Registration Dossier

Administrative data

Description of key information

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20.1. - 3.2.1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Version / remarks:
OECD Guidelines for Testing of Chemicals, Paris, 1992
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Remarks:
male/female
Details on test animals and environmental conditions:
- Source: Spolek chovatelů. Hradec Králové with veterinary health certificate; clinical examination confirmed the good state of health- Age at study initiation: not specified- Weight at study initiation: more than 2.5 kg- Housing: individually in metal cages (Velaz Praha)- Diet: ad libitum (standard granulated diet for rabbits TM-MAK1; Bergman)- Water: ad libitum (tap water acc. ČSN 757111)- Acclimation period: not specifiedENVIRONMENTAL CONDITIONS- Temperature (°C): 18±3- Humidity (%): 30 – 70%- Air changes (per hr): not specified- Photoperiod (hrs dark / hrs light): 12 /12
Type of coverage:
occlusive
Preparation of test site:
shaved
Vehicle:
water
Remarks:
only a bit moisturized with water before aplication
Amount / concentration applied:
0.5 g
Duration of treatment / exposure:
4 h
Observation period:
1, 24, 48, 72 hours
Number of animals:
3 animals
Details on study design:
TEST SITE - Area of exposure: 6x6 cm - Type of wrap if used: A sample a bit moisturized with water was covered with gauze, Alu-foil and cellulose wadding as last layer and fixed by technical tape for contact of a substance with shaved skin. REMOVAL OF TEST SUBSTANCE - Washing (if done): not specified - Time after start of exposure: 4 hoursOBSERVATION TIME POINTS1, 24, 48, 72 hoursSCORING SYSTEM: see Any other information ...
Irritation parameter:
overall irritation score
Basis:
animal: 20
Time point:
other: 1/24/48/72 h
Score:
0
Irritation parameter:
overall irritation score
Basis:
animal #1
Time point:
other: 1/24/48/72 h
Score:
0
Irritation parameter:
erythema score
Basis:
animal #2
Time point:
other: 1 h
Score:
1
Irritation parameter:
edema score
Basis:
animal #2
Time point:
other: 1 h
Score:
1
Irritation parameter:
overall irritation score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Interpretation of results:
GHS criteria not met
Conclusions:
The sample, Ostazinová žluť H8-G, is not irritating to the skin of the rabbit.
Executive summary:

In the sample Ostazinová žluť H8-G, the skin irritation test was carried out on New Zealand white rabbit breeds.

Examination of skin irritation has shown that the sample is not irritating to the skin of the rabbit.

The skin irritation test was performed according to OECD methodology No. 404.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
6.1.2017 - 31.3.2017
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
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
Commission Regulation (EC) No. 761/2009, 23rd July 2009
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: tissue for research purposes from accredited institutions
Source strain:
other: Keratinocyte strain 00267
Vehicle:
other: dosed directly on tissue moistened with 25 µL of PBS in MTT test
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE- Model used: The reconstructed human epidermal model EpiDerm™ (EPI-200, MatTek, Ashland, USA)- Tissue batch number(s): Lot No. 25802 kit CTEMPERATURE USED FOR TEST SYSTEMculture conditions 37±1°C, 5±1% CO2, humidified incubator REMOVAL OF TEST MATERIAL AND CONTROLS- Volume: thoroughly rinsed with PBS- Observable damage in the tissue due to washing: no- Modifications to validated SOP: noMTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE- MTT concentration: 1 mg·mL-1- Incubation time: 180 mins- Spectrophotometer: Libra S22 at 570 nm; Isopropyl alcohol served as a blank. Allowed band width is 2-3 nm. No external filter was used.FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATABased on Certificate of Analysis the model passed all parametres for viability, barrier function, sterility.NUMBER OF REPLICATE TISSUES: 3CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCEThe test substance does not reduce MTT directly.NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:1. Direct MTT reduction - functional check in tubes2. Colour interference3. MTT testPREDICTION MODEL / DECISION CRITERIAThe values stated in OECD Test Guideline No. 439, par. 36:- In case the test chemical is found to be non-corrosive (e.g., based on TG 430, 431 or 435), and shows tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50 %, the test chemical is considered to be irritant to skin in accordance with UN GHS Category 2. - The test chemical may be considered as non-irritant to skin in accordance with UN GHS No Category if the tissue viability after exposure and post-treatment incubation is more than (>) 50 %.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
test substance C2: 25 mg of the test substance was mixed with 2 mL of water for injection; 25 μL was dosed directly on tissue (moistened with 25 µL of PBS)NC: sterile PBS MatTek 101816ZSAPC: 5 % SDS (sodium dodecyl sulphate) in water, MatTek, Lot No. 031617MGKA
Duration of treatment / exposure:
60±1 minutes (25 minutes at room temperature and the remaining 35 minutes at culture conditions)
Duration of post-treatment incubation (if applicable):
18 hours, 12 minutes
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
83.2
Negative controls validity:
valid
Positive controls validity:
valid

Direct MTT reduction: functional check in tubes

25 μg ofthe test substance was added to 1.0 mL of MTT medium. Solution 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 0.037 what is < 0.08.The test substance is not very soluble in isopropyl alcohol. Average OD570 value from 2 wells was 0.001 what is < 0.08. It means that the test substance will go well washed from the tissue and small residue only is dissolved in isopropyl alcohol. Furthermore, the test substance does not absorb light at 570 nm. On the basis of results obtained, it was decided do not use concurrent colorant control in the MTT test.

