Registration Dossier

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Description of key information

An in vitro study was conducted to evaluate the skin irritation potential of the read-across substance bisamide (UVCB) using the EpiskinTMreconstituted human epidermis model according to EU Method B.46 and OECD Guideline 439 in compliance with GLP. The test substance and both the negative and positive controls were applied topically on triplicate tissues and incubated at room temperature for 15 (± 1) minutes. At the end of the treatment period, each tissue was rinsed with Dulbecco’s phosphate buffered saline (D-PBS) and incubated for 42 (± 1) h at 37°C / 5% CO2in a humidified incubator. Cell viability was then assessed by means of the colorimetric MTT ((3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) reduction assay. Relative viability values were calculated for each tissue and expressed as a percentage of the mean viability of negative control tissues which was set at 100% (reference viability). Also, the concentration of the inflammatory mediator IL-1α was assessed in the culture medium retained following the 42 h recovery period.All acceptance criteria for the negative and positive controls were fulfilled and hence the study was considered to be valid. Following a 15 minutes exposure and a 42 h recovery period, the relative mean viability of the tissues treated with the test substance was 109%, with a standard deviation of 5%. As the mean relative viability was > 50% after MTT reduction, culture media samples retained from the three negative controls and the three test substance-treated tissues were analysed by ELISA. The mean concentration of IL-1α within the retained medium was 53.5 pg/mL. As this value was below 60 pg/mL, the test substance was considered to be non-irritant to skin (Gerbeix, 2012a).

A second in vitro study was conducted to evaluate the skin corrosion potential of the read-across substance bisamide (UVCB) using the EpiskinTMreconstituted skin model according to EU Method B.40 and OECD Guideline 431 in compliance with GLP. The test substance, negative and positive controls were applied on duplicate tissues and incubated at room temperature as follows: positive and negative controls for 240 (± 5), test substance for 3 (± 5 seconds), 60 (± 5 seconds) and 240 (± 5) minutes. At the end of the designated incubation period, each tissue insert was rinsed with D-PBS and then incubated with 0.3 mg/ml MTT solution for 180 minutes at room temperature. At the end of the incubation period, the tissues were removed from their plastic inserts. Tissue discolouration was evaluated with the naked eye (discoloured surface area and intensity). The epidermis was then separated from the collagen matrix and both parts were put into acidified isopropanol overnight to extract the formazan (reduced MTT) out of the MTT‑loaded tissues. At the end of the extraction period, the optical density of each extract was measured at 570 nm. Relative viability values were calculated for each tissue and expressed as percentages of the negative control tissue viability which was set at 100% (reference viability).

The relative mean viabilities of the test substance-treated tissues were as follows:

-        3-minute exposure: 86% with a standard deviation of 1%,

-        60-minute exposure: 79% with a standard deviation of 4%,

-        240-minute exposure: 104% with a standard deviation of 1%.

The blue discolouration of the test substance-treated tissues following the 3, 60 and 240 minute exposure periods was representative of viable tissue. Hence, the acceptance criteria were fulfilled and the study was therefore considered to be valid. Under the conditions of thein vitrostudy, the test substance was considered to be non-corrosive to skin (Gerbeix, 2012b).

The in vitro testing was followed by an in vivo study in New Zealand White rabbits run according to OECD Guideline 404 and EU Method B.4 in compliance with GLP. Here, 0.5 g of the undiluted test substance (moistened with drinking water treated by reverses osmosis) was applied to a skin area of approximately 6 cm2under semi-occlusive conditions for 4 h. Each animal was observed once a day for mortality and clinical signs. The cutaneous reactions were evaluated approximately 1, 24, 48 and 72 h after removal of the dressing and then daily until the reversibility of cutaneous reactions. The mean values of the scores for erythema and edema were calculated for each animal. Body weight was recorded at the beginning and the end of the observation period. No unscheduled deaths occurred during the study and no clinical signs were noted in any animal. The body weight of the animals was unaffected by treatment. After a 4 h exposure, a well-defined erythema was noted from Day 1 until Day 2 in 2/3 animals or until Day 3 in 1/3 animals, followed by a very slight erythema up to Days 3, 4 or 7. Mean scores calculated for each animal over 24, 48 and 72 h were as follows:

-        Erythema: 1.7, 1.0, 1.3; showing no significant inflammation;

-        Edema: 0.0, 0.0; 0.0; showing no significant inflammation.

Under the study conditions, the test substance produced slight skin irritation in rabbits which was reversible within 7 d (Gerbeix, 2012c).

