 6-well plate containing 2 ml of isopropyl alcohol in each well so that no isopropyl alcohol is flowing into the insert. The plates are sealed with parafilm, and are either stored overnight at 2-8°C in the dark or immediately extracted. To extract the MTT, the plates are placed on an orbital plate shaker and shaken for 2 to 3 hours at room temperature. After extraction extracts are collected, mixed and two 200 μl aliquots are transferred to the appropriate wells of a pre-labeled 96-well plate for OD570 reading. OD570 measuring:OD570 is measured on a plate reader Biotek Epoch. Isopropyl alcohol serves as a blank. No external filter is used.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 two individual tissues exposed to the test substance is calculated – this value is, after correction, used for the comparison with limit value. Corrected negative control OD: negative control OD – ODBlk (blank) = 100% viabilityCorrected TS (test substance) OD: test substance OD - ODBlk

Irritation parameter:

other: % tissue viability

Value:

3.59

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritant / corrosive response data:

The percentage tissue viability cut-off value distinguishing classified from non-classified test chemicals is 60%. The test chemical is identified as not requiring classification and labelling according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60%. In this case no further testing in other test methods is required. The test chemical is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post exposure incubation is less than or equal (≤) to 60%.As the corrected viability of treated tissues was 3.59 %, i.e. viability was ≤ 60 %, the test substance should be evaluated as a potential skin irritant.

Table 1: Test results

Treatment		OD570 of tissues				Average viability (% NC)
		Tissue 1		Tissue 2
NC	water % NC	1.628	1.526	1.608	1.619	100.00
		102.05	95.66	100.80	101.49
C2	270/15 % NC	0.071	0.064	0.083	0.082	4.70
		4.45	4.01	5.20	5.14
C2-CC	270/15-CC % NC	0.017	0.015	0.019	0.020	1.11
		1.07	0.94	1.19	1.25
PC	99% MA % NC	0.339	0.313	0.380	0.355	21.74
		21.25	19.62	23.82	22.25

PC: positive control

NC: negative control

C2: tested material

CC: colorant control

Interpretation of results:

study cannot be used for classification

Conclusions:

Average viability of tissues treated by the test substance, Direct Black 51, was 4.70 % of negative control average value. The measured value was then corrected, due to red colour of the test substance. Corrected viability of treated tissues was 3.59 %, i.e. viability was ≤ 60 %. The effect of the test substance was positive in EpiOcular model (tissues were damaged). According to the classification criteria, the test substance, Direct Black 51, is identified as substance potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1).

Executive summary:

The test substance, Direct Black 51, was assayed for the in vitro eye irritation in human epidermal model EpiOcular. The test was performed according to the OECD Test Guideline No. 492: Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage. Details of the procedure are given in Protocol: EpiOcular Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals (MatTek 06/29/2015).

After pre-incubation and wetting of tissues, 50 mg of the test substance was placed directly atop to the tissue and it was spread on the entire tissue surface. Length of exposition was 6 hours at 37±1°C in humidified CO2 incubator (5±1% CO2). Two tissues were used for the test substance and every control. Two tissues more were used as colorant control to correction of possible colour interference, which was undergo the entire testing procedure excepting of incubation with MTT medium.

After removal of the test substance, tissues were post-soaked in medium for approximately 25 minutes and post-incubated for about 18 hours at culture conditions. 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 plate reader. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues. Direct MTT reduction by test substance was excluded in another study.

The assumed colour interference was solved with using of two colorant control tissues incubated by the same way as the other tissues except of colouring in MTT medium. Instead, these tissues were kept in assay medium. Average “viability” of the two tissues was then subtracted from viability of tissues incubated with MTT medium.

The first experiment was not successful due to high positive control value. The test must have been repeated. The presented results are based on the second experiment.

Average viability of treated tissues was 4.70 % (3.59 % after correction), i.e. viability was ≤ 60 %.

The effect of the test substance was positive in EpiOcular model (tissues were damaged).

According to the classification criteria, the test substance, Direct Black 51, is identified as substance potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1).

Further testing with other test methods will be required.

