Registration Dossier

Administrative data

Description of key information

Skin irritation (EU, B.4): not irritating

Eye irritation (OECD 438): non-corrosive

Eye irritation (OECD 492): irritating potential

Overall conclusion: irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 - 29 August 1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Version / remarks:
1984
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Ranch Rabbits, Crawley Down, Sussex
- Sex: female
- Age at study initiation: 11-13 weeks
- Weight at study initiation: 2 kg
- Housing: individually in grid bottomed metal cages
- Diet: antibiotic free rabbit diet (SQC standard rabbit pellets produces by Special Diets Services, Witham, Essex) ad libitum
- Water: drinking water via automatic nozzels ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 23
- Humidity (%): 51 - 70
- Photoperiod (hrs dark / hrs light): 12/12
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 0.5 g powder with 0.5 mL destilled water. 0.5 g of the test material was placed evenly over a 2.5 cm square of surgical lint.
Duration of treatment / exposure:
4 hours
Observation period:
7 days after removal of the patches
Number of animals:
4
Details on study design:
TEST SITE
- Area of exposure: left flank, immediately caudal to the last rib
- % coverage: not specified
- Type of wrap if used: Elastoplast, elastic adhesive bandage 10 cm wide

REMOVAL OF TEST SUBSTANCE
- Washing: yes, gentle swabbing with cotton wool soaked in warm water
- Time after start of exposure: 4 hours

OBSERVATION TIME POINTS
1, 24, 48 and 72 hours and 7 days after removal of the patches

SCORING SYSTEM:
- Method of calculation: Irritation was assessed and allocated a numerical value based on erythema and eschar formation or oedema formation (Draize scoring system).
Irritation parameter:
erythema score
Basis:
animal: 1, 2 &4
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritation parameter:
erythema score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0.166
Max. score:
4
Reversibility:
fully reversible within: 48 h
Irritation parameter:
edema score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritant / corrosive response data:
Irritation of the skin (erythema score 0.5 of 4) was only apparent in animal 3 after 1 and 24 h, after 48 h no effect was recorded.
Other effects:
- Other adverse local effects: not specified
- Other adverse systemic effects: not specified
Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
No skin irritating effects of the test item were recorded after 48h.
Executive summary:

The test was based on the descriptions for skin irritation, EEC Commission Directive of 25 April 1984 (84/449/EEC), page 106 to 108 of document L251. 0.5 mL aliquots were applied over an areea of 6 cm² on the dorsal skin, clipped free of fur, of four albino rabbits. The material was held in contact with the skin under a semi-occlusice dressing for a four hour period after which the patches were removed. Skin reaction to the materials was assessed after 1, 24, 48 and 72 h and 7 days. Irritation of the skin was only apparent after 1 and 24 h, after 48 h no effect was recorded.

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

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 2017 - February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Version / remarks:
July, 2015
Qualifier:
according to
Guideline:
other: MatTek Corporation Protocol: EpiOcular™ Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; for use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model
Version / remarks:
29 June 2015
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Species:
other: Human Cornea Model
Details on test animals or tissues and environmental conditions:
EpiOcular™ kits and MTT-100 kits are purchased from MatTek Corporation (82105 Bratislava, Slovakia). The EpiOcular™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. The EpiOcular™ tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELL, 10 mm diameter).
EpiOcular™ tissues were shipped at 2 - 8 °C on medium-supplemented agarose gels in a 24-well plate on Tuesday. On day of receipt of the EpiOcular™ tissues, the equilibration step (15 minutes at room temperature in the 24-well shipping container) started. 1.0 mL of the medium was aliquoted into the appropriate wells of pre-labeled 6-well plates.
Each 24-well shipping container was removed from its plastic bag under sterile conditions and its surface disinfected by wiping with 70% isopropanol- or ethanol-soaked tissue paper. The sterile gauze was removed and each tissue was inspected for air bubbles between the agarose gel and insert. The tissues were carefully removed from the 24-well shipping containers using sterile forceps. Any agarose adhering to the inserts was removed by gentle blotting on sterile filter paper or gauze. The insert was then transferred aseptically into the 6-well plates and pre-incubated at standard culture conditions for one hour in the Assay Medium. After one hour, the Assay Medium was replaced by 1 mL fresh Assay Medium at 37 °C and the EpiOcular™ tissues were incubated at standard culture conditions overnight (16 -24 hours).

