m,m'-[carbonylbis[imino(3-methoxy-p-phenylene)azo]]bis(benzenesulphonic) acid, compound with 2,2'-iminodiethanol (1:2)

Endpoint summary

Description of key information

The skin irritancy of the test item Bayscript Yellow GGN was assessed with reconstructed human epidermis (RhE). The experiment was carried out in vitro using the commercially available test method epiCS® according to OECD TG 439 and EU Test Method B.46.

The corneal damage potential of the solid test item Bayscript Yellow GGN was assessed with the Bovine Corneal Opacity and Permeability test (BCOP) using fresh bovine cornea according to OECD TG 437.

Additionally an in vitro study for assessing ocular irritation properties of the test item Bayscript Yellow GGN was performed using the reconstructed human cornea-like epithelium (RhCE) cell model EpiOcular™. The model used is standardized and commercially available. The EpiOcular™ Eye Irritation Test (EIT) was conducted in accordance with OECD 492.

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

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)

Principles of method if other than guideline:

The study was performed for the assessment of the skin irritancy of the test item Bayscript Yell ow GGN with reconstructed human epidermis (RhE). The experiment was carried out in vitro using the commercially available test method epiCS®.

GLP compliance:

yes

Specific details on test material used for the study:

Test item: Bayscript Yellow GGN
Chemical name: Benzenesulfonic acid, 3,3' -( carbonylbis(imino(3-methoxy-4, 1-
phenylene)-2, 1-diazenediyl)bis-, compd. with 2,2'iminobis(ethanol) (1 :2)
CAS number: 71550-21-5
Molecular mass: 850,9 g/mol
pH-value, diluted 1:9 in water: 4.6
Content: 89.6%
Appearance: solid, crystalline, russet

Test system:

human skin model

Source species:

other: artificial 3D-Skin model

Cell type:

other: artificial 3D-Skin model (reconstructed human epidermis)

Cell source:

other: reconstructed human epidermis model epiCS® (CellSystems, Troisdorf, Germany)

Source strain:

other: artificial 3D-Skin model

Details on animal used as source of test system:

not applicable: artificial 3D-Skin model

Justification for test system used:

The model used for this study has a functional stratum corneum with an underlying layer of living cells as recommended by the test guidelines (e.g. OECD Test guideline 439).

Vehicle:

other: Test item was used undiluted.

Details on test system:

Test System/ Study Design
The model used for this study has a functional stratum corneum with an underlying layer of living cells as recommended by the test guidelines. The barrier function of the stratum corneum is adequate, as has been shown by the supplier.
The viability of the living cells in the model must be sufficiently high to discriminate well between the positive and negative control substances. Cell viability is measured by the amount of MTT reduction, i.e. an OD value, following exposure to the negative control substance or the test item.

Methods and Parameters
Reconstructed tissues
The experiment was carried out on the reconstructed human epidermis model epiCS® (CellSystems, Troisdorf, Germany). The tissue equivalents were shipped in 24 well cell culture plates on agarose supplemented with maintenance medium (Kit contents epiCS® CellSystems, Cat.-No.CS-1001). Inserts were of 0.6 cm² size.

Adaptation to cell culture conditions
Inserts with epiCS® reconstructed human epidermis (0.6 cm2) were packed under sterile conditions and were shipped refrigerated on supplemented agarose. Upon arrival, 6 well culture plates were pre-filled with 1 ml of fresh and cool maintenance medium. The reconstructed tissues were placed into the prepared cell culture plates (1 insert/well) and were adapted to the recommended tissue culture conditions (5% C02, 37°C, max humidity) afterwards for at least 6 hours before use.

4.4.3 Environmental conditions
The environmental conditions in the incubator were standardized as follows:
Incubator temperature 37 +/- 2°C
CO2 gas concentration 5%
Humidity maximum
Occasional deviations from these conditions occurred e.g. as a result of opening the incubators door. However, these deviations had no effect on the course or outcome of the study. All Incubation steps were performed in a CO2 atmosphere incubator.

Test item formulation
Test item was used undiluted.

