Registration Dossier

Administrative data

Description of key information

Eye irritation: not irritating
Skin irritation: not irritating

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:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Qualifier:
according to
Guideline:
other: "Hazardous Substances Regulations" under the U.S. Federal Hazardous Substances Labelling Act Sect. 191.11
Version / remarks:
February 1965
GLP compliance:
no
Remarks:
pre GLP
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 12-16 weeks.
- Weight at study initiation: average body weights of 2.80 males and 2.60 kg females.
- Housing: rabbits were caged singly.
- Diet: commercial irradiated diet (Styles-Oxoid), ad libitum.
- Water: sterile filtered water, ad libitum.
- Acclimation period: one week prior to the start of the trial.

ENVIRONMENTAL CONDITIONS
- Temperature: 20 ± 1 °C
- Relative humidity: 50-70 %
- Photoperiod: animals were exposed to artificial light for 10 hours daily from 08.00 - 18.00 hours
Type of coverage:
occlusive
Preparation of test site:
other: intact shaved skin and abraded skin
Vehicle:
water
Controls:
yes
Amount / concentration applied:
10 g of the test compound was mixed with 10 ml of water to make a solution of 15 ml, 0.75 ml of which was applied to each test site on a 2.5 cm square gauze pad.
Duration of treatment / exposure:
24 hours
Observation period:
6 days
Number of animals:
3 males and 3 females
Details on study design:
TEST SITE
- Area of exposure: 24 hours prior to the dermal application, the backs of the rabbits were shaved. Two test sites lateral to the mid line of the back were used on each rabbit. Immediately before the application of the test compound, the right hand site was abraded uith the point of a sterile hypodermic needle. The abrasions were sufficiently deep to penetrate the stratum corneurn but not to damage the dermis. The left hand site remained intact.
- % coverage: area consisting of at least 10 % of the total body surface.
- Type of wrap if used: aluminium foil secured with "Sleek" adhesive tape. The test sites were then covered by a 6 wide "Coban" self adhesive bandage in order to retain the test substance in close contace with the skin.

SCORING SYSTEM
The assessment of the macroscopic skin reaction was made according to the following grading system:
Primary Irritation Score
0 Non irritating
0.1 - 0.5 Minimally irritating
0.6 - 1.5 Slightly irritating
1.6 - 3.0 Mildly irritating
3.1 - 5.0 Moderately irritating
5.1 . 6.5 Severely irritating
6.6 - 8.0 Extremely irritating

Erythema and Eschar Formation - Description
No erythema 0
Slight erythema (barely perceptible) 1
Well defined erythema 2
Moderate to severe erythema 3
Severe erythema (beet red) to slight eschar formation 4

Oedema Formation - Description
No oedema 0
Very slight oedema (barely perceptible) 1
Slight oedema (edges of area well defined by definite raising) 2
Moderate oedema (raised approximately 1 mm.) 3.
Severe-oedema (raised more than 1 mm. and extending beyond the area of exposure) 4
Irritation parameter:
erythema score
Basis:
animal: 6/6, both intact and abraded skin
Time point:
other: 24/72 hrs
Score:
< 2.3
Reversibility:
fully reversible within: 6 days
Irritation parameter:
edema score
Basis:
animal: 6/6, both intact and abraded skin
Time point:
other: 24/72 hrs
Score:
< 2.3
Reversibility:
fully reversible within: 6 days
Irritant / corrosive response data:
The primary irritation score was 1.8.
Slight to well defined erythema and wery slight to moderate oedema were seen on all rabbits 24 hours after application of the compound. By 72 hours there was only one abraded site still showing slight erythema and very slight oedema. This was normal by 6 days.
There was uery little difference in the reactions seen between abraded and intact sites.

