Registration Dossier

Administrative data

Description of key information

Not skin irritating

Not eye 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:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
January from 10th to 13th, 2002
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:
The read across approach is detailed into the document attached to the IUCLID section 13.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Qualifier:
according to
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Type of coverage:
semiocclusive
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 0.5 g
Duration of treatment / exposure:
Single 4 hours application
Observation period:
72 hours
Number of animals:
3 males
Details on study design:
OBSERVATION TIME POINTS
1, 24, 48 and 72 hours.

SCORING SYSTEM
From Draize J H (1959) "Dermal Toxicity" in Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics. Assoc. of Food and Drug Officials of the US, Austin, Texas p47.
Irritation parameter:
erythema score
Basis:
animal: 3/3
Time point:
24/48/72 h
Score:
< 2.3
Reversibility:
fully reversible within: 72 hrs
Irritation parameter:
edema score
Basis:
animal: 3/3
Time point:
24/48/72 h
Score:
< 2.3
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
Primary irritation index and classification: 0.3, mild irritant
Very slight erythema was noted at two treated skin sites one and 24 hours after patch removal and persisted at one treated skin site at the 48-hour observation. An isolated incident of very slight oedema was noted at one treated skin site one hour after patch removal. No other signs of skin irritation were noted. Orange coloured staining was noted at al ltreated skin sites throughout the study.

Individual Skin Reactions Following 4 -hour Exposure Period

Animal N. Reaction 1 hr 24 hrs 48 hrs 72 hrs
68M Erythema 0 STA 0 STA 0 STA 0 STA
84M Erythema 1 STA 1 STA 0 STA 0 STA
83M Erythema 1 STA 1 STA 1 STA 0 STA
68M Oedema 0 0 0 0
84M Oedema 0 0 0 0
83M Oedema 0 0 0 0

STA: Orange coloured staining

Interpretation of results:
other: not classified, according to the CLP Regulation (EC) No 1272/2008
Conclusions:
Not classified, according to the CLP Regulation (EC) No 1272/2008
Executive summary:

A study was performed to assess the initation of the test material to the skin of the New Zealand White rabbit (SPL Standard Test Method 540.06). The method followed OECD Guidelines for Testing of Chemicals (1992) No. 404 "Acute Dermal lrritation/Corrosion" and Method 84 of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC).

Primary irritation index and classification: 0.3, mild irritant. Very slight erythema was noted at two treated skin sites one and 24 hours after patch removal and persisted at one treated skin site at the 48-hour observation. An isolated incident of very slight oedema was noted at one treated skin site one hour after patch removal. No other signs of skin irritation were noted. Orange coloured staining was noted at al ltreated skin sites throughout the study.

Conclusion

Mean values from gradings at 24, 48 and 72 hours after patch removal were lower than 2.3 in all animals for both erythema/eschar and oedema reactions, thus the test item does not meet the criteria to be classified as irritating, according to the CLP Regulation (EC) No 1272/2008.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
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:
The read across approach is detailed into the document attached to the IUCLID section 13.
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Remarks:
reconstructed human epidermal model EpiDermTM
Details on animal used as source of test system:
24 EPI-200 tissues (reconstructed epidermis) were obtained from MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia.
The EpiDerm™ model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm™ tissues (surface 0.6 cm²) are cultured on cell culture inserts (MILLICELLs, 10 mm diameter) and are commercially available as kits (EpiDerm™ 200), containing 24 tissues on shipping agarose.
Vehicle:
physiological saline
Details on test system:
MATERIALS AND TECHNICAL EQUIPMENT
- Laminar flow bench: HERAsafe KS 18 (Thermo ELECTRON CORPORATION)
- CO2 incubator: Heraeus BBD 6220, Incubation conditions: 37 °C ± 1 °C, 5 % ± 1 % CO2, 90 % ± 5 % humidity.
- Tissue for MTT-reduction control: EPI-200 tissue that is killed by freezing at –20°C.
- Assay medium: Dulbecco's modified eagle's medium (DMEM); for the assay and for diluting MTT (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia and Sigma, Germany).
- Wash buffer: Dulbecco's phosphate buffered saline (PBS), w/o Ca 2+, Mg 2+ (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia and Biochrom, Germany).
- Detection agent: 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia and Sigma, Germany), 1.0 mg / mL assay medium.
- Extracting agent: Isopropanol p.a.