Eye irritation

An in vitro study was performed to assess the ocular irritancy potential of the read-across substance bisamide (UVCB) in rabbit following application onto the cornea of enucleated eyes in compliance with GLP. A volume of 0.1 mL of test substance, which was found to weigh approximately 48 mg, was applied onto the cornea of each of three enucleated rabbit eyes which had been maintained at a temperature of 32 ± 1.5°C within a superfusion chamber. A further two enucleated eyes were treated, for control purposes, with saline solution (0.9% sodium chloride). Effects on corneal opacity, condition of the corneal epithelium, fluorescein uptake (240 minutes following treatment) and the percentage change in corneal thickness (corneal swelling) were observed 60, 120, 180 and 240 minutes after treatment. No corneal effects (opacity) were noted in the test eyes or control eyes during the study period (i.e. 240 minutes after treatment). Corneal swelling of the test eyes during the study period was comparable to that observed in the control eyes over the same period. No fluorescein uptake was seen in test and control eyes and the condition of the corneal epithelium in the test eyes or controls appeared normal during the study period. Under the study conditions, the test substance was therefore considered unlikely to have the potential to cause severe ocular irritancyin vivo(Sanders, 2012a).

A follow-up in vivo study was conducted to evaluate the acute eye irritation potential of the read-across substance bisamide (UVCB) in New Zealand White rabbits according to the EU Method B.5 and OECD Guideline 405 in compliance with GLP. A volume of 0.1 mL of test substance (i. e, 66 mg) was placed into the conjunctival sac of the right eye of two New Zealand White rabbits without irrigation. The left eye remained untreated and was used for control purposes. Assessment of ocular damage/irritation was made approximately 1, 24, 48 and 72 h following treatment according to the Draize scoring method. No corneal or iridial effects were noted during the study. Minimal conjunctival irritation was seen in both animals 1 and 24 h after treatment and in one animal at the 48 h observation. These effects were reversible within 48 h in one animal and within 72 h in the other. In conclusion, under the conditions of the study, the test substance produced minimal conjunctival irritation in rabbits which was reversible within 72 h (Sanders, 2012).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
From 25 September 2012 to 04 October 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Read across study hence maximum reliability rating of 2 assigned according to ECHA guidance.
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
unchanged (no vehicle)
Details on test system:
Species: Human reconstructed epidermis (tissues).
Supplier: TM Model Kit (0.38 cm2 tissues) supplied by SkinEthic Laboratories, Nice, France.
Selection: At receipt, the medium quality and temperature indicators were checked to ensure the good quality of the tissues before use.
Storage conditions: The living tissues were kept at room temperature from receipt at CiToxLAB France until the end of the in vitro phase.
Description: The EpiskinTM model consists of an airlifted, living, multilayered epidermal tissue construction (surface 0.38 cm2), reconstituted from normal human epidermal keratinocytes for 13 d and produced in polycarbonate inserts in a serum-free and chemically defined medium. The model features a normal ultra-structure and is functionally equivalent to human in vivo epidermis.

Medium and Incubation T°C: 37°C
Dates of experimental phase: From 25 September 2012 to 04 October 2012.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL ADMINISTRATION:
- Amount(s) applied (volume or weight with unit): 20 mg +/- 2 mg per tissue.
The test substance was spread over the whole tissue surface without damaging the tissue sample. As the test substance was a solid, 100 μL of 0.9% NaCl were applied over the test substance to ensure good contact with the epidermis.
Duration of treatment / exposure:
Exposure period of 3, 60 and 240 minutes, followed by rinsing.
Observation period:
MTT-loading after rinsing. Observation of MTT-> formazan transformation by viable cells, after 3-h MTT incubation.
Number of animals:
Duplicate tissues for each timepoint and test substance, negative control, positive control.
Details on study design:
POSITIVE CONTROL: Glacial acetic acid.

NEGATIVE CONTROL: 0.9% NaCl.

PRELIMINARY TEST:
As a test substance may directly reduce MTT, thus mimicking mitochondrial succinate deshydrogenase activity, it is necessary to test the ability of the dose formulation to directly reduce MTT before performing the main test. This property of the dose formulation would only be a problem if, at the time of the MTT viability test (after rinsing), there is still a sufficient amount of dose formulation present on or in the tissue. In this case, the true metabolic MTT reduction and the false direct MTT reduction should be differentiated and quantified.
To identify any test substance interference, the following preliminary test was performed:
0. 20 mg of dose formulation was added to 2.2 mL of a 0.3 mg/mL freshly prepared MTT solution. The mixture was incubated in darkness at 37°C for 3 h (± 5 minutes). A negative control was tested concurrently by adding 50 μL of 0.9% NaCl to 2.2 mL of a 0.3 mg/mL freshly prepared MTT solution. If the MTT solution colour turns blue/purple when compared to the negative control (complete or partial discolouration), the dose formulation is presumed to reduce MTT.

MAIN TEST:
One 12-well plate was used for each of the three exposure times (3, 60 and 240 minutes) for test substance-treated tissues. Positive and negative controls were placed on separate plates (one plate for each). Test substance, and negative and positive controls were applied on duplicate tissues.

TREATMENT OF TISSUES:
Firstly, 2.2 mL assay medium, pre-warmed (at 37°C), were added to two wells per 12-well plate as follows: Duplicate wells for each test substance exposure time and for the 240-minute positive and negative control exposure times.
EpiskinTM kits were opened and one tissue was transferred into each assay medium pre-filled well, and then test substance, positive control or negative control dose formulations were applied on each designated tissue. The lids were replaced on each plate before incubation at room temperature as follows: positive and negative controls for 240 minutes (± 5 minutes); test substance for 3 minutes (± 5 seconds), 60 minutes (± 5 minutes) and 240 minutes (± 5 minutes).
Positive and negative controls incubated for 240 minutes (± 5 minutes) acted as controls for all the incubation times.
Tissues were processed (application and rinsing) in the same order and at regular time-intervals so the tissue exposure times were equal.