Reference 3

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

Adopted October 2, 2012

Deviations:

yes

Remarks:

In the study only non-opioid analgesic was used for systemic analgesia. This change was performed because of avoiding a negative side effects of opioid analgesic. No other deviations from study plan or test guidelines occurred during the study.

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS- Source: breeding farm VELAZ, Lysolaje, Czech Republic- Weight at study initiation: 2.40 - 2.90 kg- Housing: conventional animal room – individually in metallic cages - Diet (e.g. ad libitum): pelleted standard diet for experimental animals ad libitum- Water (e.g. ad libitum): tap water ad libitum ENVIRONMENTAL CONDITIONS- Temperature (°C): 20 ± 3°C, permanently monitored- Humidity (%): 30 – 70%, permanently monitored- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

0.1 g

Duration of treatment / exposure:

1 hour

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

3 animals

Details on study design:

REMOVAL OF TEST SUBSTANCE - Washing (if done): physiological saline solution - Time after start of exposure: 1 hour SCORING SYSTEM:CorneaOpacity: degree of density (readings should be taken from most dense area)0 - No ulceration or opacity1 - Scattered or diffuse areas of opacity; details of iris clearly visible2 - Easily discernible translucent area; details of iris slightly obscured3 - Nacrous area, no details of iris visible, size of pupil barely discernible4 - Opaque cornea, iris not discernible through the opacityIris0 - Normal1 - Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia; or injection; iris reactive to light (a sluggish reaction is considered to be an effect)2 - Hemorrhage, gross destruction, or no reaction to lightConjunctivae Redness (refers to palpebral and bulbar conjunctivae; excluding cornea and iris) 0 - Normal1 - Some blood vessels hyperaemic (injected)2 - Diffuse, crimson colour, individual vessels not easily discernible3 - Diffuse beefy redChemosisSwelling (refers to lids and/or nictating membranes)0 - Normal (no alterations)1 - Some swelling above normal2 - Obvious swelling with partial eversion of lids3 - Swelling with lids about half closed4 - Swelling with lids more than half closed TOOL USED TO ASSESS SCORE: hand-slit lamp

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

>= 0 - <= 1

Max. score:

3

Reversibility:

fully reversible within: 48 h

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

>= 0 - <= 1

Max. score:

3

Reversibility:

fully reversible within: 48 h

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

>= 0 - <= 1

Max. score:

3

Reversibility:

fully reversible within: 48 h

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Other effects:

No symptoms of systemic toxicity were observed in the animals during clinical observation in the test period and no mortality occurred.

Interpretation of results:

GHS criteria not met

Conclusions:

The following changes were observed on eye at 1 hour after application: conjunctivae – some blood vessels hyperaemic or diffuse and cornea - coloured by the test substance was observed in all rabbits. Coloured cornea and some blood vessels hyperaemic or diffuse of conjunctivae was observed in all rabbits 24 hours after application. These changes faded away after 48 hours after evaluation. No clinical signs of systemic intoxication were detected.Examination of eye irritation after single application demonstrated, that the test substance, Direct Black 51, is not irritating to eye of rabbit.

Executive summary:

The test substance, Direct Black 51, was tested for the assessment of eye irritation/corrosion effects using albino rabbit (New Zealand Albino breed). The test was performed according to the OECD Test Guideline No. 405 Acute Eye Irritation/Corrosion. Adopted October 2, 2012.

Before in vivo testing the sequential testing strategy as it is recommended in supplement to TG 405(2012) was respected.

The test was performed initially using one animal. Because no corrosive or severe irritating effects were observed in initial test, the response was confirmed using two additional animals.

The following changes were observed on eye at 1 hour after application: conjunctivae – some blood vessels hyperaemic or diffuse and cornea - coloured by the test substance was observed in all rabbits. Coloured cornea and some blood vessels hyperaemic or diffuse of conjunctivae was observed in all rabbits 24 hours after application. These changes faded away after 48 hours after evaluation. No clinical signs of systemic intoxication were detected.

Evaluation of results after single application demonstrated that the test substance, Direct Black 51, is not irritating to the eye of rabbit.

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

Based on the skin and eye irritation/corrosion test results, the test substance is not irritating to eyes and skin.