Registration Dossier

Administrative data

Description of key information

The results of an in vitro skin irritation test in the EPISKIN model as well as the results of the acute dermal toxicity study indicated that the test item is not a skin irritant.

According to the results of an in-vitro study in chicken eye and an acute eye irritation study performed in New Zealand White rabbits, Reactive Yellow F01-0555 should not be classified as an eye irritant.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 November 2011 to 11 November 2011
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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Remarks:
reconstructed human epidermis (RhE)
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
The EPISKIN model has been validated for corrosivity testing in an international trial, it is considered to be suitable for this study.
Vehicle:
unchanged (no vehicle)
Details on test system:
TEST SYSTEM
EpiSkinTM Small Model (EpiSkinTMSM) (Source: SkinEthic, France, Batch No.:11-EKIN-041, Expiry date: 14 November 2011) is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum. Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.

Source: Skinethic, Nice, France.
Batch No.: 11-EKIN-041
Expiry date: 14 November 2011

Quality Control
EPISKIN-SM kits are manufactured according to defined quality assurance procedures (certified ISO 9001). All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking an MTT cell viability test and a cytotoxicity test with sodium dodecyl sulphate (SDS).

DEMONSTRATION OF PROFICIENCY
Prior to routine use of the method CiToxLAB Hungary Ltd. demonstrated technical proficiency, using the ten Proficiency Chemicals according to OECD Test Guideline No. 439.

Kit Contents
Units: EPISKIN-SM plate containing up to 12 reconstructed epidermis units (area: 0.38 cm2) each reconstructed epidermis is attached to the base of a tissue culture vessel with an O-ring set and maintained on nutritive agar for transport.
Plate: 12-well assay plate
Punch: EPISKIN-SM biopsy punch for easy sampling of epidermis

Medium: A flask of sterile “Maintenance Medium” for incubations. (Batch No.: 11-MAIN3-053; Exp. Date: 16 November 2011)
A flask of sterile “Assay Medium” for use in for use in MTT assays. (Batch No.: 11-ESSC-043; Exp. Date: 16 November 2011)

Kit Reception Quality Check
The colour of the agar medium used for transport was checked for its pH:
-orange colour = good
-yellow or violet colour = not acceptable

The colour of the temperature indicator was inspected to verify that the kit has not been exposed to a temperature above 40°C:
-the indicator changes from white to grey at 40°C
The kit was found to be in good order at reception.

Storage
The EPISKIN-SM kit was kept in its packaging at 37°C and the assay and maintenance medium supplied with the kit was stored at 2-8°C until the initiation of the test.

ADDITIONAL MATERIALS
MTT stock solution
MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS number 298-93-1] was dissolved to a final concentration of 3 mg/mL in saline buffer (PBS). The obtained stock solution can be stored in a refrigerator (2-8°C), protected from light up to 15 days.

MTT ready to use solution
The MTT stock solution was diluted with pre-warmed “assay medium” to a final concentration of 0.3 mg/mL. The obtained solution was used within one hour.

Acidified Isopropanol
Isopropanol was diluted with HCl acid to a final concentration of 0.04N HCl.
The obtained solution can be stored in a refrigerator (2-8°C), protected from light for one month.

INDICATOR FOR POTENTIAL FALSE VIABILITY
Optical properties of the test material or its chemical action on MTT may interfere with the assay leading to a false estimate of viability. This may occur when the test substance is not completely removed from the tissue by rinsing or when it penetrates the epidermis. If the test material acts directly on MTT (MTT-reducer), is naturally coloured, or becomes coloured during tissue treatment, additional controls should be used to detect and correct for test substance interference with the viability measurement.

Check-method for possible direct MTT reduction with test substance
An amount of 10 mg test item was added to 2 mL MTT ready to use solution and mixed. The mixture was incubated for three hours at room temperature protected from light and then any colour change was assessed:
- Test substances which do not interact with MTT: yellow
- Test substances interacting with MTT: blue or purple
If the MTT solution colour becomes blue or purple, the test substance interacts with the MTT. It is then necessary to evaluate the part of optical density (OD) due to the non specific reduction of the MTT (i.e. by using killed epidermis).
The test item showed no direct interaction with MTT.

Check-method to detect the colouring potential of test-substances
As the test item has an intrinsic colour, further evaluation to detect colouring potential was necessary. Non Specific Colour % (NSC %) was determined in order to evaluate the ability of test substance to stain the epidermis by using additional control tissues.


