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Administrative data

Description of key information

Based on an in vitro skin irritation test, conducted according to OECD 439 and GLP principles, it was concluded that the substance is not irritating to the skin. Based on an in vivo eye irritation study, conducted in accordance with OECD 405 and according to GLP principles, it was concluded that the substance is also not irritating to the eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January 20, 2014 - January 27, 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
(2013)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
(2012)
Deviations:
no
GLP compliance:
yes
Species:
human
Details on test animals and environmental conditions:
ENVIRONMENTAL CONDITIONS
- All incubations, with the exception of the test substance incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 30 - 97%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 29.6 – 37.7°C).
- Temporary deviations from the temperature and humidity were caused by opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity, as the OD570 of the negative control and the mean relative tissue viability of the positive control were all within the acceptability criteria of the assay.
Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
TEST MATERIAL
- Amounts applied: 10.8 to 11.5 mg, 5 μL Milli-Q water to moisten skin.

NEGATIVE CONTOL:
- Amount applied: 25 µL Phosphate buffered saline

POSITIVE CONTROL
- Amount applied: 25 µL
- Concentration: 5% (aq) Sodium dodecyl sulphate in Phosphate buffered saline
Duration of treatment / exposure:
15 minutes
Details on study design:
TEST SITE
- EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 14-EKIN-001). This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

TEST FOR REDUCTION OF MTT
The substance was checked for possible direct MTT reduction before the study was started. To assess the ability of the test substance to reduce MTT, 10.1 mg of the test substance was added to 2 ml MTT solution (0.3 mg/ml in PBS). The mixture was incubated for 3 hours at 37°C. A negative control, sterile Milli-Q water was tested concurrently.

APPLICATION/TREATMENT
The test was performed on a total of 3 tissues per test substance together with negative and positive controls. The skin was moistened with 5 μl Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test substance to the tissue and the solid test substance (10.8 to 11.5 mg; with a small glass weight boat) was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μl PBS (negative control) and 3 tissues with 25 μl 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time.

REMOVAL OF TEST SUBSTANCE
- Washing: phosphate buffered saline
- Time after start of exposure: 15 minutes

POST INCUBATION PERIOD
- 42 hours

SCORING SYSTEM:
- Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues.
Irritation / corrosion parameter:
other: other: tissue viability
Value:
96
Remarks on result:
other:
Remarks:
Basis: other: percentage of control. Time point: 15 minutes. Remarks: Negative control = 100%; Positive control = 27%. (migrated information)
Irritant / corrosive response data:
Mean tissue viability for the test substance was > 50%, therefore the test substance is considered not to be irritant to the skin.

The test substance was checked for possible direct MTT reduction by adding the test substance to MTT medium. Because no colour change was observed it was concluded that the test substance did not interact with MTT.

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The in vitro skin irritation test was conducted according to OECD 439 guideline and GLP principles. It is concluded that this test is valid and that the test substance is not irritating in the in vitro skin irritation test.
Executive summary:

In an in vitro skin irritation test using a human skin model ( EPISKIN Small Model), conducted according to OECD 439 and GLP principles,

the influence of the test substance on the viability of human skin was tested. The test substance was applied directly to 0.38 cm2 cultured skin (10.8 to 11.5 mg, in presence of 5 μL Milli-Q water). After 15 minutes, the substance was removed and cells were cultured for 42 hours. The viability of the cells was tested by reduction of MTT. Survival of unexposed skin was set at 100%, the positive control had a mean cell viability of 27% whereas the test substance showed cell viability of 96%. Since the mean relative tissue viability after exposure to the test substance was above 50%, it can be concluded that the test substance is not irritating in the in vitro skin irritation test.

Based on the results of the in vitro skin irritation study, the substance does not need to be classified for skin irritation/corrosion in accordance with the CLP Regulation.