- RhCE tissue construct used, including batch number: EpiOcular™ (MatTek Corporation, Bratislava, Slovakia), Lot No.: 27012
- Tissue viability: The quality of the final product was assessed by undertaking an MTT cell viability test. The determined OD (540 - 570 nm) was 1.504 ± 0.057 (acceptance criteria: 1.1 - 3.0).
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 µL of 0.3% Triton X-100. The ET-50 value was determined to be 14.5 min (acceptance criteria: 12.2 - 37.5 min).
- Contamination: The cells used to produce the EpiOcular tissue were screened for the presence of viruses, bacteria, yeast and other fungi.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 50 mg

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL
Duration of treatment / exposure:
6 h
Duration of post- treatment incubation (in vitro):
25 min immersion incubation after rinsing, followed by 18 h incubation in humidified atmosphere
Number of animals or in vitro replicates:
2
Details on study design:
Assessment of Direct MTT Reduction by the Test Item:
Test items may have the ability to directly reduce MTT and to form a blue/purple reaction product which could have an impact on the quantitative MTT measurement. Therefore, it was necessary to assess this ability for the test item prior to conducting any assays with viable tissues. For this purpose approximately 50 mg of the test item were added to a 1 mL of a 1.0 mg/mL MTT solution (in DMEM) in a glass tube and the mixture was incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air for three hours. A control (50 μL of deionised water in 1 mL of 1.0 mg/mL MTT solution) was run concurrently. If the MTT solution colour turned blue/purple, the test item was presumed to have reduced the MTT. Since the MTT solution colour did not turn blue/purple, the test item was not presumed to be a MTT reducer, and an additional test with freeze-killed tissues was not necessary.

Assessment of Coloured or Staining Materials:
Coloured test items or test items which become coloured after application to the tissues may interfere with the quantitative photometric MTT measurement, if the colourant binds to the tissue and is extracted together with MTT. Therefore, each test item has to be checked for its colouring properties. Since the test item was non-coloured, additional tests had to be performed to assess, if it becomes coloured after contact with water or isopropanol. For this purpose each approximately 50 mg of the test item was added to 1.0 mL of water and to 2 mL isopropanol in a glass tube. The water mixture was incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air for one hour, the isopropanol mixture for 3 hours at room temperature.Since the test item did not become coloured either in water or isopropanol, it was not considered as possibly interacting with the MTT measurement and an additional test with viable tissues (with medium instead of MTT addition) did not have to be performed.

Experimental Performance:
After the overnight incubation, the tissues were pre-wetted with 20 μL of Ca2+Mg2+free-DPBS. The tissues were incubated at standard culture conditions for 30 minutes. After the 30 minute Ca2+Mg2+free-DPBS pre-treatment, the test and control item were tested by applying approximately 50 mg (test item) or 50 μL (controls) topically on the EpiOcular™ tissues. The tissues were incubated at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH) for 6 hours. At the end of the 6 hours treatment time, the test item was removed by extensively rinsing the tissues with Ca2+Mg2+-free DPBS (brought to room temperature). Three clean beakers containing a minimum of 100 mL each of Ca2+Mg2+-free DPBS were used per test item. The test item utilized a different set of three beakers. The inserts containing the tissue were lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. To assure throughput, the tissues were rinsed two at a time by holding replicate inserts together by their collars using forceps. The test or control items were decanted from the tissue surface onto a clean absorbent material (paper towel, gauze, etc.) and the cultures dipped into the first beaker of DPBS, swirled in a circular motion in the liquid for approximately 2 seconds, lifted out so that the inserts are mostly filled with DPBS, and the liquid was decanted back into the container. This process was performed two additional times in the first beaker. The culture was then be rinsed in the second and third beakers of DPBS three times each in the same fashion. Finally, any remaining liquid was decanted onto the absorbent material. Decanting was most efficiently performed by rotating the insert to approximately a 45° angle (open end down) and touching the upper lip to the absorbent material (to break the surface tension). After rinsing, the tissues were immediately transferred to and immersed in 5 mL of previously-warmed assay medium (room temperature) in a pre-labelled 12-well plate for a 25 minutes immersion incubation (post-soak) at room temperature. This incubation in assay medium was intended to remove any test item or control absorbed into the tissue. At the end of the post-soak immersion, each insert was removed from the assay medium, the medium was decanted off the tissue, and the insert was blotted on absorbent material and transferred to the appropriate well of the pre-labelled 6-well plate containing 1 mL of warm assay medium. The tissues were incubated for 18 hours at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 (post-treatment incubation).