Skin irritation reactions

N./sex Intact skin Abraded skin
24 hrs 72 hrs 6 days 24 hrs 72 hrs 6 days

Erythema

11M 1 0 0 2 0 0
13M 1 0 0 2 0 0
15M 1 0 0 1 0 0
12F 1 0 0 2 1 0
14F 1 0 0 2 0 0
16F 2 0 0 2 0 0
Mean 1.17 0.00 0.00 1.83 0.17 0.00

Oedema

11M 2 0 0 2 0 0
13M 2 0 0 2 0 0
15M 1 0 0 2 0 0
12F 2 0 0 3 1 0
14F 3 0 0 2 0 0
16F 1 0 0 1 0 0
Mean 1.83 0.00 0.00 2.00 0.17 0.00
Conclusions:
Mild irritating
Executive summary:

Method

The skin irritation was determined in an irritation test on rabbits according the "Hazardous Substances Regulations" under the U.S. Federal Hazardous Substances Labelling Act Sect. 191.11 (February 1965)., similar to OECD guideline 404.

Result

The primary irritation score was 1.8.

Mild irritating

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
April from 19th to 29th, 2016
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Justification for type of information:
Justification for Read Across is detailed in the report attached to the IUCLID section 13.
Reason / purpose:
read-across source
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
Cell source:
other: human-derived epidermal
Vehicle:
other: phosphate buffered saline (PBS)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: the reconstructed human epidermal model EpiDerm™ (EPI-200 ver. 2.0, MatTek, Bratislava, Slovakia).
- Tissue batch number: lot No. 23329.
- Sulturing: reconstructed human epidermal model have been cultured to form a multilayered highly differentiated model of the human epidermis.
- Surface: 0.63 cm².

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- Application: 25 mg of the test substance was placed directly atop the tissue moistened with 25 µl of PBS.
- Exposure: length of exposition was 60 minutes, of which 25 minutes were tissues placed at room temperature and the remaining 35 minutes at 37 ± 1 °C, 5 ± % CO2.
- Post-incubation: 23 hours and 53 minutes and 19 hours and 14 minutes
- MTT assay: after post-incubation, the MTT assay was performed by transferring the tissues to 24-well plates containing MTT medium (1 mg/ml). After 3 hour MTT incubation, the blue formazan salt formed by cellular mitochondria was extracted with 2.0 ml/tissue of isopropyl alcohol for 2 hours, room temperature and shaking.
-Optical density determination: the optical density of the extracted formazan was determined using a spectrophotometer at 570 nm.

REMOVAL OF TEST MATERIAL AND CONTROLS
- Removal: test substance was removed, tissues were rinsed and post-incubated.
- Washing: many washing cycles have been performed due to removal of the test substance from tissue surface. The test substance coloured medium during both post-incubations. Nevertheless, tissues (and walls of inserts) remained coloured even after extraction with isopropyl alcohol.

NUMBER OF REPLICATE TISSUES: three

DIRECT MTT REDUCTION
- Test item: 25 mg of the test substance is added to 1 ml MTT medium (red).
- Incubation: in the incubator (37 ± 1 °C, 5 ± 1 % CO2, moistened) for 1 hour.
- Check: if the solution changes colour from red to blue, other steps to correction have to be done.

COLOUR INTERFERENCE – first step
- Amount of test item: 50 mg of the test substance was added to 2 ml isopropyl alcohol.
- Contact time: it was allowed to extract for 135 minutes at room temperature at shaking.
- Interference check: test chemical in water and/or isopropanol absorbs light in the range of 570±30 nm.

COLOUR INTERFERENCE – second step
- Amount of test item: 25 mg was applied to two freeze-killed tissues.
- Incubation: 60 min of incubation (37 ± 1 °C, 5 ± 1 % CO2, moistened).
- Control: in addition, two freeze-killed tissues were treated with PBS for 60 min.
- Removal of test substance: the test substance was rinsed off and tissues were for approximately 42 hours in the same manner as viable tissues in MTT test.