ANALYSES
No analysis of test-substance preparation was performed, because the test substance was applied minimally moistened with PBS.

EXPERIMENTAL PROCEDURE
- Direct MTT reduction: The test substance was added to 0.9 mL of the MTT solution. The mixture was incubated in the dark at about 37°C for 3 hours. A negative control (de-ionized water) was tested concurrently. Due to the intense color of the test substance it was not possible to evaluate whether or not the test substance is able to reduce MTT directly, therefore freeze-killed control tissues (KC) were treated with the test article and the negative control in the same way as the unfrozen cells.
- Basic procedure: On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour the pre-incubation medium was replaced with fresh medium and preconditioning continued for 18 ± 3 hours. Three tissues were treated with the test substance, the PC and NC, respectively. In addition three killed tissues were used for the test substance and NC, respectively, in order to detect direct MTT reduction. 25 µL sterile PBS was applied first. Thereafter, a bulk volume of ca. 25 µL of the solid test material was applied with a sharp spoon and homogeneously distributed together with the fluid. Control tissues were concurrently applied with 30 µL of sterile PBS (NC, NC, or KC) or with 30 µL of 5% SDS (PC) or test substance (KC). A nylon mesh was placed carefully onto the tissue surface of the NC, NC, KC, and PC afterwards. The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator. The tissues were washed with sterile PBS to remove residual test material 1 hour after start of application. Rinsed tissues were blotted on sterile absorbent paper and transferred into new 6-well plates, pre-filled with 0.9 mL fresh medium. When all tissues were rinsed, the surface of each tissue was carefully dried with a sterile cotton swab. Subsequently, the tissues were incubated in the incubator at 37°C for 24 ± 2 hours. After 24 ± 2 hours the tissues were transferred into new 6-well plates pre-filled with 0.9 mL of fresh medium and placed into the incubator for additional 18 ± 2 hours post-incubation period. After the postincubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours. After incubation, the tissues were washed with PBS to stop the MTT-incubation. The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in isopropanol. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 4 microtiter wells filled with isopropanol for each microtiter plate.
- Data evaluation: The OD570 values determined for the various tissues are measures of their viability. The quotient of the OD570 of tissues treated with the test material and the mean OD570 values of the NC (percent of control) is used for evaluating whether or not a test material is an irritant. The individual tissue OD570 is calculated by subtracting the mean blank value of the respective microtiter plate from the respective individual tissue OD570 value. The mean OD570 for a test group of three tissues treated in the same way is calculated. In case of direct reduction of MTT by the test substance, the OD570 values measured in the freeze-killed control tissues (KC) will be used to correct the mean OD570 of the test-substance treated tissues (mean OD570 KC corrected). Since killed tissue might still have a residual enzyme activity that is able to produce some formazan net OD570 KC is calculated by subtracting the OD570 KC of the NC from the OD570 KC of the test substance. In case the net OD570 KC is greater than zero it is subtracted from the respective mean OD570 to result in the mean OD570 KC corrected. The mean OD570 KC corrected represents the formazan production linked to the tissue viability and therefore indicates the cytotoxic potency of the test substance. The quantification of tissue viability is presented as the quotient of the mean OD570 (or mean OD570 KC corrected, if applicable) divided by the respective OD570 NC value in percent for each exposure time.