MTT Assay:
At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into the 24-well plate containing 0.3 mL of MTT solution. Once all the tissues were placed into the 24-well plate, the plate was incubated for 180 minutes at standard culture conditions. Inserts were removed from the 24-well plate after 180 minutes; the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labelled 6-well plate containing 2 mL isopropanol in each well so that no isopropanol is flowing into the insert. The plates were sealed with parafilm (between the plate cover and upper edge of the wells) or a standard plate sealer, and were immediately extracted (shaken for 2 to 3 hours at room temperature). For this procedure it was necessary to seal the plates particularly thorough since a higher evaporation rate had to be expected due to the larger surface of wells in 6-well plates. The extract solution was mixed and two 200 μL aliquots were transferred to the appropriate wells of a pre-labelled 96-well plate(s). The absorbance at 570 nm (OD570) of each well was measured with a plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro Enterprise, version 4.7.1). No reference wavelength measurement was used.

Acceptability of the Assay:
The results are acceptable according to MatTek Protocol, if:
1) The negative control OD is > 0.8 and < 2.5,
2) The mean relative viability of the positive control is below 50% of the negative control viability.
3) The difference of viability between the two relating tissues of a single test item is < 20% in the same run (for positive and negative control tissues and tissues of test items). This applies also to the freeze-killed tissues (items and negative control) and the additional viable tissues (without MTT addition) which are calculated as percent values related to the viability of the relating negative control.
Irritation parameter:
other: cell viability (relative absorbance value)
Run / experiment:
1
Value:
2.8
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control OD is > 0.8 and < 2.5 (1.494 and 1.569)
- Acceptance criteria met for positive control: The mean relative viability of the positive control is below 50% of the negative control viability (28.6%)
- The difference of viability between the two relating tissues of a single item is < 20% (values between 0.3% and 2.7%) in the same run (for positive and negative control tissues and tissues of single test items)
Irritant / corrosive response data:
Relevant irritating effects were observed following 6 hours incubation with the test item. The mean relative absorption value of the tissues corresponding to the cornea viability decreased to 2.8% compared with the value of the negative control (threshold for irritancy: ≤ 60%)

Table 2: Results after treatment for 6 hours with the test item and the controls

Treatment Group

Tissue No.

OD 570 nm Well 1

OD 570 nm Well 2

Mean OD of 2 Wells

Mean OD

of 2 Wells blank

corrected

Mean

OD

of Treatment Group

blank corrected

Rel. Viability [%] Tissue 1, 2

Absolute Value of the Difference of Rel.Viability Tissue 1,2[%]

Mean Rel. Viability

[%]

Blank

0.035

0.034

0.035

 

Negative Control

1

1.543

1.494

1.518

1.484

1.507

98.5

3.0

100.0

2

1.569

1.560

1.564

1.530               

101.5

Positive Control

1

0.493

0.479

0.486

0.451

0.431

30.0

2.7

28.6

2

0.446

0.446

0.446

0.411

27.3

Test Item

1

0.076

0.075

0.076

0.041

0.043

2.7

0.3

2.8

2

0.080

0.080

0.080

0.045

3.0

Interpretation of results:
other: Eye irritating potential according to OECD TG 492
Conclusions:
This in vitro study was performed to assess the eye irritation potential of the test item by means of the Human Cornea Model Test according to OECD 492. The cornea viability decreased to 2.8 %, therefore an eye irritation cannot be excluded.
Executive summary:

This in vitro study was performed to assess the eye irritation potential of the test item by means of the Human Cornea Model Test according to OECD 492 and GLP. The test item did not prove to be an MTT reducer in the MTT pre-test. Also, its intrinsic colour was not intensive and it did not prove to dye water or isopropanol in the colour interference pre-test. Therefore, additional tests with freeze-killed tissues or viable tissues (without MTT addition) did not have to be performed. Each 50 mg of the test item were applied to each of duplicate tissue for 6 hours. Each 50 μL of the negative control (deionised water) and of the positive control (methyl acetate) were also applied to duplicate tissues each. After treatment with the negative control the absorbance values were well within the required acceptability criterion of OD > 0.8 and < 2.5, thus showing the quality of the tissues. Treatment with the positive control induced a decrease below 50% viability compared with the negative control value in the relative absorbance, thus ensuring the validity of the test system. The difference of viability between the two relating tissues was < 20% in the same run (for test item tissues, positive and negative control tissues). Irritating effects were observed following incubation with the test item. Compared with the value of the negative control, the relative mean absorption value corresponding to the viability of the tissues decreased below 60% (2.8%). In conclusion, it can be stated that in this study and under the experimental conditions reported, the test substance possesses an eye irritating potential.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 2017 - February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
26 July 2013
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Species:
chicken
Strain:
other: Gallus Gallus e.g. Ross 308 Broiler
Details on test animals or tissues and environmental conditions:
TEST SYSTEM
- Source: chicken heads of Baileys Turkeys Ltd., Cheshire, UK
- Age at study initiation: 56 days
- Weight at study initiation: 3kg