ASSAY ACCEPTANCE CRITERIA
- Negative Control: the absolute OD of the negative control (NC) tissues (treated with sterile PBS) in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after shipping and storing procedures and under specific conditions of use. The assay meets the acceptance criterion if the mean OD570 of the NC tissues is ≥ 0.8 and ≤ 2.8. OD570 historical negative control range is 1.470-2.342.
Positive Control: a 5 % SDS (in H2O) solution is used as positive control (PC) and tested concurrently with the test chemicals. Concurrent means here the PC has to be tested in each assay, but not more than one PC is required per testing day. Viability of positive control should be within 95±1 % confidence interval of the historical data. The assay meets the acceptance criterion if the mean viability of PC tissues expressed as % of the negative control tissues is ≤ 20 %. OD570 historical positive control range is 0-0.203.
Standard Deviation (SD): since in each test skin irritancy potential is predicted from the mean viability determined on 3 single tissues, the variability of tissue replicates should be acceptably low. The assay meets the acceptance criterion if the SD calculated from individual % tissue viabilities of the 3 identically treated replicates is < 18 %.

When any of the acceptance criteria is not met the experiment has to be repeated.

DECISION CRITERIA
The cut-off values for the prediction of irritation are given below; these values are stated in OECD Test Guideline No. 439 (1), 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 %.
A single testing run composed of three replicate tissues should be sufficient for a test chemical when the classification is unequivocal. However, in cases of borderline results, such as non-concordant replicate measurements and/or mean percent viability equal to 50 ± 5 %, a second run should be considered, as well as a third one in case of discordant results between the first two runs.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 25 mg of substance/surface ratio 39.7 mg/cm2.
- Concentration: tyest item is placed directly atop to the tissue moistened with 25 µl of PBS.

POSITIVE CONTROL
- Concentration: 5 % in water solution.
Duration of treatment / exposure:
1 hour
Duration of post-treatment incubation (if applicable):
24 ± 2 hours
Number of replicates:
Three tissues are used per the test substance, for the positive and negative controls.
Irritation / corrosion parameter:
% tissue viability
Value:
> 50
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
Under the experimental design conditions, average viability of tissues treated by the test substance was 86.6 % of negative control average value, i.e. viability was > 50 %. The effect of the test substance was negative in EpiDermTM model.
According to the decision criteria, the test substance can be considered as non-irritant to skin.

COLOUTR INTERFERENCE
After extraction with isopropanol, tissues as well as walls of insert remained coloured yellow.
Based on the test results, it is evident that no contribution of colour was added to OD570 of tissues in MTT test.

DIRECT MTT REDUCTION - FUNCTIONAL CHECK IN TUBES
The test substance did not reduce MTT directly.

ACCEPTANCE CRITERIA
All the assay acceptance criteria were fulfilled.

MTT TEST - First OD570 value obtained at the MTT test.

Code Treatment  OD570 Mean SD Average viability (% NC)
1 2 3
NC PBS 1.790 1.543 1.557 1.630 0.113 100.0 
% 109.8 94.7 95.5 100.0 6.950
C5 370/14 1.179 1.466 1.592 1.412 0.173 86.6 
% 72.3 89.9 97.7 86.646 10.603
PC 5% SDS 0.054 0.051 0.05 0.052 0.002 3.2
% 3.3 3.1 3.1 3.170 0.104

COLOUR INTERFERENCE - Colour interference in frozen tissues

Treatment Absorbance of tissues Average SD NC%
1 2
PBS -0.001 -0.002 -0.0015 0.001 100.0 
% of avg NC 66.7 133.3 100 33.333
370/14 0.000 -0.001 -0.0005 0.001 33.3
% of avg NC 0.000 66.7 33.3 33.333
Interpretation of results:
other: not classified, according to the CLP Regulation (EC 1272/2008)
Conclusions:
Non-irritant
Executive summary:

The test item 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 (2015), and Protocol for: In Vitro EpiDermTM Skin Irritation Test For use with MatTek Corporation’s Reconstructed Human Epidermal Model EPI-200-SIT (2014).

After pre-incubation of tissues, 25 mg of the test substance was placed directly atop to the previously moistened tissue and it was spread on the entire tissue surface. Length of exposition was 60 minutes. Three tissues were used for the test substance and every control. After removal of the test substance, tissues were post-incubated for approximately 42 hours due to leave of damage reparation. 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.

