Registration Dossier

Administrative data

Description of key information

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:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
adopted: 28th July, 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Test substance name: Acid Brown 235
- Other name: Korostan Brown DGR
- Source and lot/batch No. of test material: 7012/2007
- Expiration date of the lot/batch: May 2018
- Appearance: dark brown powder
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
physiological saline
Details on test system:
TEST SYSTEM
The reconstructed human epidermal model EpiDerm (EPI-200, MatTek, Bratislava) 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.

DIRECT MTT REDUCTION - FUNCTIONAL CHECK IN TUBES
Some test substances may interfere with the MTT endpoint if it is able to directly reduce MTT. Therefore, functional check for this possibility was performed as follows: 25 mg of the test substance was added to 1 mL MTT medium (red) and incubated in the incubator (37±1°C, 5±1 % CO2, moistened) for 1 hour. At the end of the exposure time, the presence and intensity of the staining (if any) were observed. If the solution changed colour from red to blue, other steps (test in frozen tissues) to correction have to be done.

COLOUR INTERFERENCE
To identify potential interference by coloured test chemical and decide on the need for additional controls, OD570 (optical density at 570 nm) of the test chemical in water (environment during exposure) and/or isopropyl alcohol (extracting solution) is measured.
25 mg of the test substance was mixed with 2 mL of water for injection and shaken for 2-3 hours. The same procedure was performed for isopropyl alcohol. After that, test tubes were centrifuged and decanted. OD570 of supernatant was then measured. If, after subtraction of the OD for isopropanol, the OD of the test article solution is > 0.08 (approximately 5% of the mean viability of the negative control) the test substance has to be considered as possibly interacting with the MTT measurement and an additional test on colorant controls has to be performed.

MTT TEST
Application:
25 mg of the test substance (25 mg of substance/surface ratio 39.7 mg/cm2) was dosed directly on tissue moistened with 25 µL of PBS (phosphate buffered saline). The substance was spread on the tissue surface. A single test, composed of three replicate tissues, was run.
Procedure:
On the day of receipt, EpiDerm tissues were conditioned by incubation to release transport stress related compounds and debris. Duration of pre-incubations before treatment was 60 minutes.
After pre-incubations, tissues were topically exposed to the test chemicals for 60±1 minutes (25 minutes at room temperature and the remaining 35 minutes at culture conditions). Three tissues were used per the test substance, three for the positive (PC) and three for the negative (NC) controls. Tissues are then thoroughly rinsed with PBS. Inserts were then transferred to fresh medium.
After 23 hours and 48 minutes of post-incubation period, the medium was replaced by fresh one. Tissues were incubated for another 18 hours, 21 minutes. Afterwards, the MTT assay was performed by transferring the tissues to 24-well plates containing MTT medium (1 mg/mL). After 185±5 minutes MTT incubation, the blue formazan salt formed by cellular mitochondria was extracted with 2.0 ml/tissue of isopropyl alcohol (for minim. 120 minutes, room temperature, shaking) and the optical density of the extracted formazan was determined using a spectrophotometer at 570 nm.
OD570 measuring:
OD570 was measured on a spectrophotometer Libra S22. Isopropyl alcohol served as a blank. Allowed band width is 2-3 nm. No external filter was 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 three individual tissues exposed to the test substance is calculated – this value is used for the comparison with limit.
Control samples:
yes, concurrent vehicle
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg Acid Brown 235

VEHICLE
- Amount(s) applied (volume or weight with unit): 25 µL phosphate buffered saline

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): phosphate buffered saline

POSITIVE CONTROL
- Concentration (if solution): 5% sodium dodecyl sulphate
Duration of treatment / exposure:
60 min
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Value:
94
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
DIRECT MTT REDUCTION - FUNCTIONAL CHECK IN TUBES
25 mg of the test substance was added to 1.0 mL of MTT medium. Suspension was incubated for 1 hour at culture conditions. After incubation, the medium was brown-red coloured. The test substance does not reduce MTT directly.