ACCEPTANCE CRITERIA
In case one of the below given acceptance criteria is not met repetition of the test is considered.
- Barrier function and Quality control (QC): The supplier demonstrates that each batch of the model used meets the defined production release criteria. MatTek determines the ET50 value following exposure to Triton X-100 (1%) for each EpiDermTM batch. The ET50 must fall within a range established based on a historical database of results. The following acceptability range (upper and lower limit) for the ET50 is established by the supplier as described in the cited OECD Guidelines: Lower acceptance limit: ET50 = 4.0 hours; Upper acceptance limit: ET50 = 8.7 hours
- Acceptance criteria for the negative control (NC): The absolute OD570 of the negative control tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is acceptable if the mean OD570 of the NC is ≥ 0.8. The mean OD570 of the NC should not exceed 2.8.
- Acceptance criteria for the positive control (PC): 5% SDS is used as PC and reflects the sensitivity of the tissues used in the test conditions. A viability of ≤ 20% is acceptable.
- Acceptance criteria for tissue variability: For every treatment three tissues are treated in parallel. The inter-tissue variability is considered to be acceptable if the SD of %-viability is ≤ 18%.
- Acceptance criteria for killed controls (KC): The OD570 of the tissues for the KC of the NC should be ≤ 0.35. The OD570 value for direct MTT-reduction of a test substance should be ≤ 30% of the OD570 of the NC.

EVALUATION OF RESULTS
Irritant potential of the test materials is predicted from the mean relative tissue viabilities compared to the negative control tissues concurrently treated with sterile PBS. A chemical is considered as "irritant", if the mean relative tissue viability with a test material is less than or equal to 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
ca. 25 µl bulk volume (about 24 mg) of the undiluted test substance.
Duration of treatment / exposure:
1 hours
Duration of post-treatment incubation (if applicable):
42-hours post-incubation period
Number of replicates:
Three tissues were tested in parallel.
Irritation / corrosion parameter:
% tissue viability
Value:
89
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Mean OD570 values, individual and mean viability values, standard deviations and coefficient of variation

Test Substance

 

 

mean

SD

CV [%]

Negative Control

viable tissues

Mean OD570

2.151

0.011

 

Viability [% of NC]

100.0

0.5

0.5

KC tissues

Mean OD570

0.058

0.002

 

Viability [% of NC]

2.7

0.1

3.0

Test item

viable tissues

Mean OD570

1.923

0.099

 

Viability [% of NC]

89.4

4.6

5.1

KC tissues

Mean OD570 KC NC corrected

0.008

0.003

 

Viability [% of NC]

0.4

0.1

36.9

Final mean viability of tissues after KC correction [% of NC]

89.0 (%)

 

 

Positive control

viable tissues

Mean OD570

0.080

0.010

 

Viability [% of NC]

3.7

0.5

13.0

Interpretation of results:
other: not classified, according to the CLP Regulation (EC) No 1272/2008
Conclusions:
Not classified, according to the CLP Regulation (EC) No 1272/2008
Executive summary:

The skin irritation potential of the test item was investigated in reconstructed human epidermal model EpiDermTM, according to the OECD guideline 439. ca. 25 µl bulk volume (about 24 mg) of the undiluted test substance were applied to each replicate.

Average viability was 89 %, i.e.viability was higher than 50 %. The effect of the test substance was negative in EpiDermTM model (tissues were not damaged).

Thus, the test substance does not require classification and labelling for skin irritation in accordance with UN GHS.

Conclusion

Not classified, according to the CLP Regulation (EC) No 1272/2008

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:
November from 6th to 11th, 1984
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
adopted May 12, 1981
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
GLP compliance:
no
Specific details on test material used for the study:
The test article was applied undiluted.
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Kleintierfarm Madoerin AG CH 4414 Fuellinsdorf/ Switzerland.
- Age at study initiation: 14 - 15 weeks
- Weight at study initiation: 2.2 - 2.8 kg
- Housing: individually in stainless steel cages equipped with an automatic cleaning and drinking system.
- Diet: pelleted standard Kliba 341, Batch 1/84 rabbit maintenance diet, ad libitum.
- Water: community tap water from ltingen, ad libitum.
- Acclimation period: 4 days under laboratory conditions after veterinary examination.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Relative humidity: 55 ± 10 %
- Air changes: 10-15 air changes per hour.
- Photoperiod: 12 hours artificial fluorescent light/12 hours dark.
- Other: at least 8 hours music/light period.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
A single dose was administered to the left eye of each animal. The application volume was 0.1 g per animal.
Observation period (in vivo):
72 hours
Number of animals or in vitro replicates:
2 males and 1 female
Details on study design:
OBSERVATIONS
- Viability Mortality: Daily.
- Body Weights: pre-test, day 1 and at termination of test.