The test substance was found to be not direct reducing, so none correction of results for this property must have been done. As the test substace is coloured (yellow), steps for determination of appropriate contribution of absorbace to viability of tissues were done. It was found, that no measurable colour passed to final extract. Average viability of treated tissues was 86.6 %, i.e.viability was > 50 %. The effect of the test substance was negative in EpiDermTM model (tissues were not damaged).

Conclusion

The test substance is considered to have no category in regard to skin irritation.

Endpoint:
skin irritation: in vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Justification for type of information:
Justification for Read Across is detailed in the report attached to the IUCLID section 13.
Reason / purpose:
read-across source
Principles of method if other than guideline:
Two rabbits were treated with 500 mg/µl/animal of test substance. After 24 hours of exposure, the substance was removed by water and soap / vegetable oil. The irritation degree was evaluated on the basis of the recommended methods for the toxicological investigation of dyes and auxiliaries.
GLP compliance:
no
Remarks:
pre GLP
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 3-4 kg.
- Housing: individuality.
Amount / concentration applied:
500 mg/µl/animal
Duration of treatment / exposure:
24 hours
Observation period:
7 days
Number of animals:
One male and one female
Details on study design:
TEST SITE
- Type of wrap if used: fixation of the test pattern by means of bandages on the ears of the rabbits.

REMOVAL OF TEST SUBSTANCE
- Washing: with water and soap / vegetable oil.
- Time after start of exposure: 24 hours

SCORING SYSTEM
ETAD - subcommittee for toxicology. Recommended methods for the toxicological investigation of dyes and auxiliaries.
Irritation parameter:
primary dermal irritation index (PDII)
Basis:
mean
Score:
0
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
Primary irritation index: 0
Conclusions:
Not skin irritating
Executive summary:

Method

Two New Zealand White rabbits, both sexes, were treated with 500 mg/µl/animal of test substance. The wrap used was fixed to the test pattern by means of bandages on the ears of the rabbits. After 24 hours of exposure, the substance was removed by water and soap / vegetable oil. The irritation degree was evaluated on the basis of the recommended methods for the toxicological investigation of dyes and auxiliaries.

Results

Primary irritation index: 0

Not skin irritating

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:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Qualifier:
according to
Guideline:
other: "Hazardous-Substances Regulations" under the U.S.A. Federal Hazardous Substances Labelling Act Sect. 191.12
Version / remarks:
February 1965
Deviations:
yes
Remarks:
slight modification
GLP compliance:
no
Remarks:
pre GLP
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Age at study initiation: 12-16 weeks.
- Weight at study initiation: average body weights of 2.80 kg males and 2.60 kg females.
- Housing: rabbits were caged singly.
- Diet: commercial irradiated diet (Styles-Oxoid), ad libitum.
- Water: sterile filtered water, ad libitum.
- Acclimation period: one week prior to the start of the trial.
- Health check: the eyes of the experimental animals were examined and found normal prior to the test.

ENVIRONMENTAL CONDITIONS
- Temperature: 20 ± 1 °C
- Relative humidity: 50-70 %
- Photoperiod: artificial light for 10 hours daily from 03.00 - 18.00 hours.

Controls:
yes
Amount / concentration applied:
100 mg of the test compound were instilled into the conjunctival sac of the left eye. The eyelids were then held closed for 1 second.
Duration of treatment / exposure:
30 seconds in three animals; in the remaining animals eyes were not washed.
Number of animals or in vitro replicates:
3 males and 3 females
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing: with approximately 200 ml of warm water.
- Time after start of exposure: 30 seconds.

TOOL USED TO ASSESS SCORE
Corneal damage was assessed after staining with fluorescein.

SCORING SYSTEM
The rabbits were examined 1, 24, 48 and 72 hours after "application of the test compound and for any further period that was considered necessary. The ocular reactions were scored by "the method described in "Appraisal of the Safety of Chemicals in Food Drugs and Cosmetics", published by the Association of Food and Drug Officials of the U.S.A.