COLOUR INTERFERENCE
The test substance is soluble in water for injection. OD570 of solution in water for injection was >3. The test substance is not soluble in isopropyl alcohol. Average OD570 value from 2 wells was 0.008 what is < 0.08. On the basis of results obtained, it was decided do not use concurrent colorant control in the MTT test.

ACCEPTANCE CRITERIA FULFILMENT
The mean OD570 of the NC tissue was 2.373 ± 0.115 which meets the acceptance criteria of ≥ 0.8 and ≤ 2.8.
The mean viability of the PC tissues expressed as % of the negative control tissues is 3.3 % which meets the acceptance criterion of ≤ 20 %.
The SD calculated from individual % tissue viabilities of the 3 identically treated replicates for the positive control, negative control and test substance was < 18 %.
All study acceptance criteria were fulfilled.

Table 1: Optical density and viability

 

Treatment

OD570

Avg

SD

Average viability

(% NC)

1

2

3

NC

PBS

2.450

2.458

2.211

2.373

0.115

100.0

%

103.2

103.6

93.2

100.0

4.8

C4

390/16

2.412

2.187

2.096

2.232

0.133

94.0

%

101.6

92.2

88.3

94.0

5.6

PC

5% SDS

0.063

0.087

0.082

0.077

0.010

3.3

%

2.7

3.7

3.5

3.3

0.4

NC - negative control

PC - positive control

C4 - test substance

SD - standard deviation

SDS - sodium dodecyl sulphate

Interpretation of results:
GHS criteria not met
Conclusions:
Under the test conditions, average viability of tissues treated by the test substance, Acid Brown 235, was 94.0 % of negative control average value i.e. viability was > 50 %.
The effect of the test substance was negative in EpiDerm model.
According to the classification criteria given in this assay, the test substance, Acid Brown 235, is considered to have no category in accordance with UN GHS and is therefore considered a non-irritant.
Executive summary:

The test substance, Acid Brown 235, was assayed for in vitro skin irritation in the human epidermal model EpiDerm.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 Skin Irritation Test For use with MatTek Corporation’s Reconstructed Human Epidermal Model EPI-200-SIT.

In preliminary experiment neither colour interference with the endpoint nor direct MTT reduction were found.

After pre-incubation of tissues, 25 mg of the test substance was placed directly on previously moistened tissue and spread on the entire tissue surface. The length of exposure was 60 minutes. Three tissues were used for the test substance and for positive and negative controls.

After removal of the test substance, tissues were post-incubated for approximately 42 hours. 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.

Under the experimental design average viability of treated tissues was 94.0%, i.e. viability was >50 %.

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

According to the classification criteria, thetest substance, Acid Brown 235, 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

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21.09.-22.09.2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
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. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Test substance name: Acid Brown 235
- Other name: Korostan Brown DGR
- Source and lot/batch No. of test material: 7012/2007
- Expiration date of the lot/batch: May 2018
- Appearance: dark brown powder
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 Republic
Eyes were collected by slaughterhouse employees. The eyes were enucleated as soon as possible after death. No detergent was used. Only healthy animals (12 to 30 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 a baseline opacity >7 opacity units were discarded.
From 25 eyes the 5 eyes were eliminated after inductive incubation, because the baseline opacity values were >7. Nine corneas were used for the study, 8 eyes were superfluous and the remaining 3 eyes were used for the testing of another substance.
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): 2 g
- Concentration (if solution): 20 % w/v

VEHICLE
- Amount(s) applied (volume or weight with unit): 10 mL (0.9% NaCl in water)
Duration of treatment / exposure:
4 hours
Number of animals or in vitro replicates:
Exposed group (test substance) – 3 corneas
Positive control group (20% Imidazole in 0.9% NaCl) – 3 corneas
Negative control group (0.9% NaCl) – 3 corneas
Details on study design:
PROCEDURE SCHEME
- Selection of corneas, mounting in holders
- Incubation with EMEM (Eagle's Minimum Essential Medium) 1hour (32 ± 1°C)
- Removed EMEM, measurement of baseline opacity
- Treatment by positive and negative control substances and test substance (incubation 4 hours)
- Washing of 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 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.

