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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:
2017
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)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
physiological saline
Details on test system:
TEST SYSTEMThe 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 was dosed directly on tissue moistened with 50 µL of PBS (phosphate buffered saline). Volume of the PBS was increased (from 25 to 50 µL) to wet more test substance. Not even after that the test substance was not completely wetted, but it was wetted on tissue surface. This possibility is permitted also in MatTek Protocol for: In vitro EpiDermTM Skin Irritation Test. 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 59 minutes and 19 hours and 11 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 24 hours and 15 minutes of post-incubation period, the medium was replaced by fresh one. Tissues were incubated for another 18 hours, 45 minutes. Afterwards, the MTT assay was performed by transferring the tissues to 24-well plates containing MTT medium (1 mg/mL). After 185 minutes MTT incubation, the blue formazan salt formed by cellular mitochondria was extracted with 2.0 ml/tissue of isopropyl alcohol (for 148 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 Direct Yellow 28VEHICLE- Amount(s) applied (volume or weight with unit): 50 µL phosphate buffered salineNEGATIVE CONTROL- Amount(s) applied (volume or weight): phosphate buffered salinePOSITIVE CONTROL- Concentration (if solution): 5% sodium dodecyl sulphate
Duration of treatment / exposure:
60 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Value:
71.7
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
DIRECT MTT REDUCTION - FUNCTIONAL CHECK IN TUBES25 μg 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 coloured orange. 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 what is > 0.08. The test substance is not soluble in isopropyl alcohol. Average OD570 value from 2 wells was 0.005 what is < 0.08. It means that the test substance will go well washed from the tissue and any residue is not dissolved in isopropyl alcohol. On the basis of results obtained, it was decided do not use concurrent colorant control in the MTT test.ACCEPTANCE CRITERIA FULFILMENTThe mean OD570 of the NC tissue was 2.004 ± 0.047 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 2.4 % 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 14.0 what is < 18 %.All study acceptance criteria were fulfilled.

Table 1: Optical density and viability

 

Treatment

OD570

Average

SD

Average viability

(% NC)

1

2

3

NC

PBS

2.022

1.940

2.050

2.004

0.047

100.0

%

100.9

96.8

102.3

100.0

2.33

C2

390/16

1.439

1.779

1.090

1.436

0.281

71.7

%

71.8

88.8

54.4

71.7

14.04

PC

5% SDS

0.044

0.051

0.050

0.048

0.003

2.4

%

2.2

2.5

2.5

2.4

0.15

NC - negative control

PC - positive control

C2 - 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, Direct Yellow 28, was 71.7 % of negative control average value, i.e. viability was > 50 %. The effect of the test substance was negative in EpiDerm model. The test substance, Direct Yellow 28, is considered to have no category in accordance with UN GHS and is therefore considered a non-irritant.
Executive summary:

The test substance, Direct Yellow 28, 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 experiments 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 test conditions, average viability of treated tissues was 71.7 %, i.e. viability was > 50 %. The effect of the test substance was negative in EpiDerm model (tissues were not damaged).

The test substance, Direct Yellow 28, is considered to have no category in accordance with UN GHS and is therefore considered a non-irritant.

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

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
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)
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. No detergent was used. 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 a baseline opacity >7 opacity units were discarded. From 22 eyes the 5 eyes were eliminated after inductive incubation, because the baseline opacity values were >7. Nine corneas were used for the study, 5 eyes were superfluous and the remaining 3 eyes were used for the testing of another substance.
Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
20% solution of the test substance in physiological saline
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 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 EYESCorneas 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 SUBSTANCETreatment 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. Open-chamber method was used, because the test substance at 20% concentration was a viscous paste. The test substance (the test substance in quantity enough to completely cover the cornea) was applied directly to the epithelial surface of the cornea using the spatula. After dosing, the glass window was replaced on the anterior chamber to recreate a closed system.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-EXPOSUREAfter the exposure period, the negative control and the positive control substance was removed from the anterior chamber with EMEM (containing phenol red - the effectiveness of rinsing acidic or alkaline materials). The corneas were given a final rinse with EMEM (without phenol red). The EMEM (without phenol red) was used as a final rinse to ensure removal of the phenol red from the anterior chamber prior to the opacity measurement. The anterior chamber was then refilled with fresh EMEM without phenol red. The opacity and permeability of each cornea were recorded. The test substance was removed from the anterior chamber with EMEM (containing phenol red) and sequentially was removed mechanically using a cotton swab – very gently (the test substance was coloured); also the test substance was removed from the anterior chamber with EMEM (containing phenol red) once more. The corneas (applied the test substance) were also repeatedly rinsed with EMEM (without phenol red). Rinsing was finalized after complete removal of the test substance. The anterior chamber was then refilled with fresh EMEM without phenol red. The opacity and permeability of each cornea were recorded.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
Value:
1.2
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid

Table 1: Opacity

Group

Baseline opacity

Opacity after treatment

Opacity difference

Mean

opacity difference

Mean Opacity (corrected)

Negative Control

(0.9% NaCl)

6

8

2

1.67

-

6

7

1

6

8

2

Positive Control

(20% Imidazole in 0.9% NaCl)

7

63

56

53.67

(53.67 – 1.67)

52.00

5

61

56

6

55

49

Test Substance

(Direct Yellow 28)

5

8

3

2.67

(2.67 – 1.67)

1.00

7

9

2

7

10

3

Table 2: Optical density (permeability)

Group

Values of Permeability (OD490)

Mean Permeability

Mean Permeability

(corrected)

Negative Control

(0.9% NaCl)

0.031

0.034

-

0.042

0.030

Positive Control

(20% Imidazole in 0.9% NaCl)

1.605

1.831

(1.831 – 0.034)

1.797

2.133

1.754

Test Substance

(Direct Yellow 28)

0.056

0.047

(0.047 – 0.034)

0.013

0.042

0.042

 

Table 3: IVIS

Group

IVIS

Calculation

Result

Negative Control

(0.9% NaCl)

1.67 + 15 × 0.034 

2.18

Positive Control

(20% Imidazole in 0.9% NaCl)

52.00 + 15 × 1.797 

78.96

Test Substance

(Direct Yellow 28)

1.00 + 15 × 0.013

1.20

Interpretation of results:
GHS criteria not met
Conclusions:
The In Vitro Irritancy Score (IVIS) for Direct Yellow 28 was 1.20. This value of IVIS is ≤ 3 therefore the classification of test substance effect according to UN GHS criteria for eye irritation or serious eye damage is: No category.
Executive summary:

The test substance, Direct Yellow 28, 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.

Open-chamber method was used, because the test substance was highly viscous. 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 Yellow 28 was 1.20.

This value of IVIS is ≤ 3 therefore the classification of test substance effect according to UN GHS criteria for eye irritation or serious eye damage is: No category.

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