Application of the test item and incubation
The epiCS® inserts were exposed to 30 mg of the test item (plus 30 J.tl 0.9% NaCl to moisten and ensure good contact with the epidermis surface) for 20 min. (RT, three inserts). 0.9% NaCl and 5% SDS (each 30 J.tl) treated epidermal models were used as negative and positive controls, respectively (determination in triplicates). A piece of mesh was used as a spreading aid for the test item.

Determination of cell viability (MTT assay)
After the exposure to the test item the inserts were washed carefully in PBS. After a post - treatment incubation period of 42 h of the rinsed tissue in the incubator a MTT assay was performed. For viability testing the inserts were placed in new 24 well plates containing 300 µl ofMTT solution (37°C, 1 mg/ml in MTT-assay medium, delivered by Cell Systems®). The tissues were incubated for about 3 hours under cell culture conditions (5% CO2, 37°C, max humidity). The extraction of blue formazan was performed in isopropanol (24 well plates, 2 ml per insert) on a vertical shaker (2 hours). The concentration of formazan was measured by determining the OD of the isopropanol-extracts in duplicates at 570 nm in an automatic reader.
The MTT reduction assay is the most frequently used assay for the determination of cell viability. The assay depends on the intracellular capacity ofliving cells to chemically reduce the yellow 3- [4,5-Dimethythiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) to blue formazan crystals. The test has shown to give accurate and reproducible results in various laboratories and has practically been modified for accurate analysis of cell viability in three dimensional skin models.

Control samples:

other: Negative control: NaCl 0.9%, Positive control: 5% SDS solution

Amount/concentration applied:

The epiCS® inserts were exposed to 30 mg of the test item (plus 30 µl 0.9% NaCl to moisten and ensure good contact with the epidermis surface) for 20 min. (RT, three inserts).

Duration of treatment / exposure:

The epiCS® inserts were exposed to 30 mg of the test item (plus 30 µl 0.9% NaCl to moisten and ensure good contact with the epidermis surface) for 20 min. (RT, three inserts).

Duration of post-treatment incubation (if applicable):

After the exposure to the test item the inserts were washed carefully in PBS. After a post - treatment incubation period of 42 h of the rinsed tissue in the incubator a MTT assay was performed. For viability testing the inserts were placed in new 24 well plates containing 300 µl ofMTT solution (37°C, 1 mg/ml in MTT-assay medium, delivered by Cell Systems®). The tissues were incubated for about 3 hours under cell culture conditions (5% CO2, 37°C, max humidity).

Number of replicates:

3 replicates.

Species:

other: not applicable: artificial 3D-Skin model

Strain:

other: not applicable: artificial 3D-Skin model

Type of coverage:

other: not applicable: artificial 3D-Skin model

Preparation of test site:

other: not applicable: artificial 3D-Skin model

Vehicle:

other: not applicable: artificial 3D-Skin model

Amount / concentration applied:

not applicable: artificial 3D-Skin model

Duration of treatment / exposure:

not applicable: artificial 3D-Skin model

Observation period:

not applicable: artificial 3D-Skin model

Number of animals:

not applicable: artificial 3D-Skin model

Details on study design:

not applicable: artificial 3D-Skin model

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Bayscript Yellow GGN

Value:

97.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

All acceptance criteria were met.
The test item Bayscript Yellow GGN did not show a significant impact on cell viability and is thus identified as non-irritant substance in this test model.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item Bayscript Yellow GGN did not show a significant impact on cell viability and is thus identified as non-irritant substance in this test model.

Executive summary:

The skin irritancy of the test item Bayscript Yellow GGN was assessed with reconstructed human epidermis (RhE). The experiment was carried out in vitro using the commercially available test method epiCS®.

The study was conducted in accordance with OECD TG 439 and EU Test Method B.46. The test item was applied undiluted topically to the RhE tissue construct in triplicates and incubated for 20 minutes, followed by a 42 hours post-treatment incubation period.

Cell viability was measured in a photometer by the amount ofMTT (methylthiazole tetrazolium) reduction. The optical density value obtained for the test item was used to calculate the percentage of viability relative to the negative control, which is set at 100%

The results of the concurrent negative control (NC, 0.9% NaCl) and positive control (PC, 5% SDS) demonstrated the viability (NC) and sensitivity (PC) of the test model.

The following value of cell viability was recorded for the test item: 97 % (rounded).