Additional control(s) for dyes and chemicals able to colour the tissue
In addition to the normal procedure, one additional chemical-treated tissue was used for the non specific OD evaluation. This tissue followed the same test item application and all steps as for the other tissues, except for the MTT step: MTT incubation was replaced by incubation with fresh assay medium. This is to mimic the amount of colour from the test item that may be present in the test disks. OD readings were made following the same conditions as for the other tissues.


PERFORMANCE OF THE STUDY
Procedures described in sections 3.6.1., 3.6.2. and 3.6.3. were performed under axenic conditions.

Pre-incubation (Day [-1])
The “maintenance medium” was pre-warmed to 37°C. The appropriate number of assay plate wells were filled with the pre-warmed medium (2 mL per well). The epidermis units were placed, with the media below them in contact with the epidermis, into each prepared well and then incubated overnight at 37°C in an incubator with 5% CO2.

Application and rinsing (Day 0)
First 10 µl distilled water was applied to the epidermal surface to ensure good contact with the epidermis, then 20 mg of test item was applied evenly to the epidermal surface of each of the three test skin units.
10 µl PBS was added to each of the three negative control skin units
10 µl SDS was added to each of the three positive control skin units
For additional controls for staining effects of the test item, 10 µl distilled water was applied to the epidermal surface to ensure good contact with the epidermis, then 20 mg of test item was applied evenly to the epidermal surface.

The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature (18-28°C).

After the incubation time the EPISKIN-SM units were removed and rinsed thoroughly with PBS to remove all of the test material from the epidermal surface. The rest of the PBS was removed from the epidermal surface with a suitable pipette tip linked to a vacuum source care was taken to avoid the damage of epidermis.

After rinsing the units were placed into the plate wells with fresh pre-warmed “maintenance medium” (2 mL/well) below them and then incubated for 42 hours (± 1h) at 37°C in an incubator with 5% CO2.

MTT test after 42 hours incubation (day 2)
After the 42 hours incubation all EPISKIN-SM units except the one staining control were transferred into the MTT solution filled wells (2 mL of 0.3 mg/mL MTT per well). The one additional control for coloured substances was transferred to wells filled with fresh assay medium. Then, all transferred EPISKIN-SM units were incubated for 3 hours (± 5 min) at 37°C in an incubator with 5% CO2 protected from light.

Formazan extraction (Day 2)
At the end of incubation with MTT a formazan extraction was undertaken:

A disk of epidermis from each replicate was cut from the unit (this involves the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube of 500 µL acidified isopropanol (one tube corresponding to one well of the tissue culture plate).
The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material with the acidified isopropanol then incubated for about two hours at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.

Cell viability measurements (Day 2)
Following the formazan extraction, 2×200 µL samples from each tube were placed into the wells of a 96-well plate (labelled appropriately). The OD (Absorbance / Optical Density) of the samples in a 96-well plate spectrophotometer was read at 540 nm using acidified isopropanol solution blank (6×200 µL).
The validity of the microplate reader was verified with a standard verification plate daily before use. The standard plate was calibrated yearly by the manufacturer.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
First 10 µl distilled water was applied to the epidermal surface to ensure good contact with the epidermis, then 20 mg of test item was applied evenly to the epidermal surface of each of the three test skin units.
10 µl PBS was added to each of the three negative control skin units
10 µl SDS was added to each of the three positive control skin units
For additional controls for staining effects of the test item, 10 µl distilled water was applied to the epidermal surface to ensure good contact with the epidermis, then 20 mg of test item was applied evenly to the epidermal surface.
Duration of treatment / exposure:
The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature (18-28°C).
Duration of post-treatment incubation (if applicable):
After the 42 hours incubation all EPISKIN-SM units except the one staining control were transferred into the MTT solution filled wells (2 mL of 0.3 mg/mL MTT per well). The one additional control for coloured substances was transferred to wells filled with fresh assay medium. Then, all transferred EPISKIN-SM units were incubated for 3 hours (± 5 min) at 37°C in an incubator with 5% CO2 protected from light.
Number of replicates:
In this assay 3 replicates for the test item and 3 negative controls + 3 positive controls were used. Furthermore one additional control for coloured substance was used.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean of 3 replicates
Value:
98
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
VALIDITY OF THE TEST

The mean OD value of the three negative control tissues was 0.949. The positive control result showed 14% viability. Each standard deviation value (SD) of the % viability was below 18. All validity criteria were within acceptable limits and therefore the study can be considered as valid.