TOOL USED TO ASSESS SCORE: eye examinations were made with a slit-lamp 30 SL and a Varta Cliptrix diagnostic-lamp.

SCORING SYSTEM
The eyes of each animal were examined 1-, 24-, 48- and 72 hours after administration. The irritation was assessed according to the OECD Guidelines for testing of Chemicals, Section 4, number 405 "Acute Eye Irritatian/Carrosion" adopted May 12, 1981.
The corrosive properties of the test article and the color of the treated eye were described and recorded.

CORNEAL IRRITATION
Opacity: degree of density (densest area used for assessment)
No ulceration or opacity 0
Scattered or diffuse areas of opacity (other than slight dulling of normal luster), details of iris clearly visible 1
Easily discernible translucent area 1 details of iris slightly obscured 2
Nacrous area, no details of iris visible, size of pupil barely discernible 3
Opaque cornea, iris not discernible through the opacity 4

IRIDIC IRRITATION
Normal 0
Markedly deepened rugaeI, congestion, swelling, moderate circumcorneal hyperemia, or injection, any of these or combination thereof, iris still reacting to light (sluggish reaction is positive) 1
No reaction to light, hemorrhage, gross destruction (any or all of these) 2

CONJUNCTIVAL IRRITATION
Redness (refers to palpebral and bulbar conjunctivae, cornea and iris).
Blood vessels normal 0
Some blood vessels definitely hyperemic (injected) 1
Diffuse, crimson color, individual vessels not easily discernible 2
Diffuse beefy red 3

Chemosis: lids and/or nictating membranes
No swelling 0
Any swelling above normal (includes nictating membranes) 1
Obvious swelling with partial eversion of lids 2
Swelling with lids about half closed 3
Swelling with lids more than half closed 4
Irritation parameter:
cornea opacity score
Basis:
animal: 3/3
Time point:
24/48/72 h
Score:
< 1
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal: 3/3
Time point:
24/48/72 h
Score:
< 1
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal: 3/3
Time point:
24/48/72 h
Score:
< 2
Irritation parameter:
chemosis score
Basis:
animal: 3/3
Time point:
24/48/72 h
Score:
< 2
Irritant / corrosive response data:
The substance showed a primary irritation score of 1.2 when applied to the rabbit eye mucosa.
In the area of application an orange discoloration of the cornea and conjunctivae which could be related to effects of the test article was observed.
No corrosion of the cornea was observed at each of the measuring intervals.

TOXIC SYMPTOMS I MORTALITY
No acute toxic symptoms were observed in the animals during the test period, and no mortality occurred.

BODY WEIGHTS
The body weight gain of all rabbits was similar.

Reaction observed

Animal Reaction 1 hr 24 hrs 48 hrs 72 hrs Mean 24/48/72 hrs
359 M Cornea opacity 0 0 0 0 0.0
360 M Cornea opacity 0 0 0 0 0.0
361 F Cornea opacity 0 0 0 0 0.0
359 M Iris 0 0 0 0 0.0
360 M Iris 0 0 0 0 0.0
361 F Iris 0 0 0 0 0.0
359 M Conjunctival redness 0 0 0 0 0.0
360 M Conjunctival redness 0 1 1 1 1.0
361 F Conjunctival redness 0 1 1 1 1.0
359 M Conjunctival chemosis 2 1 0 0 0.3
360 M Conjunctival chemosis 1 1 0 0 0.3
361 F Conjunctival chemosis 2 1 0 0 0.3
Interpretation of results:
other: Not classified, according to the CLP Regulation (EC) No 1272/2008
Conclusions:
Not classified, according to the CLP Regulation (EC) No 1272/2008
Executive summary:

The study was performed to assess the possible irritation potential when single doses of test item are placed in the conjunctival sac of rabbit eyes. The experiment was conducted in accordance with the method and procedures described into the OECD guideline 404.

Under the conditions of the experiment the substance was found to cause a primary irritation score of 1.2 when applied to the rabbit eye mucosa.

In the area of application an orange discoloration of the conjunctivae was observed which could be related to effects of the test article.

No corrosion was observed at each of the measuring intervals.