Heads were removed immediately after the chickens had been humanely killed at the source, for use on the same day. The time interval between collection of chicken heads and placing the eyes in the superfusion chamber following enucleation was minimized although all eyes had to fall within the acceptance criteria identified in the test guideline. Following slaughter, the intact chicken heads were placed into individual plastic compartments within a plastic box in order to minimize any damage to the eyes. The base of each compartment was lined with a paper towel moistened with isotonic saline. The heads were transported to the test facility at ambient temperature.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
0.03 g of test item, 0.03 g of the positive control item, 0.03 mL of the negative control item
Duration of treatment / exposure:
10 seconds
Duration of post- treatment incubation (in vitro):
30, 75, 120, 180 and 240 minutes (±5 minutes)
Number of animals or in vitro replicates:
The test item and positive control item groups consisted of three eyes and the negative control item group consisted of two eyes.
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
Eyes that had a high baseline fluorescein staining (>0.5) or corneal opacity score (>0.5) after the enucleation process were rejected. Eyelids were carefully excised whilst taking care not to damage the cornea. The integrity of the cornea was measured with a drop of 2% (w/v) sodium fluorescein to the surface of the cornea and then rinsed with isotonic saline after a few seconds. The treated eyes were examined with the use of the Haag-Streit BQ 900 (Switzerland) microscope, to examine for damage to the cornea. An acceptable eye for the ICE test was one where the fluorescein retention and corneal opacity scores are ≤ 0.5. Acceptable eyes were dissected from the skull and pulled from the orbit by holding the nictitating membrane firmly with surgical forceps. The tissue behind the eye was carefully removed with bent, blunt-tipped scissors. Once the eye was removed from the orbit a portion of the optic nerve remained. Other connective tissue was removed from the eye on an absorbent tray liner. Enucleated eyes were transferred to an appropriate clamp keeping the cornea vertical. They were then transferred to chambers within the superfusion apparatus ensuring the corneas received sufficient isotonic saline from the saline drip. The temperature of the chambers was at 32 ±1.5 °C. Once all eyes were placed in the superfusion apparatus, the eyes were examined again with the Haag-Streit BQ 900 to ensure the eyes had not been damaged by the dissection procedure. Corneal thickness measurements are taken with a depth measuring device no. 1 on the Haag-Streit BQ 900 slit-lamp microscope at the center of each cornea. Eyes were replaced when: (i) the fluorescein score was > 0.5; (ii) the corneal opacity score was > 0.5; or (iii) there was any additional signs of damage, (iv) the corneal thickness measurements for individual eyes deviated more than 10% from the mean value for all eyes. After the approval process the eyes were incubated for at least 45 minutes for equilibrium purposes. Time zero measurements for corneal thickness and opacity were taken to serve as a baseline. The baseline for the fluorescein measurements were taken at dissection.

EQUILIBRATION AND BASELINE RECORDINGS
Eyes that had a high baseline fluorescein staining (>0.5) or corneal opacity score (>0.5) after the enucleation process were rejected.

NUMBER OF REPLICATES
The test item and positive control item groups consisted of three eyes and the negative control item consisted of two eyes.

NEGATIVE CONTROL USED
Sodium chloride 0.9% w/v

POSITIVE CONTROL USED
Imidazole

APPLICATION DOSE AND EXPOSURE TIME
Immediately following the zero reference measurements, each test eye (including clamp) was removed from the superfusion apparatus and placed horizontally (cornea facing upwards) into a petri dish. Approximately 0.03 g of test item was applied to the cornea. The entire surface of the cornea was evenly covered. The test item remained in place for 10 seconds and was then rinsed from the eye using 20 mL of isotonic saline. The treated eye (including clamp) was subsequently returned to the superfusion apparatus in the original upright position. Approximately 0.03 g of the positive control item, Imidazole, was similarly applied to the cornea of each positive control eye and 0.03 mL of the negative control item was applied to the cornea of each negative control eye.