In conclusion the results of the assay used show no skin irritant properties of the test item Bayscript Yell ow GGN and thus, the test item requires no classification according to UN GHS (Category 2 or Category 1).

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Principles of method if other than guideline:

This study was performed to assess the corneal damage potential of the solid test item Bayscript Yellow GGN with the Bovine Corneal Opacity and Permeability test (BCOP) using fresh bovine cornea.
The study was conducted in accordance with international accepted Guidelines (e.g. OECD TG 437).

GLP compliance:

yes

Specific details on test material used for the study:

Test item: Bayscript Yellow GGN
Chemical name: Benzenesulfonic acid, 3,3' -( carbonylbis(imino(3-methoxy-4, 1-
phenylene)-2, 1-diazenediyl)bis-, compd. with 2,2'iminobis(ethanol) (1 :2)
CAS number: 71550-21-5
Molecular mass: 850,9 g/mol
pH-value, diluted 1:9 in water: 4.6
Content: 89.6%
Appearance: solid, crystalline, sorrel

Species:

other: Bovine Corneal Opacity and Permeability test (BCOP) using fresh bovine cornea.

Strain:

other: Bovine Corneal Opacity and Permeability test (BCOP) using fresh bovine cornea.

Details on test animals or tissues and environmental conditions:

Test System
Test system: isolated cornea from eyes of slaughtered cattle
Source: Slaughterhouse Laame, Buchenhofen 26, 42329 Wuppertal, Germany
Extraction: Staff of the slaughterhouse
Transport: 1 L containers with 500 mL HSS and 1 % penicillin I streptomycin solution, transport of the containers in coolers on ice. The test system matches with the guideline.

Vehicle:

other: The test item was suspended prior to application in physiologic saline solution to achieve a concentration of 20 % (w/v).

Controls:

other: Positive control: Imidaole; Negative control: Saline solution (0.9% NaCl).

Amount / concentration applied:

750 µL of the test material formulations (20% (w/v) in physiological saline) were applied to the corneas, each.

Duration of treatment / exposure:

4 hours

Duration of post- treatment incubation (in vitro):

None

Number of animals or in vitro replicates:

20% (w/v) concentration ofthe test item and the positive control imidazole in physiologic saline solution were tested on 3 bovine corneas each in comparison to the negative control (physiologic saline solution).

Details on study design:

Methods and Parameters
Preparation of Corneas
Eyes were examined after delivery to the laboratory for any damage (like opacity, scratches or neovascularization) on the day of slaughter (1 day before the experiment). Eyes without any visible defects were transferred into new containers with fresh HSS solution supplemented with 1 %penicillin I streptomycin solution and 1 % FBS and stored overnight at refrigerator temperature (2-8 °C). Eyes with defects were discarded.
On the next day (day of experiment) the containers with the eyes were transferred in an incubator at 32°C (± 1 °C) for about 2 hours.
For the preparation of the cornea the sclera of each eye was incised with a scalpel and cut by scissors. A 2-3 mm scleral edge was left around the cornea for further handling. The isolated corneas were placed with the epithelium side down into a prepared beaker filled with MEM medium supplemented with 1 %penicillin I streptomycin solution and 1 % FBS. Each cornea was placed into a cornea holder with the endothelial side facing the sealing ring of the posterior chamber. The anterior chamber was then fixed by screws on the other side. The chambers were filled with MEM medium, avoiding air bubbles. The holders so prepared were transferred for at least 1 hour in the incubator at 32°C (± 1 °C).

Selection of corneas for application
After approximately 1 hour, the MEM medium was aspirated and the chambers were filled with fresh MEM medium. For each cornea the reference opacity value was measured then. The mean and standard deviation of the measured values were calculated by using Microsoft Excel. The corneas with values within the range of mean ± 1 standard deviation were selected for the actual test and assigned to the test groups.
The numbers of the selected corneas selected were documented in an appropriate protocol. The holders were labeled with a new serial number for the further testing.

Positive control and Test item Formulation
The positive control imidazole was formulated (20 % imidazole in physiologic saline solution (wlv)) immediately before administration. The formulation was visually described as solution. The test item was suspended with a glass rod shortly prior to application in physiologic saline solution to achieve a concentration of 20 % (w/v). The formulation was visually described as suspension. Before each application the suspension was mixed thoroughly.