Conclusion

The mean values from gradings at 24, 48 and 72 hours were lower than 1 for corneal opacity, lower than 1 for iritis, lower than 2 for both conjunctival redness and oedema, in all of the three tested animals.

Therefore, the substance does not meet the criteria to be classified as eye irritating, according to the CLP Regulation (EC) No 1272/2008.

Endpoint:
eye irritation: in vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
From February 05th to March 20th, 2002
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:
The read across approach is detailed into the document attached to the IUCLID section 13.
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: single application of 0.1 ml (68 mg)
Duration of treatment / exposure:
Single application
Observation period (in vivo):
21 days
Number of animals or in vitro replicates:
3 males
Irritation parameter:
cornea opacity score
Basis:
animal: 3/3
Time point:
24/48/72 h
Score:
< 1
Reversibility:
fully reversible within: 72 hrs
Irritation parameter:
iris score
Basis:
animal: 3/3
Time point:
24/48/72 h
Score:
< 1
Reversibility:
fully reversible within: 48 hrs
Irritation parameter:
conjunctivae score
Basis:
animal: 1/3
Time point:
24/48/72 h
Reversibility:
fully reversible within: 7 days
Remarks on result:
not determinable
Remarks:
due to staining
Irritation parameter:
conjunctivae score
Basis:
animal: 1/3
Time point:
24/48/72 h
Score:
< 2
Reversibility:
fully reversible within: 48 hrs
Irritation parameter:
conjunctivae score
Basis:
animal: 1/3
Time point:
24/48/72 h
Score:
2
Reversibility:
fully reversible within: 7 days
Irritation parameter:
chemosis score
Basis:
animal: 3/3
Time point:
24/48/72 h
Score:
< 2
Reversibility:
fully reversible within: 14 days
Irritant / corrosive response data:
The test material produced scattered or diffuse corneal opacity, iridial inflammation and moderate to severe conjunctival initation. All treated eyes appeared normal at the 21 -day observation.

Individual and Mean Scores for Cornea, Iris and Conjunctivae for EU Labelling Regulations

Animal N. Reaction 24 hrs 48 hrs 72 hrs 24/48/72 hrs
37 M Corneal opacity 0 0 0 0.00
57 M Corneal opacity 1 1 0 0.67
59 M Corneal opacity 0 0 0 0.00
37 M Iridial inflammation 0 0 0 0.00
57 M Iridial inflammation 1 0 0 0.33
59 M Iridial inflammation 0 0 0 0.00
37 M Conjunctival Redness ?s 2s 2s 2.00
57 M Conjunctival Redness ?s ?s ?s ?s
59 M Conjunctival Redness 1 0 0 0.33
37 M Conjunctival Chemosis 1 1 1 1.00
57 M Conjunctival Chemosis 2 1 1 1.33
59 M Conjunctival Chemosis 1 0 0 0.33

?s : Red colou¡ed staining prevented evaluation ofocular effect

Interpretation of results:
other: Not classified, according to the CLP Regulation (EC) No 1272/2008
Conclusions:
Not classified, according to the CLP Regulation (EC) No 1272/2008
Executive summary:

A study was performed to assess the irritation of the test material to the eye of the New Zealand White rabbit (SPL Standard Test Method 560.09), The method followed OECD Guidelines for Testing of Chemicals (24 April 2002) No. 405 "Acute Eye Initation/Corrosion" and Method B5 of Commission Directive 2004/73/EC (which constitutes Annex V of Council Directive 67/548/EEC). The test material produced scattered or diffuse corneal opacity, iridial inflammation and moderate to severe conjunctival initation. All treated eyes appeared normal at the 21 -day observation. Classification: at leat mild irritant (class 4 on a 1-8 scale).