OBSERVATION PERIOD
Treated corneas were evaluated prior to treatment and at 30, 75, 120, 180 and 240 minutes (±5 minutes) after the eyes had been decontaminated with the isotonic saline.

METHODS FOR MEASURED ENDPOINTS
Corneal opacity was calculated with the most densely opacified areas for scoring. The mean value for all test eyes was calculated for all time points. The highest mean score, as observed at any time point was given an overall category for each test item.
The mean fluorescein retention scores for all test eyes are calculated at the 30 minute time interval only. These measurements are used for the overall classification for each test item. Pitting, sloughing, roughening of the corneal surface, and adhering of test item are all morphological effects that can be noted on the cornea. The classification of these findings was subject to interpretation.

SCORING SYSTEM:
- Mean corneal swelling (%):
Percentage corneal swelling was assessed from corneal thickness measurements. The calculation was expressed in the following formula:
(corneal thickness at time (t) - corneal thickness at time =0) / (corneal thickness at time =0) x 100
Mean percentage of corneal swelling for all test eyes was calculated for all the time points. The overall category score was determined from the highest mean score for epithelial swelling as observed at any time point.

- Mean maximum opacity score:
Corneal opacity was calculated with the most densely opacified areas for scoring. The mean value for all test eyes was calculated for all time points. The highest mean score, as observed at any time point was given an overall category for each test item.

Opacity scores:
0 = No opacity
0.5 = Very faint opacity
1 = Scattered or diffuse areas; details of the iris are clearly visible
2 = Easily discernible translucent area; details of the iris are slightly obscured
3 = Severe corneal opacity; no specific details of the iris are visible; size of the pupil barely discernible
4 = Complete corneal opacity; iris invisible

- Mean fluorescein retention score at 30 minutes post-treatment:
The mean fluorescein retention scores for all test eyes are calculated at the 30 minute time interval only. These measurements are used for the overall classification for each test item.

Fluorescein retention scores:
0 = No fluorescein retention
0.5 = Very minor single cell staining
1 = Single cell staining scattered throughout the treated area of the cornea
2 = Focal or confluent dense single cell staining
3 = Confluent large areas of the cornea retaining fluorescein.


DECISION CRITERIA
Once each endpoint had been established, ICE classes were determined based on a predetermined range. Interpretation of the endpoints was classified as in the ICE Classification Criteria.
A test was considered acceptable if the concurrent negative or vehicle/solvent items and the concurrent positive controls were identified as GHS Non-Classified and GHS Category 1, respectively.
Irritation parameter:
cornea opacity score
Run / experiment:
1
Value:
2
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein retention score
Run / experiment:
1
Value:
2
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
percent corneal swelling
Remarks:
maximal mean
Run / experiment:
1
Value:
22.86
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: Corneal Epithelium Condition
Run / experiment:
1
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Result: Normal
Other effects / acceptance of results:
Easily discernible translucent area; details of the iris clearly visible were noted in all test item treated eyes. Complete corneal opacity; iris invisible was noted in all positive control treated eyes. Very faint opacity was noted in the negative control treated eyes. No morphological effects were noted in the test item or control item treated eyes. Sloughing was noted in one positive control treated eye.
Focal or confluent dense single cell staining was noted in all test item treated eyes. Confluent large areas of the cornea retaining fluorescein were noted in all positive control treated eyes. Very minor single cell staining was noted in one of the negative control treated eyes.

Table 3: Individual Scores and Mean Scores for Corneal Effects – Test Item

End Point

Eye Number

Time (after decontamination)

0 minutes

30 minutes

75 minutes

120 minutes

180 minutes

240 minutes

Corneal Opacity

3A

0.5

0.5

1

2

2

2

6A

0.5

1

2

2

2

2

8A

0

1

2

2

2

2

Mean

0.3

0.8

1.7

2.0

2.0

2.0

ICE Class

III

Fluorescein Retention

3A

 

2

 

 

 

 

6A

 

2

 

 

 

 

8A

 

2

 

 

 

 

Mean

 

2.0

 

 

 

 

ICE Class

III

Corneal Thickness

3A

0.68

0.75

0.78

0.92

0.82

0.85

6A

0.70

0.76

0.78

0.82

0.82

0.86

8A

0.72

0.78

0.80

0.82

0.94

0.86

Mean

0.70

0.76

0.79

0.85

0.86

0.86

Mean Corneal Swelling (%)

