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Description of key information

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:
1999-01-26 to 1999-01-29
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Version / remarks:
1992
Qualifier:
according to guideline
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Version / remarks:
1992
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: No details on the source of the test material were provided. Batch number: 323.
- Expiration date of the lot/batch: 16 December 1999 (allocated by testing facility, 1 year after reciept of the test substance).
- Purity test date: No details reported.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature in the dark.
- Stability under test conditions: Stable
- Solubility and stability of the test substance in the solvent/vehicle: The vehicle was selected nased on a pretest performed at the testing facitlity. The stability of the test substance in the vehicle was not indicated.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was moistened with Milli-U water immediately before application, to ensure close contact with the animal's skin.
- Preliminary purification step (if any): No details reported.
- Final dilution of a dissolved solid, stock liquid or gel: No details reported.
- Final preparation of a solid: No details reported.
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: At least 6 weeks old
- Weight at study initiation: Less than 3.5 kg
- Housing: Individually in cages with perforated floors (Scanuber, Denmark)
- Diet (e.g. ad libitum): approx.100 gram per day. In addition, hay was provided once a week.
- Water (e.g. ad libitum): Free Access
- Acclimation period: at least 5 days under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C
- Humidity (%): 50%
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours dark per day



Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
water
Remarks:
Volume not reported.
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 g
- Concentration (if solution): Not reported

VEHICLE
- Amount(s) applied (volume or weight with unit): Not reported.
- Concentration (if solution): Not reported. The powdery test substance was moistened with water (Milli-U), immediately before application, to ensure close contact with the animal´s skin.
- Lot/batch no. (if required): Not reported.
- Purity: Not reported.

NEGATIVE CONTROL
No details reported.

POSITIVE CONTROL
Not applicable.
Duration of treatment / exposure:
4 h
Observation period:
Mortality/Viability: Twice daily
Toxicity: At least once daily
Body weight: Day of treatment (prior to application)
Skin reactions after removal of dressing and test substance: 1, 24, 48 and 72 h
Number of animals:
3 (male)
Details on study design:
TEST SITE
- Area of exposure: 150 cm^2 (10x15 cm^2), dorsal
- Type of wrap if used: Metalline patch (Lohmann GmbH, Neuwied, Germany) of 2x3 cm. The patch was mounted on Micropore tape (3M, St. Paul, U.S.A.), which was wrapped around the abdomen and secured with Coban elastic bandage (3M, St. Paul, U.S.A.)

REMOVAL OF TEST SUBSTANCE
- Washing (if done): with water
- Time after start of exposure: 4 hours

SCORING SYSTEM: Draize scoring system, detailed in the report as follows:

Erythema and Eschar Formation:
No erythema = 0
Very slight erythema (barely perceptible)= 1
Well defined erythema = 2
Moderate to severe erthema = 3
Severe erythema (beet redness) = 4
In cases where signs of necrosis or corrosion prevent erythema scoring, the maximum grade for erythema (=4) is given.

Oedema Formation:
No oedema = 0
Very slight oedema (barely perceptbile) = 1
Slight oedema (edges of area well defined by definite raising) = 2
Moderate oedema (raised approximatley 1 mm) = 3
Severe oedema (raised more than 1 mm and extending beyond area of exposure) = 4

Irritation parameter:
edema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
edema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
edema score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
erythema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
erythema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
erythema score
Basis:
animal #3
Time point:
other: 1, 24, 48 and 72 hours
Score:
0
Max. score:
4
Irritant / corrosive response data:
No skin irritation was caused by 4 hours of exposure to the test substance. There was no evidence of a corrosive effect on the skin.
Other effects:
Yellowish staining of the treated skin was observed on day 1 (no further details reported).

Irritation

No skin irritation was caused by 4 hours of exposure

Corrosion

There was no evidence of a corrosive effect on the skin

Colouration

Yellowish staining of the treated skin by the test substance was observed on day 1.

Toxicity/Mortality

No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Interpretation of results:
GHS criteria not met
Conclusions:
No skin irritation was caused by 4 hours of exposure to the test substance. There was no evidence of a corrosive effect on the skin. Based on the results of this study, the test substance is classified as not skin irritating according to CLP criteria.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-01-31 to 2017-02-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
2013
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch Number: 600CC
- Expiration date of the lot/batch: 28 February 2017
- Purity test date: Not reported.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: Not reported
- Solubility and stability of the test substance in the solvent/vehicle: Not applicable. The test substance was administered directly.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was administered directly. Physiological saline (0.9% NaCl) was used as a moistner.
- Preliminary purification step (if any): Not reported.
- Final dilution of a dissolved solid, stock liquid or gel: Not reported.
- Final preparation of a solid: Not reported.
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Abattoir A. Moksel AG, Buchloe, Germany (isolated corneas obtained as by -product from animals freshly slaughered)
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): transported in HBSS containing Pen/strep on ice to the laboratory
- Time interval prior to initiating testing: Immediatley fter arrival of the eyes, cornea preparation was initiated.
- Indication of any existing defects or lesions in ocular tissue samples: Eyes presenting defects were discarded. The corneas was excised leaving a 2 to 3 mm rim of sclera and stored in a petri dish containig HBSS and posterior mounted on corneal holders . The corneas (which are free of defects) were incubated for one hour at 32 °C +-1.
Vehicle:
physiological saline
Remarks:
0.9 % Sodium Chloride Solution (aq)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 mg of the test substance. 750 uL of the positive control.