CORNEA
A Opacity and degree of density (most dense area scored) .
No opacity: 0
Scattered or diffuse area, details of iris clearly visible: 1
Easily discernible translucent areas, details of iris slightly obscured: 2
Opalescent areas, no details of iris visible, size of pupil barely discernible: 3
Opaque, iris invisible: 4

B Area of cornea involved
One quarter (or less) but not zero: 1
Greater than one quarter, but less than half: 2
Greater than half, but less than three quarters: 3
Greater than three quarters, up to whole area: 4
A x B x 5; Maximum possible score = 80

IRITIS
A Values
Normal: 0
Folds above normal, congestion, swelling, circumcornea injection (any or all of these or combination of any thereof) iris still reacting to light (sluggish reaction is positive: 1
No reaction to light, hemorrhage, gross destruction (any or all of these): 2
A x 5; Maximum possible score = 10

CONJUNCTIVAE
A Redness (refers to lids and bulbar conjunctivae excluding cornea and iris)
Vessels normal: 0
Vessels definitely injected above normal: 1
More diffuse, deeper crimson red, individual vessels not easily discernible: 2
Diffuse beefy red: 3

B Chemosis
No swelling: 0
Any swelling above normal (includes nictitating membrane): 1
Obvious swelling with partial eversion of lids: 2
Swelling with lids about half closed: 3
Swelling with lids about half closed to completely closed: 4

C Discharge
No discharge: 0
Any amount different from normal (does not include small amounts observed in inner cantus of normal animals): 1
Discharge with moistening of the lids and hairs just adjacent to lids: 2
Discharge with moistening of the lids and hairs, and considerable area around the eye: 3
(A + B + C) x 2; Maximum possible score = 20

The eye reactions were assessed as follows:
0 Non irritant
0 - 10 Minimally irritant
11 - 25 Slightly irritant
26 - 56 Moderately irritant
57 - 84 Markedly irritant
> 84 Extremely irritant
Irritation parameter:
cornea opacity score
Basis:
animal: 6/6, both rinsed and not rinsed eyes
Time point:
24/48/72 h
Score:
< 1
Irritation parameter:
iris score
Basis:
animal: 6/6, both rinsed and not rinsed eyes
Time point:
24/48/72 h
Score:
< 1
Reversibility:
fully reversible within: 72 hrs
Irritation parameter:
conjunctivae score
Basis:
animal: 3/3, rinsed eyes
Time point:
24/48/72 h
Score:
< 2
Reversibility:
fully reversible within: 6 days
Irritation parameter:
conjunctivae score
Basis:
animal: 3/3, not rinsed eyes
Time point:
24/48/72 h
Score:
2
Reversibility:
fully reversible within: 10 days
Irritation parameter:
chemosis score
Basis:
animal: 6/6, both rinsed and not rinsed eyes
Time point:
24/48/72 h
Score:
< 2
Reversibility:
fully reversible within: 10 days
Irritant / corrosive response data:
Minimal irritant (score 0 - 10)
A slight to mild conjunctival reaction was seen in all treated eyes one hour after application of the compound. Generally this had improved very slightly by 24 hours and thereafter continued to do so until all eyes were normal by 6 days (3/3 washed eyes) and 10 days (3/3 unwashed eyes).
Slight opacity of the cornea with damage to the surface epithelium was seen in one unwashed eye at one hour only Increased folding of the iris was noted in one washed eye at 24 and 48 hours.
There was very little difference in the reactions seen between washed and unwashed eyes, apart from the washed eyes recovering slightly sooner.