INDICATOR FOR POTENTIAL FALSE VIABILITY

Possible direct MTT reduction with Test Substance

No colour change was observed after three hours of incubation of the test item in MTT solution. The test material did not interact with the MTT, therefore additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

Colouring potential of test substances

As the test item has an intrinsic colour, one additional chemical-treated tissue was used for the non specific OD evaluation. Mean OD (measured at 540 nm) of this tissue was determined as 0.024, Non Specific Colour % was calculated as 2.5%. Therefore additional data calculation was not necessary.

CELL VIABILITY

 The results of the optical density (OD) measured at 540 nm of each replicate and the calculated % viability of the cells is presented below:

 

Substance

Optical Density (OD)

Viability (%)

Negative Control:

PBS

 

1

0.954

101

2

0.899

95

3

0.994

105

mean

0.949

100

standard deviation (SD)

5.03

Positive Control:

SDS 5% aq. solution

 

1

0.169

18

2

0.111

12

3

0.110

12

mean

0.130

14

standard deviation (SD)

3.46

Test Item:

Reactive Yellow F01-0555

 

1

0.892

94

2

0.894

94

3

1.019

107

mean

0.935

98

standard deviation (SD)

7.51

Interpretation of results:
GHS criteria not met
Conclusions:
Disks of EPISKIN (three units / chemical) were treated with Reactive Yellow F01-0555 and incubated for 15 minutes at room temperature. Exposure of test material was terminated by rinsing with PBS. Epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in 5% CO2 protected from light. The formazan extract in acidified isopropanol was then spectrophotometrically evaluated for optical density (OD) and quantified.
SDS 5% and PBS treated epidermis were used as positive and negative controls respectively. For each treated tissue viability was expressed as a % relative to negative control.
The test substance is considered to be irritant to skin, if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control.


In this in vitro skin irritation test in the EPISKIN model with Reactive Yellow F01-0555 the results indicated that the test item is Non Irritant (NI) [UN GHS: No Category].
All validity criteria were within acceptable limits and therefore the study can be considered as valid.
Executive summary:

Disks of EPISKIN (three units / chemical) were treated with Reactive Yellow F01-0555 and incubated for 15 minutes at room temperature. Exposure of test material was terminated by rinsing with PBS. Epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in 5% CO2 protected from light. The formazan extract in acidified isopropanol was then spectrophotometrically evaluated for optical density (OD) and quantified. SDS 5% and PBS treated epidermis were used as positive and negative controls, respectively. For each treated tissue viability was expressed as a % relative to negative control.

The test substance is considered to be irritant to skin, if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control.

In this in vitro skin irritation test in the EPISKIN model with Reactive Yellow F01-0555 the results indicated that the test item is Non Irritant (NI) [UN GHS: No Category].

All validity criteria were within acceptable limits and therefore the study can be considered as valid.

Endpoint:
skin irritation: in vivo
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
the study does not need to be conducted because an acute toxicity study by the dermal route does not indicate skin irritation up to the relevant limit dose level (2 000 mg/kg body weight)
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 August 2011 to 21 September 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
yes
Remarks:
The relative humidity (max 89%) was out of the target range (30-70%) during the study.
Qualifier:
according to guideline
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
yes
Remarks:
The relative humidity (max 89%) was out of the target range (30-70%) during the study.
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2400 (Acute Eye Irritation)
Deviations:
yes
Remarks:
The relative humidity (max 89%) was out of the target range (30-70%) during the study.
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
Species and strain: New Zealand White rabbits
Source: S&K-LAP Kft. 2173 Kartal, Császár út 135, Hungary
Justification of strain: The New Zealand White rabbit is one of the standard strains used for acute irritation toxicity studies.
Animal health: Only animals in acceptable health condition were used for the test. Both eyes of each animal provisionally selected for testing were examined approximately one hour before starting the study. Animals showing eye irritation, ocular defects or pre-existing corneal injury were not used.
Number of animals: 3 animals
Age of animals at treatment: ~11 weeks old (adult)
Sex: Male
Body weight range at the
beginning of the life phase: 2754 – 2837 g
end of the life phase: 3152 – 3233 g
Date of receipt: 24 August 2011
Acclimatization time: 7 days
Animal identification: The individual identification was by engraved ear tag. The cages were marked with individual identity cards with information about study code, sex, dose group, cage number and individual animal number.