Application of the test material and incubation
Immediately before application, the medium was aspirated from the anterior chamber. 750 µL of the test material formulations were applied to the corneas, each through the holes of the anterior chamber. The holes of both chambers were sealed with adhesive tape and the holders were kept with the front side up, so that the formulations covered the cornea sufficiently. The holders were transferred into the incubator at 32°C (± 1 °C) for the exposure time of 4 hours.
After the exposure, the formulations were aspirated from the anterior chamber and the corneas were rinsed at least 3 times with phenol red containing MEM to show effectiveness of test substance removal. During the final rinse cycle the corneas were rinsed again with pure MEM medium in order to remove residues of the dye. The anterior chamber was then filled again with MEM medium to avoid drying out of the cornea. Before measuring opacity, fresh MEM medium was filled in the chambers.

Determination of Opacity
The opacity of a cornea was measured by the diminution of light passing through the cornea. The measurements of opacity were carried out using an opacitometer BASF OP3.0 (with integrated light meter testo 545 and Comfort V3.4 SP6 software from Testo AG, Lenzkirch). Before each measurement the opacitometer was adjusted to about 1000 LUX and a filter calibration measurement was carried out by using 3 different filters.

Determination of Permeability
The medium in anterior chamber of each holder was replaced by 1 mL of fluorescein sodium solution (concentration 5 mg/mL). Afterwards the holders were incubated at 32°C (± 1 °C) for about 90 minutes.
After the incubation period, the medium of the posterior chamber was aspirated by a syringe and filled into a 10 mL tube. Three wells of a 96 well plate were filled with 300 µL of each tube (triplicate determination). In addition, a standard series of 5 mg/mL sodium fluorescein solution was prepared and also filled into the 96-well plate, in triplicates.
The permeability was determined by measuring the amount of fluorescein sodium which diffused through all cell layers of the cornea. The measurement was carried out at a wavelength of 490 nm (OD490) by an ELISA- Reader.

Irritation parameter:

other: In vitro irritancy score (IVIS)

Run / experiment:

Test item 20% Bayscript Yellow GGN

Value:

-4.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No classification

Irritant / corrosive response data:

Based on OECD TG 437 and the experimental conditions reported the test item is classified as not seriously damaging the eye (no Category).

Based on OECD TG 437 and the experimental conditions reported the test item is classified as not seriously damaging the eye (no Category).

Interpretation of results:

GHS criteria not met

Conclusions:

Based on OECD TG 437 and the experimental conditions reported the test item is classified as not seriously damaging the eye (no Category).

Executive summary:

This study was performed to assess the corneal damage potential of the solid test item Bayscript Yellow GGN with the Bovine Corneal Opacity and Permeability test (BCOP) using fresh bovine cornea. The study was conducted in accordance with international accepted Guidelines (e.g. OECD TG 437).

20% (w/v) concentration ofthe test item and the positive control imidazole in physiologic saline solution were tested on 3 bovine corneas each in comparison to the negative control (physiologic saline solution). For determination of corneal damage opacity as well as tissue permeability was measured after a 4 hour treatment time.

Test items were applied to the epithelial surface of the cornea in a special corneal holder. Corneal opacity was measured quantitatively as the amount of light transmission through the cornea before and after treatment with the test item. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea after treatment. The In Vitro Irritancy Score (IVIS) value was calculated based on these data.

In accordance with OECD TG 437 and the study results Bayscript Yellow GGN was characterized by having no potential to seriously damage the eye.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

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)

Principles of method if other than guideline:

An in vitro study for assessing ocular irritation properties of the test item Bayscript Yellow GGN was performed using the reconstructed human cornea-like epithelium (RhCE) cell model EpiOcular™. The model used is standardized and commercially available. The EpiOcular™ Eye Irritation Test (EIT) was conducted in accordance with OECD 492.