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

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 31, 2014 - April 21, 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
(2012)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Version / remarks:
(2008)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2400 (Acute Eye Irritation)
Version / remarks:
(1998)
Deviations:
no
Qualifier:
according to
Guideline:
other: Food and Agricultural Materials Inspection Centre (FAMIC), 12 Nohsan, Notification No. 8147, April 2011; including the most recent partial revisions.
Deviations:
no
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
- Source: Charles River France, L'Arbresle Cedex, France
- Age at study initiation: 12 - 13 weeks old
- Weight at study initiation: 2.9 - 3.1 kg
- Housing: Individually housed in cages with perforated floors.
- Diet: Free access to pelleted diet for rabbits (Global Diet 2030 Harlan Teklad, Italy). Hay and wooden sticks were available during the study period.
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 – 24
- Humidity (%): 40 - 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
unchanged (no vehicle)
Controls:
other: One eye of each animal remained untreated and served as the reference control.
Amount / concentration applied:
TEST MATERIAL- Amount(s) applied (volume or weight with unit):
average 98.6 mg (range: 98.4 mg – 98.8 mg) (a volume of approximately 0.1 mL)
Duration of treatment / exposure:
Single instillation on Day 1.
Observation period (in vivo):
7 days
Number of animals or in vitro replicates:
3 males
Details on study design:
STUDY DESIGN
The study was performed in a stepwise manner and was started by treatment of a single rabbit (sentinel). The two other animals were treated in a similar manner two weeks later, after considering the degree of eye irritation observed in the first animal.

PREEMPTIVE PAIN MANAGEMENT
One hour prior to instillation of the test substance, buprenorphine (Buprenodale®, Dechra Ltd., Stokeon-Trent, United Kingdom) 0.01 mg/kg was administered by subcutaneous injection in order to provide a therapeutic level of systemic analgesia. Five minutes prior to instillation of the test substance, two drops of the topical anesthetic lidocaïne eyedrops (AST Farma BV, Oudewater, The Netherlands) were applied to both eyes.

TREATMENT
After instillation of the test substance, as such, in the conjunctival sac of one of the eyes, the lids were gently held together for about one second to prevent loss of the test substance. Immediately after the 1 hour observation, the treated eye was rinsed with approximately 50 mL tepid tap water, using a velocity of flow which did not affect the eye, to remove any visible residual test substance. For reference control the other eye was also rinsed. Immediately after the 24-hour observation, a solution of 2% fluorescein in water (adjusted to pH 7.0) was instilled into both eyes of each animal to quantitatively determine corneal epithelial damage. Immediately after fluorescein examination on Day 2, in order to provide a continued level of systemic analgesia, buprenorphine 0.01 mg/kg and meloxicam 0.5 mg/kg were administered by subcutaneous injection.

REMOVAL OF TEST SUBSTANCE
-Washing (if done): Yes, immediately after the 1 hour observation, with approximately 50 mL tepid tap water

OBSERVATIONS
- Mortality/Viability: Twice daily
- Toxicity: At least once daily.
- Body Weight: Day of treatment (prior to instillation) and after the final observation
- Necropsy: No necropsy was performed
- Irritation:
The eyes of each animal were examined approximately 1, 24, 48 and 72 hours and 7 days after instillation of the test substance.
The irritation scores and a description of all other (local) effects were recorded. The irritation was assessed according to OECD 405.
Irritation parameter:
cornea opacity score
Remarks:
(opacity)
Basis:
animal: #1, #2 and #3
Time point:
other: Mean value of score at 24, 48 and 72 h after installation
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
animal: #1, #2 and #3
Time point:
other: Mean value of score at 24, 48 and 72 h after installation
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Remarks:
(redness)
Basis:
animal: #1, #2 and #3
Time point:
other: Mean value of score at 24, 48 and 72 h after installation
Score:
1.3
Max. score:
3
Reversibility:
fully reversible within: 7 days
Irritation parameter:
chemosis score
Basis:
animal: #1 and #3
Time point:
other: Mean value of score at 24, 48 and 72 h after installation
Score:
0.3
Max. score:
4
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
other: Mean value of score at 24, 48 and 72 h after installation
Score:
0.7
Max. score:
4
Reversibility:
fully reversible within: 72 hours
Irritant / corrosive response data:
Instillation resulted in effects on the iris and conjunctivae. Iridial irritation grade 1 was observed in all animals and resolved within 24 hours. The irritation of the conjunctivae consisted of redness, chemosis and discharge and completely resolved within 7 days in all animals. No corneal opacity was observed, and treatment of the eyes with 2% fluorescein 24 hours after instillation revealed no corneal epithelial damage. There was no evidence of ocular corrosion.
Other effects:
- Remnants of the test substance were present in the eye on Day 1 in all animals
- No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred
Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In an eye irritation study with rabbits, performed according to OECD/EC test guidelines, limited irritation was observed.
Executive summary:

In an eye irritation study in rabbits, conducted in accordance with OECD 405 (2012) and according to GLP principles, single samples of approx. 98 mg of the substance (a volume of approximately 0.1 mL) were instilled into one eye of each of three male rabbits. Observations were made 1, 24, 48 and 72 hours and 7 days after instillation. Instillation resulted in effects on the iris and conjunctivae. Iridial irritation grade 1 was observed in all animals and resolved within 24 hours. The irritation of the conjunctivae consisted of redness, chemosis and discharge and was completely resolved within 7 days in all animals. Based on the results of this study, the substance does not need to be classified for eye irritation/corrosion in accordance with the CLP Regulation.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation

In an in vitro skin irritation test using a human skin model ( EPISKIN Small Model), conducted according to OECD 439 and GLP principles, the influence of the test substance on the viability of human skin was tested. The test substance was applied directly to 0.38 cm2cultured skin (10.8 to 11.5 mg, in presence of 5 μL Milli-Q water). After 15 minutes, the substance was removed and cells were cultured for 42 hours. The viability of the cells was tested by reduction of MTT. Survival of unexposed skin was set at 100%, the positive control had a mean cell viability of 27% whereas the test substance showed cell viability of 96%. Since the mean relative tissue viability after exposure to the test substance was above 50%, it can be concluded that the test substance is not irritating in the in vitro skin irritation test.

Eye irritation

Using the Bovine Corneal Opacity and Permeability test (BCOP test) the substance was screened for its eye irritancy potential in accordance with OECD 437 and according to GLP principles. The substance was applied as a 20% (w/w) suspension (750 µL) directly on top of the corneas. Adequate negative and positive controls were included.

The substance induced ocular irritation through both endpoints in the category mild to moderate, resulting in a mean in vitro irritancy score (IVIS) of 3.5 after 240 minutes of treatment. Based on the results of this study, the substance does not need to be classified as Eye Irr. Cat. 1, as the mean IVIS was determined to be ≤ 55. But since the substance did induce an IVIS > 3, it cannot be concluded whether the substance needs to be classified or not and therefore an in vivo study is needed in accordance with REACH Annex VIII.

In this in vivo study in rabbits, conducted in accordance with OECD 405 (2012) and according to GLP principles, single samples of approx. 98 mg of the substance (a volume of approximately 0.1 mL) were instilled into one eye of each of three male rabbits. Observations were made 1, 24, 48 and 72 hours and 7 days after instillation. Instillation resulted in effects on the iris and conjunctivae. Iridial irritation grade 1 was observed in all animals and resolved within 24 hours. The irritation of the conjunctivae consisted of redness, chemosis and discharge and was completely resolved within 7 days in all animals.


Justification for selection of skin irritation / corrosion endpoint:
Only one study available, suitable for the endpoint conclusion.

Justification for selection of eye irritation endpoint:
There are 2 studies available, an in vitro and an in vivo study. But the selected study is the in vivo study, which is suitable for the endpoint conclusion.

Justification for classification or non-classification

Based on the results of the in vitro skin irritation study, the substance does not need to be classified for skin irritation/corrosion in accordance with the CLP Regulation, since the mean relative tissue viability after exposure to the substance was above 50%. Based on the results of the eye irritation study in rabbits, the substance does not need to be classified for eye irritation/corrosion in accordance with the CLP Regulation, since there was no evidence of ocular corrosion, all observed ocular effects reversed within the obervation period and the mean scores at 24, 48 and 72 h after installation were all below the criteria of classification as mentioned in Tables 3.3.1 and 3.3.2.