 

9.05

12.38

21.90

22.86

22.38

ICE Class

III

Corneal Epithelium Condition

3A

N

N

N

N

N

N

6A

N

N

N

N

N

N

8A

N

N

N

N

N

N

ICE Classes Combined:

3 x III

Classification:

No prediction can be made

Interpretation of results:
other: not severely eye damaging (Cat. 1) according to OECD 438
Conclusions:
The study was performed according to OECD 438 to evaluate the possible corrosivity or severe irritancy potential of the test item as measured by its ability to induce toxicity in an enucleated chicken eye. The substance proved to be not severely eye damaging (Cat. 1).
Executive summary:

This ex vivo study was performed to assess the eye irritation potential of the test item by means of the Isolated Chicken Eye Test according to OECD 438 and GLP. The study was performed to evaluate the possible corrosivity or severe irritancy potential of the test item as measured by its ability to induce toxicity in an enucleated chicken eye. 0.03 g of the test item was applied onto the cornea of each of three enucleated eyes. A further three enucleated eyes were treated with positive control item (Imidazole). A further two enucleated eyes were treated with saline for control purposes. Treatment with the positive control showed 3x ICE Class IV, thus ensuring the validity of the test system. Treatment with the test item showed a maximal mean score for corneal opacity of 2 (ICE Class III), a mean score of flourecein retention of 2 (ICE Class III) and a maximal mean corneal swelling compared to time zero of 22.86% (ICE Class III). In conclusion, it can be stated that in this study and under the experimental conditions reported, the substance proved to be not severely eye damaging (Cat. 1).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation

The test was based on the descriptions for skin irritation, EU B.4 (1984). 0.5 mL aliquots were applied over an areea of 6 cm² on the dorsal skin, clipped free of fur, of four albino rabbits. The material was held in contact with the skin under a semi-occlusice dressing for a four hour period after which the patches were removed. Skin reaction to the materials was assessed after 1, 24, 48 and 72 h and 7 days. Irritation of the skin was only apparent after 1 and 24 h, after 48 h no effect was recorded.

Eye irritation OECD 492

This in vitro study was performed to assess the eye irritation potential of the test item by means of the Human Cornea Model Test according to OECD 492 and GLP. The test item did not prove to be an MTT reducer in the MTT pre-test. Also, its intrinsic colour was not intensive and it did not prove to dye water or isopropanol in the colour interference pre-test. Therefore, additional tests with freeze-killed tissues or viable tissues (without MTT addition) did not have to be performed. Each 50 mg of the test item were applied to each of duplicate tissue for 6 hours. Each 50 μL of the negative control (deionised water) and of the positive control (methyl acetate) were also applied to duplicate tissues each. After treatment with the negative control the absorbance values were well within the required acceptability criterion of OD > 0.8 and < 2.5, thus showing the quality of the tissues. Treatment with the positive control induced a decrease below 50% viability compared with the negative control value in the relative absorbance, thus ensuring the validity of the test system. The difference of viability between the two relating tissues was < 20% in the same run (for test item tissues, positive and negative control tissues). Irritating effects were observed following incubation with the test item. Compared with the value of the negative control, the relative mean absorption value corresponding to the viability of the tissues decreased below 60% (2.8%). In conclusion, it can be stated that in this study and under the experimental conditions reported, the test substance possesses an eye irritating potential.

Eye irritation OECD 438

This ex vivo study was performed to assess the eye irritation potential of the test item by means of the Isolated Chicken Eye Test according to OECD 438 and GLP. The study was performed to evaluate the possible corrosivity or severe irritancy potential of the test item as measured by its ability to induce toxicity in an enucleated chicken eye. 0.03 g of the test item was applied onto the cornea of each of three enucleated eyes. A further three enucleated eyes were treated with positive control item (Imidazole). A further two enucleated eyes were treated with saline for control purposes. Treatment with the positive control showed 3x ICE Class IV, thus ensuring the validity of the test system. Treatment with the test item showed a maximal mean score for corneal opacity of 2 (ICE Class III), a mean score of flourecein retention of 2 (ICE Class III) and a maximal mean corneal swelling compared to time zero of 22.86% (ICE Class III). In conclusion, it can be stated that in this study and under the experimental conditions reported, no predicitons can be made about the irritating potoential of the test item. The substance has not to be classified as eye damaging cat. 1.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on skin irritation, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.

Based on available data on eye irritation, the test item is classified as eye irritant, Cat 2 (H319) according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.