APPLICATION OF THE TEST SUBSTANCE
Treatment protocol for non-surfactant solids was used. Non-surfactant solids are typically tested as solutions or suspensions at 20% concentration.
Application form preparation:
The test substance was tested at 20% concentration in a 0.9% sodium chloride solution.
2 g of the test substance was suspended in 10 mL of 0.9% sodium chloride solution.
Closed-chamber method was used, because the test substance was applicable by micropipette. The test substance (750 µL of application form) to cover the epithelial side of the cornea is introduced into the anterior chamber through the dosing holes on the top surface of the chamber, and the holes were subsequently sealed with the chamber plugs during the exposure.

CONTROL SUBSTANCES
Concurrent negative controls and positive controls were included in experiment. The controls were included in the BCOP test method so that nonspecific changes in the test system could be detected and to provide a baseline for the assay endpoints.

POST-EXPOSURE
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) – repeatedly, because the test substance is coloured. Subsequentially the test substance was removed mechanically using a cotton swab and brush. The corneas (applied the test substance) were also rinsed with EMEM (without phenol red). Lastly EMEM (without phenol red) was used for final rinsing. The test substance was complete removal, but corneas stayed coloured by the test substance. The anterior chamber was then refilled with fresh EMEM without phenol red. The opacity and permeability of each cornea were recorded.

EXPERIMENTAL MEASUREMENTS
- 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°C.
The amount of sodium fluorescein that crosses into the posterior chamber was quantitatively measured with the aid of UV/VIS spectrophotometry (Spectrophotometer GENESYS 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.
- Mean opacity:
Opacity values of treated corneas were corrected by subtracting individual background opacity values and the mean opacity is calculated.
- Mean permeability:
Mean OD value of treated corneas was corrected by subtracting the mean OD value of negative control and the mean permeability 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)
Irritation parameter:
in vitro irritation score
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable

Table 1: Opacity values

Group

Baseline opacity

Opacity after treatment

Opacity difference

Mean

opacity difference

Mean Opacity (corrected)

Negative Control

(0.9% NaCl)

5

6

1

1.00

-

5

6

1

6

7

1

Positive Control

(20% Imidazole in 0.9% NaCl)

7

61

54

52.00

(52.00 – 1.00)

51.00

4

52

48

4

58

54

Test Substance

(Acid Brown 235)

5

199

194

not evaluated

-

7

189

182

6

199

193

Interpretation of results:
study cannot be used for classification
Conclusions:
The In Vitro Irritancy Score (IVIS) for test substance, Acid Brown 235, could not be calculated. The basic values for the IVIS calculation (opacity values) could not be measured because of colouring of corneas by the test substance which were applied. For this reason the classification was not performed.
Executive summary:

The test substance, Acid Brown 235, 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 test substance, Acid Brown 235, could not be calculated. The basic values for the IVIS calculation (opacity values) could not be measured because of colouring of corneas by the test substance which were applied. For this reason the classification was not performed.

Endpoint:
eye irritation: in vitro / ex 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:
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:
06/29/2015
Deviations:
yes
Remarks:
see Any other information on materials and methods
GLP compliance:
yes (incl. certificate)
Species:
other: reconstructed human cornea-like epithelial model EpiOcular
Details on test animals or tissues and environmental conditions:
The 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.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
50 mg
Duration of treatment / exposure:
6 ± 0.25 hours
Duration of post- treatment incubation (in vitro):
18 hours
Number of animals or in vitro replicates:
3 replicates
Details on study design:
CHEMICALS AND MEDIA
Chemicals:
- phosphate buffered saline (PBS) (prepared in laboratory 04/04/2017)
- 3-(4,5-Dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Lot No. 10196058)
- isopropyl alcohol (Lot No. PP/2016/04778)
Positive control:
- methyl acetate (Lot No. 032817ISA)
Negative control:
- water for injection (Lot. No. 1508210536)
Media:
- EPI-100 (Lot No. 041717MGKA)

TEST SYSTEM
The reconstructed human cornea-like epithelial model EpiOcular (OCL-200 ver. 2.0) comes from MatTek, Bratislava, SK.
The 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: 23776 kit A.