GLP compliance:

yes

Specific details on test material used for the study:

Test item: Bayscript Yellow GGN
Chemical name: Benzenesulfonic acid, 3,3' -( carbonylbis(imino(3-methoxy-4, 1-
phenylene)-2, 1-diazenediyl)bis-, compd. with 2,2'iminobis(ethanol) (1 :2)
CAS number: 71550-21-5
Molecular mass: 850,9 g/mol
Content: 89.6%
Appearance: crystalline, russet

Species:

other: not applicable: artificial human 3D-Cornea model EpiOcular

Strain:

other: not applicable: artificial human 3D-Cornea model EpiOcular

Details on test animals or tissues and environmental conditions:

The experiment was carried out on the EpiOcular™ RhCE tissue construct (about 0.6 cm² in size; MatTek Corporation, Slovakia).
The RhCE tissue equivalents were shipped in 24 well cell culture plates on semi solid agar's medium.

Vehicle:

unchanged (no vehicle)

Controls:

other: Positive control: methyl acetate; Negative control: deionized water

Amount / concentration applied:

50 mg of the neat test item, 50 µl negative control (deionized water) and positive control (neat methyl acetate), respectively, were applied to the EpiOcular™ tissue surface in duplicate.

Duration of treatment / exposure:

The tissues were treated for about 6 hours under SCC. After the end of treatment time the test item was removed by rinsing with PBS followed by a post-soak immersion period of about 25 min. in fresh medium.

Duration of post- treatment incubation (in vitro):

Post-treatment incubation: the tissues were incubated for about 18 hours in fresh medium under SCC.

Number of animals or in vitro replicates:

50 mg of the neat test item, 50 µl negative control (deionized water) and positive control (neat methyl acetate), respectively, were applied to the EpiOcular™ tissue surface in duplicate.

Details on study design:

Test System/ Study Design
The EpiOcular™ RhCE tissue construct consists of 3 viable layers of cells and a nonkeratinized surface as recommended by the test guidelines. The cell viability and barrier function as well as sterility of each batch of the RhCE tissue construct used is adequate, as has been demonstrated by the supplier.
RhCE tissue viability in EpiOcular™ EIT is measured by enzymatic conversion of the vital dye MTT by the viable cells of the tissue into a blue MTT formazan salt that is quantitatively measured after extraction from tissues.

Methods and Parameters
Reconstructed tissues
The experiment was carried out on the EpiOcular™ RhCE tissue construct (about 0.6 cm² in size; MatTek Corporation, Slovakia).
The RhCE tissue equivalents were shipped in 24 well cell culture plates on semi solid agar's medium.

Adaptation to cell culture conditions
Upon receipt the tissue inserts were allowed to equilibrate to room temperature for 15 minutes. Subsequently each tissue insert was transferred from the packaging plate into the well of a 6 well culture plate containing 1 ml of fresh prewarmed ( approx. 37°C) maintenance medium. The EpiOcular™ inserts were incubated for 1 hour under SCC. Afterwards a medium change was performed and the tissue inserts were allowed to further adapt overnight to the recommended SCC.

Cell Culture conditions
The culture conditions in the incubator were standardized as follows:
Incubator temperature: 37 ± 2°C
CO2 gas concentration: 5%
Humidity: maximum
Occasional deviations from these conditions occurred e.g. as a result of opening the incubators door. However, these deviations had no effect on the course or outcome of the study. All Incubation steps were performed in a CO2 atmosphere incubator.

Test item formulation
Test item was used undiluted.

Pre-check for potential optical interferences of the test item
Optical properties of the test item or its chemical action on MTT may interfere with the measurement of MTT formazan leading to a false estimate of tissue viability.
The test item was therefore tested in advance for a potential direct influence on the test results not related to cytotoxic effects on tissue cells. For this pre-check the following parameters were tested:
1. Assessment of potential direct MTT -reduction of the test item In case of a direct MTT -reduction of the test item a killed tissue control (inserts, which were killed by freezing) has to be used in the main assay.
2. Assessment of potential interference of colored or staining test items, which become colored after application to the tissues, with OD read out
2.1. Assessment of the color reaction with water
2.2. Assessment of the color reaction with isopropanol
In case of an influence of test item color on OD measurement, a color control has to be used in the main assay.

The evaluation criteria for the pre-check are:
For MTT reduction (visual assessment): If the MTT solution color turns blue/purple, the test item is presumed to have reduced the MTT. A killed control must be conducted. For color reaction (measurement of the OD): If, after subtraction of the OD for water or isopropanol the OD of the test item solution is >0.08 a Color Control must be conducted.