Conclusion

The mean values from gradings at 24, 48 and 72 hours were lower than 1 for corneal opacity, lower than 1 for iritis, lower than 2 for oedema, in all of the three tested animals. Regarding conjuctival redness, in one animal the evaluation was not possible due to staining; in one rabbit the mean value resulted to be equal to 2 and in the other one resulted to be lower than 2. All treated eyes appeared normal at the 21 -day observation. Therefore, the substance does not meet the criteria to be classified as eye irritating, according to the CLP Regulation (EC) No 1272/2008.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
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:
The read across approach is detailed into the document attached to the IUCLID section 13.
Qualifier:
according to
Guideline:
other: OECD 492 EpiOcular Eye Irritation Test
GLP compliance:
yes (incl. certificate)
Species:
human
Details on test animals or tissues and environmental conditions:
The EpiOcular™ model (OCL-200) is a three-dimensional non-keratinized tissue construct composed of normal human derived epidermal keratinozytes used to model the human corneal epithelium. The EpiOcular™ tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs, 10 mm diameter) and are commercially available as kits (EpiOcular™ 200), containing 24 tissues on shipping agarose.
Vehicle:
water
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
ca. 50 µl bulk volume (about 26 mg) of the undiluted test substance
Duration of treatment / exposure:
6 hours
Observation period (in vivo):
18-hours post-incubation
Number of animals or in vitro replicates:
Two tissues were tested in parallel
Details on study design:
MATERIALS AND TECHNICAL EQUIPMENT
- Laminar flow bench: HERAsafe KS 18 (Thermo ELECTRON CORPORATION)
- CO2 incubator: Heraeus BBD 6220, incubation conditions: 37 ± 1 °C, 5 ± 1 % CO2, 90 ± 5 % humidity
- Spectrophotometer: SunriseTM Absorbance Reader, for the determination of the optical density of colored extracts. Measurement using a filter wavelength 570 nm without reference filter
- Tissue for MTT-reduction control: OCL-200 tissue that is killed by freezing at –20 °C
- Assay medium: Dulbecco's modified eagle's medium (DMEM); for the assay and for diluting MTT (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia and Sigma, Germany)
- Pre-treatment / wash buffer: Dulbecco's phosphate buffered saline (PBS), w/o Ca2+, Mg2+ (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia and Biochrom, Germany)
- Detection agent: 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia and Sigma, Germany), 1.0 mg / ml assay medium
- Extracting agent: Isopropanol p.a.

ANALYSES
No analysis of test-substance preparation was performed, because the test substance was applied undiluted.

EXPERIMENTAL PROCEDURE
- Direct MTT reduction: The test substance was added to 0.9 mL of the MTT solution. The mixture was incubated in the dark at about 37 °C for 3 hours. A negative control (de-ionized water) was tested concurrently. Due to the intense color of the test substance it was not possible to evaluate whether or not the test substance is able to reduce MTT directly, therefore freeze-killed control tissues (KC) were treated with the test article and the negative control in the same way as the unfrozen tissues.
- Pre-incubation of the tissues: On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 1 mL assay medium and preconditioned in the incubator at 37 °C. After 1 hour the pre-incubation medium was replaced with fresh medium and preconditioning continued in the incubator at standard culture conditions for 16 – 24 hours. After the pre-incubation the tissues were pre-treated with 20 µL of PBS in order to wet the tissue surface. The tissues were incubated at standard culture conditions for 30 minutes.
- Application of the test substance: Using a sharp spoon, a bulk volume of ca. 50 µl of the test material was applied covering the whole tissue surface. Control tissues were concurrently applied with 50 µl of sterile de-ionized water (NC, NC KC) or with 50 µl of methyl acetate (PC) or test substance (KC). After application, the tissues were placed into the incubator until the total exposure time of 6 hours was completed. To remove the test substance, the tissues were washed with sterile PBS. For this purpose the tissues were immersed and swiveled three times in each of three beakers filled with PBS. Washed tissues were immediately immersed into 12-well plates, pre-filled with 5 mL/well pre-warmed medium (post-soak immersion) in order to remove residual test substance. After 25 minutes of post-soak immersion, each tissue was dried on absorbent paper and transferred to fresh 6-well plates filled with 1 mL/well pre-warmed medium. Subsequently, the tissues were incubated at standard culture conditions for 18 hours (postincubation period).
- MTT incubation: After the post-incubation period, the assay medium was replaced by 0.3 ml MTT solution and the tissues were incubated in the incubator for 3 hours. After incubation, the tissues were washed with PBS to stop the MTT-incubation. The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in isopropanol at room temperature overnight or for at least 2 hours on a plate shaker. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 4 microtiter wells filled with isopropanol for each microtiter plate.