VEHICLE
- Amount(s) applied (volume or weight with unit): Physiological saline 0.9% NaCl was applied as a moistner. Volumes not reported.
Duration of treatment / exposure:
4 hours ± 5 minutes incubation at 32+-1 °C either the test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red).
Duration of post- treatment incubation (in vitro):
After the illuminance measurement was performed, the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI. 1 mL of a 5 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 ± 1 °C.
Number of animals or in vitro replicates:
3 corneas for the test item
3 corneas as negative controls treated with physiological saline 0.9% NaCl
3 corneas as positive controls treated with imidazole 20% in physiological saline 0.9% NaCl
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated. The eyes were carefully examined for defects and any defective eyes were discarded. The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders (Duratec GmbH) with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI). The posterior chamber was always filled first. The corneas were incubated for one hour at 32 ± 1 °C.

QUALITY CHECK OF THE ISOLATED CORNEAS:
Only corneas that had an initial illuminance reading I > I0/1.1651 lux were used for the assay. Eyes that were noted to have defects were discarded and not used on the study.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: Yes (treated with physiological saline 0.9% NaCl)

POSITIVE CONTROL USED: Yes (treated with imidazole 20% in physiological saline 0.9% NaCl)

APPLICATION DOSE AND EXPOSURE TIME: 750 mg of the test item and 750 µl per control substance. Exposure 4 hours ± 5 minutes.

TREATMENT METHOD: Test substance (open-chamber method), control substance (closed-chamber method)

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: the epithelium was washed at least three times with MEM (containing phenol red). Once the medium was free of test item, the cornea was finally rinsed with complete RPMI (without phenol red).
- POST-EXPOSURE INCUBATION: 90 minutes at 32 ± 1 °C


METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacimeter (BASF-OP3.0, Duratec GmbH)
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of [UV/VIS spectrophotometry] (OD490)
- Others (e.g, pertinent visual observations, histopathology): pertinent visual observation and histopathology (the corneas of the test group and control groups were fixed in a 10% formalin-buffered solution for histopathological assessment; histological processing and evaluation of all preserved tissues to haematoxylin and eosin stained microscope slides (approximate thickness of 4 µm))


SCORING SYSTEM: In Vitro Irritancy Score (IVIS). This was detailed in the study report as follows:
= 3 = No UN GHS Category
> 3; = 55 = No UN GHS Prediction can be made
>55 = Category 1.
An identification of test substances that should be classified as irritating to eyes (UN GHS Category 2 or Category 2A) or test substances that should be classified as mildly irritating to eyes (UN GHS Category 2B) cannot be made.

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used: Yes.


Irritation parameter:
in vitro irritation score
Value:
12.64
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Other effects / acceptance of results:
The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid.
The negative control responses resulted in opacity and permeability values which were less than the established upper limits for background bovine corneas treated with the respective negative control.
At the microscopic assessment, all of the 3 corneas treated with the test substance showed a slight opacity of the tissue and were reported to be distinctively different from controls. They were all consistently characterized by moderately increased cytoplasmic acidophilia (eosinophilia) of the most superficial layers of the corneal epithelium, sometimes submembranous, with occasional extension of this change in individual cells of the mid and basal layers. This main change was associated with slight to moderate desquamation and a brown cytoplasm involving the most superficial layers. Minimal to slight cytoplasmic vacuolation and cell swelling was also consistently noted. In the most affected cornea moderate desquamation was associated with slight epithelial erosion/ulcer, and slight necrosis of the corneal endothelium.

The mean in vitro irritation score was calculated to be 12.64. According to IVIS criteria, no predication could be made regarding the UN GHS classification of the test substance.

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
No prediction can be made regarding the classification of the test substance according to the evaluation criteria. However, all 3 corneas treated with the substance showed a slight opacity of the tissue.
Based on a worst case decision, the substance will be classified for CLP Category 1 (H318).
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

According to Annex VII guidelines (ECHA 2015),in vitrotesting for eye irritation is recommended as part of an integrated testing strategy. The BCOP assay (OECD 437) is suitable for this class of materials and is sufficiently robust to determine the eye irritation potential. The assay generates an In Vitro Irritancy Score (IVIS). This assay is capable of identifying materials with CLP Category 1 responses (Serious eye damage). This assay can also identify materials with little or no eye irritation potential for which no classification is necessary. Responses from the BCOP resulting in either of these results are considered conclusive and no further testing is required. If the results from the BCOP fall outside the range of either of these two classification ranges, then additional testing would be possible to better define the eye irritation classification (i.e. Category 2A or 2B).

For the test substance, a BCOP assay was conducted and a mean IVIS score of 12.64 was determined.

The in vitro irritation score was not high enough to be classified as a Category 1 eye irritant, but not low enough to be unclassified.

Therefore, no prediction can be made regarding the classification of the test substance according to the evaluation criteria of the BCOP assay as the IVIS result fall outside of the classification ranges. However, it can be assumed according to results of histopathology that the test item requires classification for an eye irritant.

Therefore, based on the available data and assuming a worst case scenario decision, the substance will be classified for CLP Category 1 Eye Irritant (H318).

Justification for classification or non-classification

Skin irritation/corrosion:

Not classified based on the available data.

Eye irritation:

Based on the available data and assuming a worst case scenario decision, the substance will be classified for CLP Category 1 Eye Irritant (H318).

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