Treatment and post-treatment incubation (Main Assay)
Treatment: 50 mg of the neat test item, 50 µl negative control (deionized water) and positive control (neat methyl acetate), respectively, were applied to the EpiOcular™ tissue surface in duplicate. The tissues were treated for about 6 hours under SCC. After the end of treatment time the test item was removed by rinsing with PBS followed by a post-soak immersion period of about 25 min. in fresh medium.
Post-treatment incubation: the tissues were incubated for about 18 hours in fresh medium under SCC.

Procedure for killed control or color control
Color control (CC): The test item was applied to two additional EpiOcular™ tissue inserts which were in principle treated as described for the main assay. However the further processing for the determination of cell viability differs in that these color control inserts were placed in Assay medium instead of placing them in MTT solution. A killed control was not required.

Determination of cell viability (MTT assay)
The MTT assay is a standardized quantitative method that is used to measure tissue viability. The assay depends on the intracellular capacity of living cells to chemically reduce the yellow 3 [4,5 Dimethythiazol2 yl] 2,5 diphenyl tetrazolium bromide (MTT) to blue formazan crystals.
Immediately after the end of post-treatment incubation the MTT reduction assay wasb performed. For viability testing the inserts were placed in new plates containing MTT solution (1 mg/ml in Maintenance medium at 37°C, or Assay medium for the color control inserts). The tissues were incubated for 180 ± 10 min. under SCC. The extraction of blue formazan was performed in isopropanol on a vertical shaker for 2-3 hours at room temperature. The concentration of formazan was measured by determination of the OD of the isopropanolextracts in duplicate at 570 nm in an automatic reader of a spectrophotometer.
Data acquisition and evaluation were performed with the software "GenS" (Bio-Tek).
The OD values obtained with the replicate tissue extracts for the test item were used to calculate the mean percent tissue viability normalized to negative control, which was set at 100%.
The final viability value for the test item was then calculated by subtracting the appropriate controls from the test item treated values (color control (CC) / killed control (KC)) as follows:
Final viability test item % viability test item - (%viability test item KC - % viability NC KC) - % viability test item CC.

Irritation parameter:

other: Final cell viability [%]

Run / experiment:

Bayscript Yellow GGN

Value:

71.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

All assay acceptance criteria were met.
As the final test item-treated tissue viability was> 60% relative to negative control, the test item was characterized as NOT having eye irritating properties (UN GHS: No Category).

Irritant / corrosive response data:

As the final test item-treated tissue viability was> 60% relative to negative control, the test item was characterized as NOT having eye irritating properties (UN GHS: No Category).

As the final test item-treated tissue viability was> 60% relative to negative control, the test item was characterized as NOT having eye irritating properties (UN GHS: No Category).

Interpretation of results:

GHS criteria not met

Conclusions:

As the final test item-treated tissue viability was> 60% relative to negative control, the test item was characterized as NOT having eye irritating properties (UN GHS: No Category).

Executive summary:

An in vitro study for assessing ocular irritation properties of the test item Bayscript Yellow GGN was performed using the reconstructed human cornea-like epithelium (RhCE) cell model EpiOcular™. The model used is standardized and commercially available.

The EpiOcular™ Eye Irritation Test (EIT) was conducted in accordance with OECD 492.

The solid test item was applied topically to the RhCE tissue surface in duplicate for 6 hours, followed by an 18 hour post-treatment incubation period. Cell viability was measured in a spectrophotometer by assessing the extent of MTT (methylthiazole tetrazolium) reduction. The optical density value obtained for the test item was used to calculate the percentage of viability relative to the negative control, which was set at 100%.

The results of the concurrent negative control (NC, deionized water) and positive control (PC, neat methyl acetate) demonstrated the viability (NC) and sensitivity (PC) of the tissue model.

The final mean percent tissue viability recorded for the test item is 72% (rounded).

According to the results of this study the test item Bayscript Yell ow GGN was identified as not requiring classification for eye irritation according to UN GHS (No Category).

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

All in-vitro tests according to OECD guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method), OECD guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants) and 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) were negative. According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is not justified.