Overview of results - Rinsed eyes


24 hrs 48 hrs 72 hrs Mean 24/48/72 hrs
Cornea
Animal 14 0 0 0 0.00
Animal 15 0 0 0 0.00
Animal 16 0 0 0 0.00
Iris
Animal 14 1 1 0 0.66
Animal 15 0 0 0 0.00
Animal 16 0 0 0 0.00
Conjunctival redness
Animal 14 2 2 1

1.66

Animal 15

2

2

1

1.66

Animal 16

2

1

1

1.33

Conjunctival chemosis

Animal 14

0

0

0

0.00

Animal 15

1

0

0

0.33

Animal 16

0

0

0

0.00

Overview of results - Not rinsed eyes


24 hrs 48 hrs 72 hrs Mean 24/48/72 hrs

Cornea

Animal 11 0 0 0 0.00
Animal 12 0 0 0 0.00
Animal 13 0 0 0 0.00
Iris
Animal 11 0 0 0 0.00
Animal 12 0 0 0 0.00
Animal 13 0 0 0 0.00
Conjunctival redness
Animal 11 2 2 2 2.00
Animal 12 2 2 2 2.00
Animal 13 2 2 2 2.00
Conjunctival chemosis
Animal 11 1 1 1 1.00
Animal 12 1 1 1 1.00
Animal 13 1 1 0 0.66

Details on results - Rinsed eyes

Time score

1 hr

24 hrs

48 hrs

72 hrs

6 days

10 days

Animal N.

14

15

16

14 15 16 14 15 16 14 15 16 14 15 16 14 15 16
Cornea

















A Opacity

















B Area involved

















a: AxBx5 (max 80)

















Iris


1

1











b: score x 5 (max 10)


5

5











Conjunctivae

















A Redness 1 1 1 2 2 2 2 2 1 1 1 1





B Chemosis 1 1 1
1












C Discharge 2 1 1














c: (A+B+C) x 2 (Max 20) 8 6 6 4 6 4 4 4 2 2 2 2





Total = a+b+c (Max: 110) 8 6 6 4 6 4 4 4 2 2 2 2





Not rinsed eyes

Time score

1 hr 24 hrs 48 hrs 72 hrs 6 days 10 days
Animal N. 11 12 13 11 12 13 11 12 13 11 12 13 11 12 13 11 12 13
Cornea

















A Opacity
1















B Area involved
1















a: AxBx5 (max 80)
5















Iris

















b: score x 5 (max 10)

















Conjunctivae

















A Redness 2 2 2 2 2 2 2 2 2 2 2 2 1 1 1


B Chemosis 1 2 2 1 1 1 1 1 1 1
1 1




C Discharge 1 1 1














c: (A+B+C) x 2 (Max 20) 5 10 10 6 6 6 6 6 6 6 4 6 4 2 2


Total = a+b+c (Max: 110) 8 15 10 6 6 6 6 6 6 6 4 6 4 2 2


Conclusions:
Minimal irritant
Executive summary:

Method

The eye irritation was determined in an irritation test on rabbits according to the" Hazardous-Substances Regulations" under the U.S.A. Federal Hazardous Substances Labelling Act Sect. 191.12 (February 1965), similar to OECD guideline 405.

Result

Minimal irritant

Endpoint:
eye irritation: in vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Justification for type of information:
Justification for Read Across is detailed in the report attached to the IUCLID section 13.
Reason / purpose:
read-across source
Principles of method if other than guideline:
Two rabbits were treated with 50 mg/100 μl/animal of test substance. The irritation degree was evaluated on the basis of the recommended methods for the toxicological investigation of dyes and auxiliaries.
GLP compliance:
no
Remarks:
pre GLP
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 3-4 kg.
- Housing: individuality.
Amount / concentration applied:
50 mg/100 µl/animal, introduced into the conjunctival sac.
Observation period (in vivo):
7 days
Number of animals or in vitro replicates:
One male and one female
Details on study design:
SCORING SYSTEM
ETAD - subcommittee for toxicology. Recommended methods for the toxicological investigation of dyes and auxiliaries.
Irritation parameter:
other: primary orritation index
Basis:
mean
Score:
7
Remarks on result:
probability of weak irritation
Conclusions:
Not eye irritating
Executive summary:

Method

Two rabbits were treated with 50 mg/100 μl/animal of test substance. The irritation degree was evaluated on the basis of the recommended methods for the toxicological investigation of dyes and auxiliaries.