DIRECT MTT REDUCTION – FUNCTIONAL CHECK IN TUBES
The test was performed as a part of Study No. 323/16/4AI: Acid Brown 235 - In vitro Skin Irritation Test (EpiDerm Model).
The test substance does not reduce MTT directly.

COLOUR INTERFERENCE
The test was performed as a part of Study No. 323/16/4AI: Acid Brown 235 - In vitro Skin Irritation Test (EpiDerm Model).
The test substance does not interfere with the endpoint.

MTT TEST
Application:
The test substance (50 mg of substance/surface ratio 39.7 mg/cm2) was placed directly atop to the tissue moistened with 20 µL of PBS. The material should cover the entire tissue surface. A single testing, composed of two replicate tissues, was run.
Procedure
On the day of receipt, EpiOcular tissues were 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 were wetted with 20 μl of PBS spread across entire tissue surface. After 30 minutes incubation tissues were topically exposed to the test chemical (50 mg per tissue) for 6 ± 0.25 hours. Three tissues were used per test substance (TS) and three for the positive control (PC) and three for the negative control (NC).
At the end of the 6 ± 0.25 hours treatment time, the test substance was removed by extensively rinsing the tissues with PBS brought to room temperature.
After rinsing, tissues were 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 was intended to remove any test substance absorbed into the tissue.
At the end of the post-soak immersion, each insert was removed from the assay medium, the medium is decanted off the tissue, and the insert was 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 were incubated for 18±0.25 hours at standard culture conditions (post-treatment incubation).
At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent material. Two tissues were placed into the 24-well plate containing 0.3 ml of MTT solution and incubated for 180±10 minutes at standard culture conditions.
In the end of staining (incubation with medium) the bottom of all inserts was blotted on absorbent material and inserts were then transferred to a pre-labelled 6-well plate containing 2 ml of isopropyl alcohol in each well so that no isopropyl alcohol was flowing into the insert. The plates were sealed with parafilm, and were extracted the same day. To extract the MTT, the plates were placed on an orbital plate shaker and shaken for 2 hours and 11 minutes at room temperature.
After extraction extracts were collected, mixed and two 200 μl aliquots from every well were transferred to the appropriate wells of a pre-labelled 96-well plate for OD570 reading.
OD570 measuring
OD570 was measured on a plate reader Biotek Epoch. Isopropyl alcohol serves as a blank. No external filter was used.

ASSAY ACCEPTANCE CRITERIA
1) The negative control OD > 0.8 and < 2.5,
2) The mean relative viability of the positive control is below 50% of control viability
3) The difference of viability between the three relating tissues of a single chemical is < 20% in the same run. This applies also to the killed controls and the colorant controls which are calculated as percent values related to the viability of the relating negative control.

EVALUATION OF RESULTS AND CLASSIFICATION
The percentage tissue viability cut-off value distinguishing classified from non-classified test chemicals is 60%. Results should thus be interpreted as follows:
- 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%.
Irritation parameter:
other: % optical density at 570 nm (OD570) compare with negative control
Value:
4.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid

Table 1: Optical density results

Treatment

OD570of tissues

average

[%]

average viability

[% NC]

Tissue 1

Tissue 2

Tissue 3

NC

1.522

1.261

1.558

1.447

100.0

% NC

105.21

87.17

107.70

100.0

test subst.