- Data evaluation: The OD570 values determined for the various tissues are measures of their viability. The ratio of the OD570 of tissues treated with the test material and the mean OD570 values of the NC (percent of control) is used for evaluating whether or not a test material was an irritant. The corrected measured OD570 value for each individual tissue was calculated by subtracting the mean blank value of the respective microtiter plate from the respective individual tissue OD570 value. The mean OD570 for a test group of two tissues treated in the same way was calculated. In case of direct reduction of MTT by the test substance, the OD570 values measured in the freeze-killed control tissues (KC) will be used to correct the mean OD570 of the test-substance treated tissues (mean OD570 KC corrected). Since killed tissue might still have a residual enzyme activity that is able to produce some formazan net OD570 KC is calculated by subtracting the OD570 KC of the NC from the OD570 KC of the test substance. In case the net OD570 KC is greater than zero it is subtracted from the respective mean OD570 to result in the mean OD570 KC corrected. The mean OD570 KC corrected represents the formazan production linked to the tissue viability and therefore indicates the cytotoxic potency of the test substance. The quantification of tissue viability is presented as the ratio of the mean OD570 (or mean OD570 KC corrected, if applicable) divided by the respective OD570 NC value in percent.

ACCEPTANCE CRITERIA
In case one of the below given acceptance criteria is not covered, repetition of the test was considered.
- Barrier function and Quality control (QC): The supplier demonstrates that each batch of the model used meets the defined production release criteria. MatTek determines the ET50 (min) value following exposure to 100 µl of 0.3 % Triton X-100 for each EpiOcular™ EIT (OCL-200) batch. The ET50 must fall within a range established based on a historical database of results. The following acceptability range (upper and lower limit) for the ET50 is established by the supplier as described in the cited OECD Guideline: Lower acceptance limit: ET50 = 12.2 min; Upper acceptance limit: ET50 = 37.5 min
- Acceptance criteria for the NC: The absolute OD570 of the NC-tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is acceptable if the mean OD570 of the NC is ≥ 1.0. The mean OD570 of the NC should not exceed 2.5.
- Acceptance criteria for the PC: Methyl acetate used as PC usually leads to a tissue viability of approx. 25 %. A viability of < 60 % is acceptable.
- Acceptance criteria for tissue variability: Two tissues were treated under the same conditions. A variability between the tissues is considered to be acceptable if the relative difference of the viability is < 20 %.
- Acceptance criteria for the KC: The OD570 of the killed control tissues treated as negative control should be ≤ 0.35. The value for direct MTT-reduction of a test substance should be ≤ 30 % of the NC.

EVALUATION OF RESULTS
The irritation potential of the test materials is predicted from the mean relative tissue viabilities compared to the negative control tissues concurrently treated with sterile water. A chemical is considered as "irritant", if the mean relative tissue viability with a test material is less than or equal to 60 %.
Irritation parameter:
other: mean tissue viability
Run / experiment:
Mean
Value:
77.4
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- Minimal (viable tissues) to moderate (KC tissues) test substance residues remained on the tissues after the washing procedure.
- Due to the intense color of the test substance, the ability of the test substance to reduce MTT directly could not be determined in a pretest, therefore KC tissues were applied in parallel.
- The results of the KC tissues indicate an increased MTT reduction (mean viability: 0.7% of NC). Thus for the test substance the final mean viability is given after KC correction.

Mean OD570values, individual and mean viability values and inter-tissue variability

Test Substance

 

 

mean

Inter-tissue variability [%]

NC

viable tissues

Mean OD570

1.793

 

Viability [% of NC]

100.0

7.0

KC tissues

Mean OD570

0.029

 

Viability [% of NC]

1.6

0.0

14/0416-1

viable tissues

Mean OD570

1.400

 

Viability [% of NC]

78.1

1.8

KC tissues

Mean OD570 KC NC corrected

0.013

 

Viability [% of NC]

0.7

0.4

Final mean viability of tissues after KC correction [% of NC]

77.4 %

 

pc

viable tissues

Mean OD570

0.434

 

Viability [% of NC]

24.2

1.1

Interpretation of results:
other: Not classified, according to the CLP Regulation (EC) No 1272/2008
Conclusions:
Not classified, according to the CLP Regulation (EC) No 1272/2008
Executive summary:

The eye irritation potential of the test item was investigated in EpiOcular™ model (OCL-200) is a three-dimensional non-keratinized tissue construct composed of normal human, according to the OECD guideline 492. ca. 50 µl bulk volume (about 26 mg) of the undiluted test substance were applied to each replicate.