Results

Primary irritation index: 7

Not eye irritating

Endpoint:
eye irritation: in vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Justification for type of information:
Justification for Read Across is detailed in the report attached to the IUCLID section 13.
Reason / purpose:
read-across source
Principles of method if other than guideline:
Draize test method
GLP compliance:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Summit View Farm, Hazelton, PA, USA.
- Weight at study initiation: 3 - 3.5 kg.
- Housing: rabbits were individually housed in stainless steel, wire mesh-floor cages in a room.
- Diet: feed (Certified Lab Rabbit Chow HF; Purina No. 5325) was limited to 125g/day.
- Water: ad libitum.
- Acclimation period: all animals were acclimatized for at least 2 wk prior to the start of the study at which time only healthy animals of the normal weight range found to be free from ocular irritation or damage were used for administration of test and control materials.
- Health check: all animals were checked for viability twice daily.

ENVIRONMENTAL CONDITIONS
- Temperature: maintained at 16-21 °C.
- Relative humidity: 38 %.
- Photoperiod: 12-hr light/dark cycle.
Vehicle:
other: 0.5 % (w/v) hydroxypropyl methylcellulose and 0.25 % (w/v) laureth-10 acetate
Controls:
yes, concurrent vehicle
Amount / concentration applied:
- Dose per application: 30 µl
- Concentration: test colourant was prepared daily as a 3 % (w/v) suspension in aqueous vehicle containing 0.5 % (w/v) hydroxypropyl methylcellulose and 0.25 % (w/v) laureth-10 acetate.
- Preparation: test mixtures were stirred continuously (magnetic stirrer) to ensure uniformity of the dosing solution.
- Control of dosing: samples of test and control mixtures administered were analysed for colourant content and microbiological contamination.
- Analysis: analyses for colourant were performed by Hilton Davis Co., (Cincinnati, OH, USA). All microbiological analyses were performed within 5 hr of sampling. All dosing solutions were within 10 % of the expected concentration and free of microbiological contamination throughout the study.
Duration of treatment / exposure:
mixtures administered on days 1, 7, 14 and 21, once daily.
Observation period (in vivo):
21 days
Number of animals or in vitro replicates:
6 males and 6 females
Details on study design:
APPLICATION
- Apllication: on instillation of test material, upper and lower lids were gently held together for approximately 1 sec before being released so as to prevent loss of test material.
- Control: the contralateral eye of each animal remained untreated and served as control.

TOOL USED TO ASSESS SCORE: both eyes of all animals were examined by slit-lamp bimicroscopy (including examinations of fluorescein stain retention to evaluate integrity of the corneal epithelium). Indirect ophthalmoscopic examinations (using 1 % tropicamide as mydriatic) were performed on both eyes.

SCORING SYSTEM
Ocular irritation was determined according to a modification of the Draize test (Draize, 1959). Interpretation of observations and assignment of scores were consistent with those described by the Consumer Product Safety Commission (1972). All eyes were scored for ocular irritation pretest (8 days, 24 hr and immediately prior to the initial dose) and approximately 24 hr after each treatment, prior to the next instillation of test material; on days 1, 3, 7, 14 and 21, the eyes were also evaluated for irritation 1 hr after treatment. In addition, all readily observable ocular structures were evaluated for eye stain and particle embedment 24 hr after each treatment.
The degree of ocular staining was scored (following rinsing of residual material where necessary) and noted according to the scheme given below. Ophthalmic observations were performed 7 days and 24 hr prior to the initial dose, on days 3, 7 and 14 (prior to daily dosing), and at the end of the study.