HUSBANDRY
Animal health: Only healthy animals were used for the test. The veterinarian certified health status.
Number of animal room: 632
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 20 ± 3°C
Relative humidity: 40 - 89 %
Housing/Enrichment: Rabbits were individually housed in AAALAC approved metal wire rabbit cages. Cages were of an open wire structure and cages were placed together to allow some social interaction with rabbit(s) in adjoining cages
Ventilation: 15-20 air exchanges/hour

The environmental parameters were recorded twice daily during the study. Variations from the target humidity (max. 89%) range were observed during the study. These deviations were considered to have no impact on the animal health, as certified by the Clinical Veterinarian, or on the outcome of the study and interpretation of the results.

FOOD AND FEEDING

Animal received PURINA Base – Lap gr. diet for rabbits produced by AGRIBRANDS Europe Hungary PLC, H-5300 Karcag, Madarasi road, Hungary, ad libitum.

WATER SUPPLY AND QUALITY CONTROL OF WATER

The animals received municipal tap water, as for human consumption, ad libitum, from an automatic system.

The quality control analysis is performed once every three months and microbiological assessment is performed monthly, by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A.u.36., Hungary). The quality control results are retained in the archives of CiToxLAB Hungary Ltd.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
A single dose of 0.1 g of the solid test item was administered to each animal.
Duration of treatment / exposure:
1 hour
Observation period (in vivo):
3 weeks
Number of animals or in vitro replicates:
3
Details on study design:
Application of the Test Item

Three male animals in acceptable health condition were selected for the test. Care was taken to select only those animals that had a normal eye condition and any with ocular lesions were rejected.

An initial test was performed using one animal. The test item was instilled into the conjunctival sac of the left eye. The eyelids were held closed for several seconds to prevent the loss of the test item. The contralateral eye served as the control. Immediately after the administration of the test item, an assessment of the initial pain reaction was made.

After consideration of the ocular responses produced in the first animal, two additional animals were treated.

Duration of Exposure

The eyes of the test animals were washed out at 1 hour after application of test item.

OBSERVATIONS AND SCORING

Clinical Observations

The eyes were examined at 1, 24, 48, 72 hours and 1, 2 and 3 weeks after treatment. The duration of the observation period was sufficient to identify reversibility or irreversibility of changes. Any clinical signs of toxicity or signs of ill- health during the study were recorded.

At the end of the observation period, each animal was sacrificed by intramuscular injections of CP-Ketamin 10% and CP-Xylazin 2% followed by iv. Euthasol® 40% anaesthesia. Death was verified by checking pupil and corneal reflex and the absence of respiration.

Scoring and Assessment of Local Reaction

The eye irritation scores were evaluated according to the scoring system by Draize (1977) and OECD 405 (24 April 2002).


Classification of the Test Items

Individual reactions of the animal were recorded at each observation time. The nature, severity and duration of all lesions observed were described.

Results were presented and interpreted according to Regulation (EC) No 1272/2008, as follows:

Irreversible effects on the eye/serious damage to eyes (Category 1)

Substances that have the potential to seriously damage the eyes are classified in Category 1 (irreversible effects on the eye). These observations include animals with grade 4 cornea lesions and other severe reactions (e.g., destruction of cornea) observed at any time during the test, as well as persistent corneal opacity, discoloration of the cornea by a dye substance, adhesion, pannus, and interference with the function of the iris or other effects that impair sight.

Category for irreversible eye effects:

If, when applied to the eye of an animal, a substance produces:
— at least in one animal effects on the cornea, iris or conjunctiva that are not expected to reverse or have not fully reversed within an observation period of normally 21 days;
and/or
— at least in 2 of 3 tested animals, a positive response of:
o corneal opacity ≥ 3 and/or
o iritis > 1.5
calculated as the mean scores following grading at 24, 48 and 72 hours after installation of the test material.

Reversible effects on the eye/irritating to eyes (Category 2):

Substances that have the potential to induce reversible eye irritation are classified in Category 2 (irritating to eyes).

