Registration Dossier

Administrative data

Description of key information

Skin irritation: Not classified, OECD 404 (equiv.), Anon, 1991.

Eye irritation (BCOP): Not classified, OECD 437, Anon, 2017.

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:
From the 4th June 1991 to the 25th April 1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The methodology used in this study was equivalent to the current guideline requirements at the time the study was conducted. Although there are some minor deficiencies in reporting compared to modern requirements, the study is considered suitable as a key study for skin irritation.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
not applicable
Qualifier:
equivalent or similar to
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Deviations:
not applicable
Principles of method if other than guideline:
The procedures used in this study were designed to meet the requirements of the test for skin irritation described within document L251, sn EEC Commission Directive of 25 April 1984.
GLP compliance:
yes
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 4 ºC / Dark
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: N/A
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: N/A
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
-Source: Recognised supplier
-Housing: In grid bottomed metal cages
-Diet: SQC standard rabbit pellets
-Water: main drinking water via automated watering system
-Acclimation period: 5 days

ENVIRONMENTAL CONDITION
-Temperature: 17 -21°C
-Humidity: 46 - 65%
-Air changes (per hour): Air conditioned
Photoperiod (hrs dark/hrs light): 12 hrs dark/12 hrs light
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
0.5 ml
Duration of treatment / exposure:
4 hours
Observation period:
observation was made after one, 24, 48, and 72 hours and 7 then 14days.
Number of animals:
4
Details on study design:
The day befoe dose the animals were placed in a restraining stocks and the dorsal surface of the trunk was clipped free of fur using an Oster Model A2 clipper with Angra blade.
On the day of doseing the animals were placed in a restraining stocks. 0.5ml aliqout of the test item was placed evenly over a 2.5 cm square of surgical lint. the lint was placed over the dorsal skin of the animal, the test item was held in contact with the skin under a semi-occlusive patch for 4 hour period. After which the patches were removed and skin reaction assesd after 1, 24, 48, and 72 hours and 7 and 14 days.
Irritation parameter:
erythema score
Basis:
mean
Time point:
other: 1h
Score:
2
Max. score:
2
Reversibility:
fully reversible
Irritation parameter:
edema score
Basis:
mean
Time point:
other: 1h
Score:
0
Max. score:
0
Irritation parameter:
erythema score
Basis:
mean
Time point:
24 h
Score:
1
Max. score:
2
Reversibility:
fully reversible
Irritation parameter:
edema score
Basis:
mean
Time point:
24 h
Score:
0
Max. score:
0
Irritation parameter:
erythema score
Basis:
mean
Time point:
48 h
Score:
1
Max. score:
2
Reversibility:
fully reversible
Irritation parameter:
edema score
Basis:
mean
Time point:
48 h
Score:
0
Max. score:
0
Irritation parameter:
erythema score
Basis:
mean
Time point:
72 h
Score:
2
Max. score:
3
Reversibility:
fully reversible
Irritation parameter:
edema score
Basis:
mean
Time point:
72 h
Score:
1
Max. score:
2
Reversibility:
fully reversible
Irritation parameter:
erythema score
Basis:
mean
Time point:
7 d
Score:
1
Max. score:
1
Reversibility:
fully reversible
Irritation parameter:
edema score
Basis:
mean
Time point:
7 d
Score:
0
Max. score:
0
Irritation parameter:
erythema score
Basis:
mean
Time point:
14 d
Score:
0
Max. score:
0
Irritation parameter:
edema score
Basis:
mean
Time point:
14 d
Score:
0
Max. score:
0
Irritant / corrosive response data:
Mild inflamation observed during 7 day period but all effected had dissipated by 14 days.

1 Hour Observation

 

 Rabbit Number

 Erythema

 Oedema

 Comments

 383

2

0

None 

 385

1

0

 None

 388

1

0

 None

 395

2

 None

 

24 Hour Observation

 Rabbit Number

 Erythema

 Oedema

 Comments

 383

0

0

None 

 385

1

0

 None

 388

1

0

 None

 395

2

 None

48 Hour Observation

 Rabbit Number

 Erythema

 Oedema

 Comments

 383

0

0

None 

 385

1

0

 None

 388

1

0

 None

 395

2

 None

72 Hour Observation

 Rabbit Number

 Erythema

 Oedema

 Comments

 383

1

0

None 

 385

3

2

 None

 388

2

1

 None

 395

2

1

 None

 

7 Day Observation

 Rabbit Number

 Erythema

 Oedema

 Comments

 383

0

0

Skin Thinkening

 385

1

0

 Skin Thickening

 388

1

0

 None

 395

0

 None

 

14 Day Observation

 Rabbit Number

 Erythema

 Oedema

 Comments

 383

0

0

None 

 385

0

0

 None

 388

0

0

 None

 395

0

 None

 

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was concluded to be non irritant to the skin.
Executive summary:

The skin irritation potential of the test substance was evaluated using a method similar to OECD 404 and in accordance with GLP. In the test, 0.5 ml of the test item was applied to the clipped dorsal skin of 4 albino rabbits under semi-occlusion for a 4 hour period. Mild inflammation on day 7 was observed, which disappeared by day 14. From the result of the study, it was concluded that the test item would not require classification as a skin irritant.