MTT test:

OD570values obtained at the MTT test, their averages, standard deviations (%) and relative viabilities

Treatment

OD570

OD570 

OD570 

Mean

SD

%NC

PBS (NC)

1.961

1.946

1.941

1.949

0.008

 

viability (%NC)

100.6

99.8

99.6

100.0

0.436

100.0

393/16 (C2)

1.699

1.383

1.786

1.623

0.173

 

viability (%NC)

87.2

70.9

91.6

83.2

8.883

83.2

5% SDS (PC)

0.059

0.053

0.056

0.056

0.002

 

viability (%NC)

3.0

2.7

2.9

2.9

0.126

2.9

Interpretation of results:
GHS criteria not met
Conclusions:
Under the above-described experimental design, average viability of tissues treated by the test substance Reactive Yellow 85 was 83.2 % 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, the test substance, Reactive Yellow 85, is considered to have no category in accordance with UN GHS and is therefore considered as non-irritant.
Executive summary:

The test item, Reactive Yellow 85, was assayed for the in vitro skin irritation in human epidermal model EpiDermTM. The test was performed according to the OECD Test Guideline No. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method test (2015) and Protocol for: In Vitro EpiDermTMSkin Irritation Test For use with MatTek Corporation’s Reconstructed Human Epidermal Model EPI-200-SIT (2012).

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 above-described experimental design average viability of treated tissues was 83.2 %, i.e. viability was > 50 %.

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

According to the classification criteria, the test substance, Reactive Yellow 85, 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:
14.12. – 15. 12. 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)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES- Source: Breeding service CHOVSERVIS a.s., division TORO® Hlavečník, Hradec Králové, Czech Republic- Characteristics of donor animals (e.g. age, sex, weight): The eyes were enucleated as soon as possible after death. 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. - Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions):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).- Time interval prior to initiating testing: 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). All eyes used in the assay were from the same group of eyes collected on a specific day.- indication of any existing defects or lesions in ocular tissue samples: Only corneas from eyes free of defects including scratched, and neovascularisation 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.
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):2g 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 viscous. 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.
Duration of treatment / exposure:
4 hrs
Duration of post- treatment incubation (in vitro):
1.5 hr
Number of animals or in vitro replicates:
The results were based on the selection criteria for the eyes, as well as the positive and negative control responses.Number of corneas per group: Exposed group (test substance) - 3 corneas (No. 7, 8, 9,) Positive control group (20% Imidazole) – 3 corneas (No. 4, 5, 6) Negative control group (0.9% NaCl) – 3 corneas (No. 1, 2, 3)
Details on study design:
SELECTION AND PREPARATION OF CORNEASSelection criteria for eyes used in BCOP: Only corneas from eyes free of defects including scratched, and neovascularisation 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. Preparation: 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. QUALITY CHECK OF THE ISOLATED CORNEASFrom 25 eyes the 2 eyes were eliminated after inductive incubation, because the baseline opacity values were >7. Nine corneas were used for the study (the corneas No. 1, 2, 3, 4, 5, 6, 7, 8 and 9), 11 eyes was superfluous and remaining 3 were used for the testing of another substance.NUMBER OF REPLICATESNumber of corneas per group:Exposed group (test substance) - 3 corneas (No. 7, 8, 9,) Positive control group (20% Imidazole) – 3 corneas (No. 4, 5, 6) Negative control group (0.9% NaCl) – 3 corneas (No. 1, 2, 3) NEGATIVE CONTROL USED0.9% NaClSOLVENT CONTROL USED (if applicable)0.9% NaClPOSITIVE CONTROL USED20% ImidazoleAPPLICATION DOSE AND EXPOSURE TIME2g of the test substance was suspended in 10 mL of 0.9% sodium chloride solution; 4 hrsTREATMENT METHOD: open-chamber method POST-INCUBATION PERIOD: REMOVAL OF TEST SUBSTANCE- Number of washing steps after exposure period: - POST-EXPOSURE INCUBATION: 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 – more repeatedly, because the test substance is coloured . The corneas (applied the test substance) were also rinsed with EMEM (containing phenol red). Lastly EMEM (without phenol red) was used for final rinsing. The test substance was complete removal, but corneas stayed mild coloured by the test substance (yellow colour). The anterior chamber was then refilled with fresh EMEM without phenol red. The opacity and permeability of each cornea were recorded.METHODS FOR MEASURED ENDPOINTS: - Corneal opacity: measured quantitatively with the aid of an opacitometer (Opacitometer, MC2 - Le spécialiste du laboratoire – France) resulting in opacity values measured on a continuous scale- Corneal permeability: 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.SCORING SYSTEM: In Vitro Irritancy Score (IVIS)IVIS = mean opacity value + (15 x mean permeability OD490 value)DECISION CRITERIA: IVISUN GHS ≤ 3 No Category> 3; ≤ 55 No prediction can be made ≥ 55 Category 1
Irritation parameter:
in vitro irritation score
Run / experiment:
1
Value:
17.65
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Other effects:
After exposure the test substance was removed from the corneas. In spite of this procedure the corneas treated by the test substance remain mildly coloured. This colouring of the corneas had influence on the measuring of opacity what could result in higher opacity values.
Interpretation of results:
other: no prediction can be made
Conclusions:
The In Vitro Irritancy Score (IVIS) for Reactive Yellow 85 was 17.65 but this result could be affected by higher opacity values (corneas were coloured by the test substance: mild yellow colour). On the basis of IVIS score the classification according to the criteria of the UN GHS resulted in category “No prediction can be made”. But because of the colouring of corneas this result is ambiguous.
Executive summary:

The test substance, Reactive Yellow 85, 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 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 Reactive Yellow 85 was 17.65 but this result could be affected by higher opacity values (corneas were coloured by the test substance: mild yellow colour).

On the basis of IVIS score the classification according to the criteria of the UN GHS resulted in category “No prediction can be made”. But because of the colouring of corneas this result is ambiguous.

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26.1. - 3.2.1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
OECD Guidelines for Testing of Chemicals, Paris, 1987
Deviations:
not specified
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Remarks:
male/female
Details on test animals or tissues and environmental conditions:
TEST ANIMALS- Source: Spolek chovatelů. Hradec Králové with veterinary health certificate; clinical examination confirmed confirmed the good state of health- Age at study initiation: not specified- Weight at study initiation: more than 2.5 kg- Housing: individually in metal cages (Velaz Praha)- Diet: ad libitum (standard granulated diet for rabbits TM-MAK1; Bergman)- Water: ad libitum (tap water acc. ČSN 757111)- Acclimation period: not specifiedENVIRONMENTAL CONDITIONS- Temperature (°C): 18±3- Humidity (%): 30 – 70%- Air changes (per hr): not specified- Photoperiod (hrs dark / hrs light): 12 /12
Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
0.1 g
Duration of treatment / exposure:
24 hours
Observation period (in vivo):
1, 24, 48, 72 hours
Number of animals or in vitro replicates:
3 animals
Details on study design:
REMOVAL OF TEST SUBSTANCE - Washing (if done): careful washing with water - Time after start of exposure: 24 hour SCORING SYSTEM:see Any other information ... TOOL USED TO ASSESS SCORE: 2% Na+ salt of fluorescein
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
animal: 20
Time point:
24/48/72 h
Score:
0
Max. score:
3
Irritation parameter:
conjunctivae score
Basis:
animal: 10
Time point:
24 h
Score:
2
Max. score:
3
Irritation parameter:
conjunctivae score
Basis:
animal: 10
Time point:
48 h
Score:
1
Max. score:
3
Irritation parameter:
conjunctivae score
Basis:
animal: 10
Time point:
72 h
Score:
1
Max. score:
3
Irritation parameter:
conjunctivae score
Basis:
animal: 11
Time point:
24 h
Score:
1
Max. score:
3
Irritation parameter:
conjunctivae score
Basis:
animal: 11
Time point:
48 h
Score:
0
Max. score:
3
Irritation parameter:
conjunctivae score
Basis:
animal: 11
Time point:
72 h
Score:
0
Max. score:
3
Other effects:
Animal 10 at 24h: efflux score 1 (max score unknown)

Irritation score 2.6 (see table in attached picture).

Interpretation of results:
GHS criteria not met
Conclusions:
The sample, Ostazinová žluť H8-G, is not irritating to the rabbit eye.
Executive summary:

For the sample Ostazinová žluť H8-G, the eye irritation test was carried out on New Zealand white rabbit breeds.

Examination of eye irritation has shown that the sample is not irritating to rabbit eye.

Eye irritation testing was performed according to OECD methodology No. 405.

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

The average viability of tissues treated by the test substance Reactive Yellow 85 was 83.2 % of negative control average value i.e. viability was > 50 %. The effect of the test substance was negative in EpiDermTM model.

The skin irritation test performed according to OECD methodology No. 404 has shown that the sample is not irritating to the skin of the rabbit.

The eye irritation test performed according to OECD methodology No. 405 with conclusion not irritating to the eye of rabbit.

According to the classification criteria, the test substance, Reactive Yellow 85, is considered to have no category in accordance with UN GHS and is therefore considered as non-irritant.