Category for reversible eye effects
If, when applied to the eye of an animal, a substance produces:
— at least in 2 of 3 tested animals, a positive response of:
o corneal opacity ≥ 1 and/or
o iritis ≥ 1, and/or
o conjunctival redness ≥ 2 and/or
o conjunctival oedema (chemosis) ≥ 2

— calculated as the mean scores following grading at 24, 48 and 72 hours after installation of the test material, and which fully reverses within an observation period of 21 days

Measurement of Body Weight

Individual body weight was recorded at the beginning and end of the experiment.


Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
all animals
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
mean
Remarks:
all animals
Time point:
24/48/72 h
Score:
0
Max. score:
2
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.67
Max. score:
3
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
24/48/72 h
Score:
1.33
Max. score:
3
Reversibility:
fully reversible within: 2 weeks
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
24/48/72 h
Score:
1.67
Max. score:
3
Reversibility:
fully reversible within: 2 weeks
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 48 hours
Irritant / corrosive response data:
The eyes were examined at 1, 24, 48, 72 hours and 1, 2 and 3 weeks after the application.

Initial Pain Reaction (IPR) (score 2) was observed in all animals after test item administration.

One hour after the application:
Conjunctival redness (score 2) was found in all rabbits, conjunctival chemosis (score 1) were observed in two animals and all rabbits showed conjunctival discharge (score 3).

At 24 hours after treatment:
Conjunctival redness (score 2 or 1) was found in all rabbits, chemosis (score 1).was observed in two animals. One rabbit displayed conjunctival discharge (score 1).

At 48 hours after treatment:
Conjunctival redness (score 1) was observed in two animals and redness (score 2) was noted in one rabbit.

At 72 hours after treatment:
Conjunctival redness (score 1) was observed in two animals.

At 1 week after treatment:
Conjunctival redness (score 1) was observed in 2 rabbits.


At 2 and 3 weeks after treatment:
No signs of eye irritation or other clinical signs were observed.

The study was terminated after the 3-week observation.
Other effects:
The conjunctivae were coloured by the test item during the study up to and including the 3-week observation.

TABLE 1: INDIVIDUAL SCORES FOR OCULAR IRRITATION

Study Code:             11/174-005N                     Species:    NZW Rabbit

Dose:                        0.1g                                  Sex:          Male

Start of Exposure:    31 August 2011                Test Item: Reactive Yellow F01-0555

 

 

Time

Animal No.

Score of irritation

IPR

Conjunctivae

Opacity of cornea

Iris

Control eye

Other sign

R

CH

D

OD

OE

R

1 hour

00427

2

0

3

0

0

0

0

*

2

00388

2

1

3

0

0

0

0

*

2

00389

2

1

3

0

0

0

0

*

2

Time of Observation: Day 0

Time

Animal No.

Score of irritation

Conjunctivae

Opacity of cornea

Iris

Control eye

Other sign

R

CH

D

OD

OE

R

24 hours

00427

1

0

1

0

0

0

0

*

00388

2

1

0

0

0

0

0

*

00389

2

1

0

0

0

0

0

*

Time of Observation: Day 1

Time

Animal No.

Score of irritation

Conjunctivae

Opacity of cornea

Iris

Control eye

Other sign

R

CH

D

OD

OE

R

48 hours

00427

1

0

0

0

0

0

0

*

00388

1

0

0

0

0

0

0

*

00389

2

0

0

0

0

0

0

*

Time of Observation: Day 2

Time

Animal No.

Score of irritation

Conjunctivae

Opacity of cornea

Iris

Control eye

Other sign

R

CH

D

OD

OE

R

72 hours

00427

0

0

0

0

0

0

0

*

00388

1

0

0

0

0

0

0

*

00389

1

0

0

0

0

0

0

*

Time of Observation: Day 3


 

Time

Animal No.

Score of irritation

Conjunctivae

Opacity of cornea

Iris

Control eye

Other sign

R

CH

D

OD

OE

R

1 week

00427

1

0

0

0

0

0

0

*

00388

0

0

0

0

0

0

0

*

00389

1

0

0

0

0

0

0

*

Time of Observation: Day 7

 

Time

Animal No.

Score of irritation

Conjunctivae

Opacity of cornea

Iris

Control eye

Other sign

R

CH

D

OD

OE

R

2 weeks

00427

0

0

0

0

0

0

0

*

00388

0

0

0

0

0

0

0

*

00389

0

0

0

0

0

0

0

*

Time of Observation: Day 14

 

Time

Animal No.