0.063

0.073

0.066

0.067

4.6

% NC

4.35

5.05

4.56

4.6

PC

0.858

0.309

0.247

0.471

32.6

% NC

59.31

21.36

17.07

32.6

NC - negative control

PC - positive control

Interpretation of results:
study cannot be used for classification
Conclusions:
Under the experimental conditions, average viability of tissues treated by the test substance, Acid Brown 235, was 4.6 % of negative control average value. The effect of the test substance was positive in EpiOcular model (tissues were damaged). According to the classification criteria given in test guidance, the test substance, Acid Brown 235, is identified as substance potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1).
Executive summary:

The test substance, Acid Brown 235, 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.

Tests for colour interference and direct reduction were performed as a part of another study. Direct reduction or colour interference were not found.

Under the experimental conditions, average viability of treated tissues was 4.6 % 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, Acid Brown 235, is identified as substance potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1).

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:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Version / remarks:
published in O.J. L. 112, 2017
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: breeding farm VELAZ s.r.o., Lysolaje, Czech Republic
- Weight at study initiation: 2.60 – 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): Drinking 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)
Amount / concentration applied:
0.1 g per animal
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:
Cornea (maximum possible: 4)
0 - no ulceration or opacity
1 - scattered or diffuse areas of opacity (other than slight dulling of normal lustre); details of iris clearly visible
2 - easily discernible translucent area; details of iris slightly obscured
3 - nacreous area; no details of iris visible; size of pupil barely discernible
4 - opaque cornea; iris not discernible through the opacity

Iris (maximum possible: 2)
0 - normal
1 - 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 light

Conjunctivae (maximum possible: 3)
0 - normal
1 - some blood vessels hyperaemic (injected)
2 - diffuse, crimson colour; individual vessels not easily discernible
3 - diffuse beefy red

Chemosis (maximum possible: 4)
0 - normal
1 - some swelling above normal
2 - obvious swelling, with partial eversion of lids
3 - swelling, with lids about half closed
4 - 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:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.333
Max. score:
3
Reversibility:
fully reversible within: 48 h
Irritation parameter:
chemosis 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:
iris score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
3
Irritation parameter:
chemosis 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:
iris score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
3
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritant / corrosive response data:
The initial irritation (1 hour after exposure) was caused by mechanical irritation effect of test substance petty particles.
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 – diffuse, crimson colour, individual vessels not easily discernible, and chemosis – obvious swelling with partial reversion of lids was observed in all rabbits.
Some hyperaemic blood vessels of conjunctivae were observed in one rabbit at 24 hours after application. No alterations were observed in other rabbits at 24 hours after application. No alterations were observed in all rabbits at 48 and 72 hours after application. All eye lesions disappeared at least at 48 hours after treatment. No clinical signs of systemic intoxication were detected.
Evaluation of results after single application demonstrated that the test substance, Acid Brown 235, is not irritating to the eye of rabbit.
The initial irritation (1 hour after exposure) is caused by mechanical irritation effect of test substance petty particles.
Executive summary:

The test substance, Acid Brown 235, was tested for the assessment of eye irritation/corrosion effects using albino rabbit (New Zealand Albino breed).

The test was performed according to EU Method B.5 (Acute Eye Irritation/Corrosion), Commission Regulation (EU) 2017/735, published in O.J.L. 112, 2017.

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. As 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 – diffuse, crimson colour, individual vessels not easily discernible, and chemosis – obvious swelling with partial reversion of lids was observed in all rabbits.

Some hyperaemic blood vessels of conjunctivae were observed in one rabbit at 24 hours after application. No alterations were observed in the other rabbits at 24 hours after application. No alterations were observed in all rabbits at 48 and 72 hours after application.

No clinical signs of systemic intoxication were detected.

Evaluation of results after single application demonstrated that the test substance, Acid Brown 235, is not irritating to the eye of rabbit. The initial irritation (1 hour after exposure) is caused by mechanical irritation effect of test substance petty particles.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Based on the test results and according to the CLP criteria for classification and labelling requirements for dangerous substances and preparations, the test substance, Acid Brown 235, did not fall into any of quoted categories of skin or eye irritation/corrosion, and has no obligatory labelling requirement in this respect.