Average viability was 77.4 %. Thus, the test substance does not require classification and labelling for eye irritation or serious eye damage in accordance with UN GHS.

Conclusion

Not classified, according to the CLP Regulation (EC) No 1272/2008

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

SKIN IRRITATION

The skin irritation potential of Direct Orange 118 was assayed in rabbits, according to the US Hazardous Substances Act, 16.09.1974 method. Unfortunately, only a brief sheet of results is available, thus details about test procedures, results and test conditions are lacking. Therefore, a reliability cannot be assigned. The substance was indicated as non skin irritating.

In order to confirm the oral acute toxicity potential of Direct Oragne 118, the available data on structural analogues Similar Substance 01 ans Similar Substance 02 have also been taken into account. The read across approach can be considered as reliable and suitable for the purpose; details and explanations are detailed in the report attached to the IUCLID section 13.

Similar Substance 01 was assessed in New Zealand White rabbit, according to the OECD Guidelines 404. Very slight erythema was noted at two treated skin sites one and 24 hours after patch removal and persisted at one treated skin site at the 48-hour observation. An isolated incident of very slight oedema was noted at one treated skin site one hour after patch removal. No other signs of skin irritation were noted. Orange coloured staining was noted at al ltreated skin sites throughout the study. The mean values from gradings at 24, 48 and 72 hours after patch removal were lower than 2.3 in all animals for both erythema/eschar and oedema reactions, thus the test item does not meet the criteria to be classified as irritating, according to the CLP Regulation (EC) No 1272/2008.

Similar Substance 02 was assessed in an in vitro experiment. The skin irritation potential of the test item was investigated in reconstructed human epidermal model EpiDermTM, according to the OECD guideline 439. Average viability was 89 %, i.e.viability was higher than 50 %. The effect of the test substance was negative in EpiDermTM model (tissues were not damaged). Thus, the test substance does not require classification and labelling for skin irritation in accordance with UN GHS.

EYE IRRITATION

The possible irritation potential of Direct Orange 118 was tested according to the method and procedures described into the OECD guideline 404. Under the conditions of the experiment the substance was found to cause a primary irritation score of 1.2 when applied to the rabbit eye mucosa. In the area of application an orange discoloration of the conjunctivae was observed which could be related to effects of the test article. No corrosion was observed at each of the measuring intervals. The mean values from gradings at 24, 48 and 72 hours were lower than 1 for corneal opacity, lower than 1 for iritis, lower than 2 for both conjunctival redness and oedema, in all of the three tested animals.

In addition, an old non-GLP experiments was performed according to the US Hazardous Substances Act, 16.09.1974. Unfortunately, only a brief sheet of results is available, thus details about test procedures, results and test conditions are lacking. Therefore, a reliability cannot be assigned. The substance was indicated as non eye irritating.

The test results are confirmed by outcomes obtained in both in in vivo and in vitro experiments perfromed on the structural analogues Similar Substance 01 and Similar Substance 02, respectively.

Justification for classification or non-classification

According to the CLP Regulation (EC) No 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.

In the key study, the mean values from gradings at 24, 48 and 72 hours after patch removal were lower than 2.3 in all animals for both erythema/eschar and oedema reactions.

According to the CLP Regulation (EC) No 1272/2008, serious eye damage means the production of tissue damage in the eye, or serious physical decay of vision, following application of a test substance, which is not fully reversible within 21 days of application.

The mean values from gradings at 24, 48 and 72 hours were lower than 1 for corneal opacity, lower than 1 for iritis, lower than 2 for both conjunctival redness and oedema, in at least two of three tested animals.

In conclusion, the substance does not meet the criteria to be classified for the eye/skin irritation, according to the CLP Regulation (EC) No 1272/2008.