Score of irritation

Conjunctivae

Opacity of cornea

Iris

Control eye

Other sign

R

CH

D

OD

OE

R

3 weeks

00427

0

0

0

0

0

0

0

*

00388

0

0

0

0

0

0

0

*

00389

0

0

0

0

0

0

0

*

Time of Observation: Day 21

 

Abbreviations:   R    = Redness                                OD =   Opacity degree of density

                           CH = Chemosis                               OE =   Extent of opaque area

D    = Discharge                              

*: The conjunctivae were coloured by the test item.

TABLE 2: MEAN VALUES OF EYE IRRITATION (24, 48, 72 hour reading)

Study Code:             11/174-005N                    Species:    NZW Rabbit

Dose:          0.1g                                   Sex:          Male

Start of Exposure:     31 August 2011                Test Item: Reactive Yellow F01-0555

 

  

Animal Number

Cornea Opacity

Iris

Conjunctivae

Redness

Chemosis

Discharge

00427

0.00

0.00

0.67

0.00

0.33

00388

0.00

0.00

1.33

0.33

0.00

00389

0.00

0.00

1.67

0.33

0.00

Interpretation of results:
GHS criteria not met
Conclusions:
The test item Reactive Yellow F01-0555, applied to rabbit eye mucosa, caused significant conjunctival irritant effects at one hour which were reduced at 24 hours after application. There were no corneal effects observed at any timepoint, all conjunctival irritation effects were completely reversed after the 2-week observation period. Some discoloration of the conjuctivae remained at the observation period of 3 weeks, but this is not considered as classifiable effect under CLP or GHS guidelines.
Reactive Yellow F01-0555 is “not classified” according to Regulation (EC) No 1272/2008.

Executive summary:

An acute eye irritation study of the test item Reactive Yellow F01-0555 was performed in New Zealand White rabbits. The irritation effects of the test item were evaluated according to the Draize method (OECD No.: 405, 2002).

The test item was placed into the conjunctival sac of the left eye of each animal. The untreated right eye served as control. An amount ofof the test item was administered as a single dose.

 

Individual body weight was recorded at the beginning and end of the study. Morbidity and clinical signs of toxicity were checked daily. The eyes were examined at 1, 24, 48, 72 hours and 1, 2 and 3 weeks after the application.

 

No adverse effects on body weight development were noted during the study period. The general state and behavior of animals were normal throughout the study period.

 

Eye examination:

 

During the study, no signs of eye irritation were observed in the control eye of all animals.

 

Initial Pain Reaction(IPR) (score 2) was observed in all animals after test item administration.

 

One hour after the application:

Conjunctival redness (score 2) was found in all rabbits, conjunctival chemosis (score 1) were observed in two animals and all rabbits showed conjunctival discharge (score 3).

At 24 hours after treatment:

Conjunctival redness (score 2 or 1) was found in all rabbits, chemosis (score 1).was observed in two animals. One rabbit displayed conjunctival discharge (score 1).

At 48 hours after treatment:

Conjunctival redness (score 1) was observed in two animals and redness (score 2) was noted in one rabbit.

 

At 72 hours after treatment:

Conjunctival redness (score 1) was observed in two animals.

 

At 1 week after treatment:

Conjunctival redness (score 1) was observed in 2 rabbits.

 

At 2 and 3 weeks after treatment:

No signs of eye irritation or other clinical signs were observed.


The conjunctivae were coloured by the test item during the study up to and including the 3-week observation.

 

The study was terminated after the 3-week observation.

 

The animal’s individual mean scores (considering readings at 24, 48 and 72 hours after the treatment) were as follows:

Animal Number

Cornea Opacity

Iris

Conjunctivae

Redness

Chemosis

Discharge

00427

0.00

0.00

0.67

0.00

0.33

00388

0.00

0.00

1.33

0.33

0.00

00389

0.00

0.00

1.67

0.33

0.00

The test item Reactive Yellow F01-0555, applied to rabbit eye mucosa, caused significant conjunctival irritant effects at one hour which were reduced at 24 hours after application. There were no corneal effects observed at any timepoint, all conjunctival irritation effects were completely reversed after the 2-week observation period. Some discoloration of the conjuctivae remained at the observation period of 3 weeks, but this is not considered as classifiable effect under CLP or GHS guidelines.

Reactive Yellow F01-0555 is “not classified” according to Regulation (EC) No 1272/2008.


Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 August 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants: OECD GUIDELINES FOR TESTING OF CHEMICALS 438
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
other: Chicken
Strain:
other: ROSS 308
Details on test animals or tissues and environmental conditions:
CHICKEN HEADS COLLECTION AND TRANSPORT

Species of chicken: ROSS 308
Source: TARAVIS KFT. 9600 Sárvár, Rábasömjéni út. 129.

Chicken heads were collected after slaughter in a commercial abattoir from chickens which are used for human consumption. Heads were collected by a slaughter house technician and heads transported to CiToxLAB Hungary Ltd. at ambient temperature at the earliest convenience.

After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were received at CiToxLAB Hungary Ltd. and processed within approximately 2 hours of collection.

SELECTION AND PREPARATION OF EYES FOR THE TEST

Eyes selection:

After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of fluorescein solution 2 (v/v) % was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 ml isotonic saline. Then the fluorescein-treated cornea was examined with hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.

Preparation of eyes:

The eye ball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.

Eyes examination and acclimatization time:

The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head). Again avoiding too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was needed to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with isotonic saline dripping from a stainless steel tube, at a rate of approximately 3 or 5 drops/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.

The appropriate number of eyes was selected and after being placed in the superfusion apparatus, were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the isotonic saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured, any eye with cornea thickness deviating more than 10 % from the mean value for the eyes, or eyes that showed any other signs of damage, were rejected and replaced. If the selected eyes were appropriate for the test, acclimatization started and was conducted for approximately 45 to 60 minutes. A temperature of the circulating water was verified to ensured in all chambers were in the range of 32±1.5 °C during the acclimatization and treatment periods.

Identification

The eyes were identified by chamber number, marked on the door of the chamber.

Vehicle:
unchanged (no vehicle)
Controls:
other: control eyes
Amount / concentration applied:
The Reactive Yellow F01-0555 was applied in a volume of 30 mg by powdering the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance, taking care not to damage or touch the cornea with the application equipment.
Duration of treatment / exposure:
The time of application was observed, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 ml isotonic saline at ambient temperature, taking care not to damage the cornea but attempting to remove all the residual test item if possible.
Observation period (in vivo):
The control eye and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within ±5 minutes were considered acceptable.
Number of animals or in vitro replicates:
Not applicable.
Details on study design:
THE BASE LINE ASSESSMENTS

The base line assessments:

At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a base line (t=0) for each individual eye. The cornea thickness of the eyes should not increase by more than 5-7 % between the
-45 and the zero time. Slight changes in thickness (-2% to 0%) were observed in the eyes, this is considered normal when maintaining enucleated eyes following the equilibration period, the fluorescein retention was measured. Base line values were required to evaluate any potential test item related effects after treatment; the location of any minor findings were marked on the record sheet as a drawing. All eyes were considered to be suitable for the assay.

TEST PROCEDURE

Treatment:

After the zero reference measurements, take the first eye out of its chamber and place it on a layer of tissue with the cornea facing upwards. The eye was held in horizontal position over a container to catch waste, while the Reactive Yellow F01-0555 was applied onto the centre of the cornea. The Reactive Yellow F01-0555 was applied in a volume of 30 mg by powdering the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance, taking care not to damage or touch the cornea with the application equipment.
The positive control eyes were treated in a similar way with 30 mg Imidazole.
The negative control eye was treated with 30µL of isotonic saline.

Test item removal:

The time of application was observed, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 ml isotonic saline at ambient temperature, taking care not to damage the cornea but attempting to remove all the residual test item if possible.

OBSERVATION

Observation and assessment of corneal effects:

The control eye and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse.
Minor variations within ±5 minutes were considered acceptable.

The cornea thickness and cornea opacity were measured at all time points.
Fluorescein retention was measured on two occasions, at base line (t=0) and 30 minutes after the post-treatment rinse.

STORAGE OF CORNEAS

Storage method of corneas

At the end of the procedures, the corneas from the eyes were carefully removed from the eyes and placed individually into labelled containers of preservative fluid (10% neutral buffered formalin) for potential histopathology and stored.

Irritation parameter:
other: maximum corneal swelling
Run / experiment:
1
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein retention score
Run / experiment:
1
Value:
1.17
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
In this in vitro eye irritation in the isolated chicken eyes test with Reactive Yellow F01-0555, the results suggest that the test item was not irritating. The test item was not a severe irritant.