Scoring scheme for ocular staining
Intensity
No reaction 0
Barely perceptible (scattered or diffuse areas of staining) +
Slight (easily discernible stained areas; details of iris clearly visible) + 1
Moderate (moderately stained conjunctivae, and/or no details of iris visible; size of pupil barely discernible) + 2
Marked (severely stained conjunctivae, and/or iris not discernible through staining) + 3

Area of cornea involved (1 or greater)
One-quarter (or less) but not zero 1
Greater than one-quarter, but less than one-half 2
Greater than one-half, but less than three-quarters 3
Greater than three-quarters, up to whole area 4
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
EVALUATION OF EYE IRRITATION
All animals were free of significant signs of ocular irritation other than slight conjunctival redness (score of 1) or discharge (score of 1 or 2) seen sporadically in the eyes of both test and control animals throughout the study.

EVALUATION OF EYE STAINING AND EMBEDMENT
All animals were free of significant signs of eye staining and partial embedment.

OPHTHALMOSCOPIC EXAMINATION
Most animals were free of ocular abnormalities in the test eye.
Other effects:
CLINCAL OBSERVATIONS/BODY WEIGHTS
All animals survived and were free of significant clinical signs throughout the study. Most animals gained weight; single animals in both control and treated groups lost weight at one or more intervals but subsequently gained weight by the end of the study. One male rabbit exhibited slight net weight losses over the 21 days of study.

Summary of ocular effects*+ in evaluation of the (repeated application) eye irritation potential colourant

Observation

Test day

-8 -1 1 1 2 3 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 21 22

Interval (h)

- -24 24 1 24 24 1 24 24 24 1 24 24 24 24 24 24 1 24 24 24 24 24 24 24 1 24
Test eye Conjunctivae
Redness - score of 1 - - - - - - 1 1 - - 3 2 - - - - - - - - - - - - - - -
Discharge - score of 1 - - - 4 - - 1 1 - - - - - - - - - - - - - - - - - - -
Degree of staining II II II II - - II ±

- - 1 - - - - - - II - - - - - - - II -
Control Conjunctivae
Redness - score of 1 - - - 1 - - - - - - - - - - - 1 - - - - - - - - - 1 -
Discharge - score of 1 - - - - 1 - - - - - - - - - - - - - - - - - - - - - -

*Interpretation of observations and assignment of conjunctival, iridial and corneal scores were consistent with those described by the Consumer Product Safety Commission (1972). Staining was scored according to the scoring system described in the text. Unless otherwise noted, control (untreated) eyes exhibited negative scores for all parameters.

+Values represent number of animals (out of 12 animals per group) for which observations were noted during the specified time interval. Test day: number or days before or after first dose.

Interval (h): ntervals are hr following previous dose (i.e. 24 hr interval is 24 hr following previous dose, and immediately before next dose). II Evaluation not required by protocol.

¶: the area of involvement was conjuctivae only.

Conclusions:
Not eye irritating
Executive summary:

A protocol to evaluate the ocular irritation, staining, and embedding potential of FD&C colours produced by repeated topical application to rabbit eyes is described. Test materials (3 %, w/v in aqueous vehicle) were administered once daily, for a total of 21 days, to the conjunctival sac of the right eye of New Zealand White Rabbits (6 of each sex per group) at a dose volume of 30 µl. Control animals (6 of each sex) received 30 µl of the vehicle daily. All animals survived and were free of significant clinical signs of toxicity throughout the study. Ophthalmoscopic examinations revealed that all animals were free of abnormalities considered to be of clinical importance; all animals were free of significant signs of ocular irritation, staining and particle embedment.

Conclusion

Not eye irritating

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

According to the CLP Regulation (EC 1272/2008), 3.2 Skin corrosion/irritation section, skin Irritation means the production of reversible damage to the skin following the application of a test substance for up to 4 hours.

Based on the available information, Acid Yellow 017 can be considered as not skin irritating.

According to the CLP Regulation (EC 1272/2008), 3.3 serious eye damage/eye irritation section, eye irritation means the production of changes in the eye following the application of test substance to the anterior surface of the eye, which are fully reversible within 21 days of application.

Based on the available information, Acid Yellow 017 can be considered as not eye irritating.

In conclusion, the substance is not classified for the eye/skin irritation,according to the CLP Regulation (EC 1272/2008).