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:
09 August - 20 October 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study was conducted in accordance with international guidelines and in accordance with GLP. All relevant validity criteria were met.
Qualifier:
according to
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)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: Assumed stable. Stability at higher temperatures not availbale.
- Solubility and stability of the test substance in the solvent/vehicle: N/A
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: N/A
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Recognised supplier
- Number of animals: 9 eyes
- Characteristics of donor animals (e.g. age, sex, weight): Not reported
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Transported in physiological saline in a suitable container under cooled conditions.
- Time interval prior to initiating testing: Not reported
- indication of any existing defects or lesions in ocular tissue samples: Eyes exhibiting defects were discarded.
- Indication of any antibiotics used: Not reported.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): Undiluted

VEHICLE
- Amount(s) applied (volume or weight with unit): N/A
- Concentration (if solution): N/A
- Lot/batch no. (if required): N/A
- Purity: N/A
Duration of treatment / exposure:
10 ± 1 minutes
Duration of post- treatment incubation (in vitro):
120 ± 10 minutes
Number of animals or in vitro replicates:
3 replicates per test group
Details on study design:
SELECTION AND PREPARATION OF CORNEAS

Eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded. Isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1 % (v/v) L-glutamine (Life Technologies) and 1 % (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1 ºC. The corneas were incubated for the minimum of 1 hour at 32 ± 1 ºC.

QUALITY CHECK OF THE ISOLATED CORNEAS

After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

NUMBER OF REPLICATES

3 replicates per test group.

NEGATIVE CONTROL USED

Yes, physiological saline.

SOLVENT CONTROL USED (if applicable)

Not applicable.

POSITIVE CONTROL USED

Yes, ethanol.

APPLICATION DOSE AND EXPOSURE TIME

750 µL, for 10 minutes.

TREATMENT METHOD: [closed chamber / open chamber]

Closed chamber.

POST-INCUBATION PERIOD: yes/no. If YES please specify duration

Yes, for 120 ± 10 minutes.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM.
- POST-EXPOSURE INCUBATION: The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120 ± 10 minutes at 32 ± 1 ºC.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter. The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each treated cornea with the test item or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test item or positive control treated cornea. The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany).

- Corneal permeability: passage of sodium fluorescein dye measured with the aid of [UV/VIS spectrophotometry / microtiter plate reader] (OD490): Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Sigma-Aldrich, Germany) was evaluated.
The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 ml of 4 mg Na-fluorescein (Sigma-Aldrich Chemie GmbH, Germany)/ml cMEM solution. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 ± 5 minutes at 32 ± 1 ºC. After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 µl of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation. The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution has been performed, the OD490 of each reading of the positive control and the test item was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.

- Others (e.g, pertinent visual observations, histopathology): (please specify: None

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used.
Irritation parameter:
cornea opacity score
Run / experiment:
Mean value
Value:
1.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
fluorescein retention score
Run / experiment:
Mean score
Value:
0.022
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean value
Value:
1.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: None

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Range of historical values if different from the ones specified in the test guideline: Reported

Table 1:       Summary of Opacity, Permeability andin vitroscores

Treatment

 Mean Opacity1

 Mean Permeability1

 Meanin vitroIrritation Score1,2

 Negative control

1.7 

0.007 

1.8 

 Positive control (ethanol)

18.5 

2.113 

50 

 Test item

1.3 

0.022 

1.6 

 

Interpretation of results:
GHS criteria not met
Conclusions:
The test substance induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.
Executive summary:

Evaluation of the eye hazard potential of the test substance was conducted in accordance with OECD 437 using the Bovine Corneal Opacity and Permeability test (BCOP test) in accordance GLP.

Undiluted test substance was applied topically (750 µL) to the cornea of bovine eyes for 10 minutes under controlled conditions. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (ethanol) was 50 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.6 after 10 minutes of contact time.

The test substance induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin Irritation (OECD 404 – Anon., 1991): The skin irritation potential of the test substance was evaluated using a method similar to OECD 404 and in accordance with GLP. In the test, 0.5 ml of the test item was applied to the clipped dorsal skin of 4 albino rabbits under semi-occlusion for a 4 hour period. Mild inflammation on day 7 was observed, which disappeared by day 14. From the result of the study, it was concluded that the test item would not require classification as a skin irritant.

 

Eye Irritation (OECD 437 – Anon., 2017): Evaluation of the eye hazard potential of the test substance was conducted in accordance with OECD 437 using the Bovine Corneal Opacity and Permeability test (BCOP test) in accordance GLP.

Undiluted test substance was applied topically (750 µL) to the cornea of bovine eyes for 10 minutes under controlled conditions. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The meanin vitroirritancy score of the positive control (ethanol) was 50 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

The test item did not induce ocular irritation through both endpoints, resulting in a meanin vitroirritancy score of 1.6 after 10 minutes of contact time.

The test substance induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Justification for classification or non-classification

The test item did not meet classification criteria under Regulation (EC) No 1272/2008, however the substance can cause transient mild skin irritation.