The mean values of the treated eyes for maximum corneal thickness change, corneal opacity and fluorescein retention are given below.

Test Item: Reactive Yellow F01-0555

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

0 %

I

Mean maximum corneal swelling at up to 240 min

2 %

I

Mean maximum corneal opacity

0.17

I

Mean fluorescein retention

1.17

II

Other Observations

None

Overall ICE Class

2xI 1xII

Positive Control: Imidazole

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

6 %

II

Mean maximum corneal swelling at up to 240 min

11 %

II

Mean maximum corneal opacity

4.00

IV

Mean fluorescein retention

2.83

IV

Other Observations

The Test item was stuck on the cornea surface after the post-treatment rinse. The cornea surface was not cleared 240 min after the post-treatment rinse.

Overall ICE Class

1xII 2xIV

The positive control Imidazole was classed as severely irritating, GHS Classification: Category 1.

 

Negative Control: Sodium chloride

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

0 %

I

Mean maximum corneal swelling at up to 240 min

0 %

I

Mean maximum corneal opacity

0.00

I

Mean fluorescein retention

0.00

I

Other Observations

None

Overall ICE Class

3xI

The negative control Sodium chloride 0.9% had no significant effects on the chicken eye in this study.

Interpretation of results:
GHS criteria not met
Conclusions:
In this in vitro eye irritation study in the Isolated Chicken Eyes model with Reactive Yellow F01-0555, the results suggests that the test item was not irritating. According to the guideline OECD 438, Reactive Yellow F01-0555 does not require a classification as a severe eye irritant; an in vivo rabbit study is required for classification.
Executive summary:

An in vitro eye irritation study of the test item Reactive Yellow F01-0555 was performed in chicken’s eyes. The irritation effects of the test item were evaluated according to the OECD No.: 438 (07 September 2009).

 

After the zero reference measurements, the eye was held in horizontal position and 30 mg of Reactive Yellow F01-0555 was applied onto the centre of the cornea such that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with saline. The positive control eyes were treated in a similar way with 30 mg Imidazole. The negative control eye was treated with 30 µL of isotonic saline.

In this in vitro eye irritation study in the Isolated Chicken Eyes model with Reactive Yellow F01-0555, the results suggests that the test item was not irritating. According to the guideline OECD 438, Reactive Yellow F01-0555 does not require a classification as a severe eye irritant; an in vivo rabbit study is required for classification.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

No skin irritating effects were seen in the acute dermal toxicity study in rats. Furthermore, the in vitro skin irritation in reconstituted human skin did not give any evidence for a skin irritating potention of Reactive Yellow F01 -0555.

For this purpose, disks of EPISKIN (three units / chemical) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of test material was terminated by rinsing with PBS. Epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in 5% CO2 protected from light. The formazan extract in acidified isopropanol was then spectrophotometrically evaluated for optical density (OD) and quantified. SDS 5% and PBS treated epidermis were used as positive and negative controls, respectively. For each treated tissue viability was expressed as a % relative to negative control.

In this in vitro skin irritation test in the EPISKIN model with Reactive Yellow F01-0555 the results indicated that the test item is Non Irritant (NI) [UN GHS: No Category].

All validity criteria were within acceptable limits and therefore the study can be considered as valid.

The results of the in vitro eye irritation study in the isolated chicken eyes model with Reactive Yellow F01-0555 indicated that the test item was not irritating.

Furthermore, the irritant effects of Reactive Yellow F01 -0555 were evaluated according to the Draize method (OECD No.: 405, 2002) in an acute eye irritation study performed in New Zealand White rabbits. The test item, applied to rabbit eye mucosa, caused conjunctival irritant effects at one hour which were reduced to minimal effects at 24 hours after application. There were no corneal effects observed at any timepoint, all conjunctival effects were completely reversed after the 2-week observation period. Some discoloration of the conjuctivae remained at the observation period of 3 weeks, but this is not considered as classifiable effect under CLP or GHS guidelines.

Justification for classification or non-classification

The above studies have all been ranked reliability 1 according to the Klimisch et al system. This ranking was deemed appropriate because the studies were conducted to GLP an in compliance with agreed protocols. Sufficient dose ranges and numbers are detailed; hence it is appropriate for use based on reliability and animal welfare grounds.

The above results triggered no classification under the Dangerous Substance Directive (67/548/EEC) and the CLP Regulation (EC No 1272/2008).