REMOVAL OF TEST SUBSTANCE
- Rinsing: For all treated tissues (test substance-treated, positive and negative controls), at the end of the designated incubation period each tissue insert was removed from the well of the treatment plate and rinsed with Dulbecco’s phosphate buffered saline (D PBS).
Rinsing was achieved by gently filling and emptying each tissue insert 12 times with 2 mL D-PBS to gently remove any residual dose formulation.

MTT VIABILITY ASSAY
Two empty wells of each 12-well plate were filled with 2.2 mL MTT solution (0.3 mg/mL) and the corresponding tissues were placed in these wells. Each plate was protected from light and incubated for 3 h (or 180 minutes) (± 5 minutes) at room temperature.
At the end of the MTT incubation period, the underside of each tissue culture insert was blotted. Then the tissues were removed from its plastic insert using a biopsy punch. Any tissue discolouration was evaluated with the naked eye.
Thereafter, for each tissue, the epidermis was separated from the collagen matrix. Both parts (epidermis and collagen matrix) were put into a stoppered plastic tube containing 0.85 mL acidified isopropanol. After vortexing, each tube was protected from light and left at room temperature overnight to extract the
formazan (reduced MTT) out of the MTT-loaded tissues.

OPTICAL DENSITY MEASUREMENTS:
At the end of the extraction period, each tube was vortexed, then any cell fragments were allowed settle so that they do not interfere with the absorbance readings.
Each tube was used to fill three consecutive wells of a 96-well plate with 200 μL of extract per well. One 96-well plate was used for the negative and positive controls (placed at opposite ends of the plate), another 96-well plate was used for the test substance dose formulation (the extracts obtained after all exposure times were placed on the same plate).
For each 96-well plate, the average Optical Density value (OD) of six wells containing 200 μL of acidified isopropanol only was used as the blank. The OD was measured at 570 nm using a Multiskan Ascent plate reader.


SCORING SYSTEM:
- Optical density (OD) was measured at 540 nm:
The mean cOD values (background corrected mean OD values of the duplicate tissues) were calculated, based on the OD triplicate values measured for each tissue. The mean blank OD values were calculated from the six replicates on each plate.
The following formulas were used:
Mean cOD Negative Control = mean ODNC – mean ODblank
Mean cOD Positive Control = mean ODPC – mean ODblank
Mean cOD Test substance = mean ODTI – mean ODblank
The cODNC was set at 100% viability and used as a reference.
Relative viability values for the test substance and positive control dose formulations were expressed as percentages of the reference viability (cODNC) and were calculated as follows:
Relative mean viability (%) = 100 x mean cOD(test substance) / mean cOD(negative control)

ACCEPTANCE CRITERIA FOR NEGATIVE AND POSITIVE CONTROLS:
The test complies when:
- The mean cOD (i.e., corrected OD values) of the negative control is between 0.115 and 0.400,
- Relative mean viability of the positive control is ≤ 20% of the relative mean viability of the negative control.

Interpretation: See below
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean
Run / experiment:
3 minutes
Value:
86
Negative controls validity:
valid
Remarks:
100%
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean
Run / experiment:
60 minutes
Value:
79
Negative controls validity:
valid
Remarks:
100
Irritation / corrosion parameter:
% tissue viability
Remarks:
mean
Run / experiment:
240 minutes
Value:
104
Negative controls validity:
valid
Remarks:
100%
Other effects / acceptance of results:
PRELIMINARY TEST
During the preliminary test, the MTT solution colour did not turn blue/purple when compared to the negative control, the dose formulation is presumed not to reduce MTT. As a result, no additional controls were performed on water-killed tissues in parallel to the main test (performed on viable tissues).

MAIN TEST
All acceptance criteria were fulfilled. (Refer to the attached PDF under attached background material for Table 1 and 2).

Evaluation of the results:
The relative mean viabilities of the test substance-treated tissues were:
- 3 minutes exposure: 86% with a standard deviation of 1%,
- 60 minutes exposure: 79% with a standard deviation of 4%,
- 240 minutes exposure: 104% with a standard deviation of 1%.
The blue discolouration of the test substance-treated tissues following the 3-minute, 60-minute and 240-minute exposure periods was representative of viable tissue.
Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the in vitro study, the test substance was considered to be non-corrosive to the skin.
Executive summary:

An in vitro study was conducted to evaluate the skin corrosion potential of the test substance using the EpiskinTMreconstituted skin model according to EU Method B.40 and OECD Guideline 431 in compliance with GLP.

The test substance, negative and positive controls were applied on duplicate tissues and incubated at room temperature as follows: positive and negative controls for 240 (± 5), test substance for 3 (± 5 seconds), 60 (± 5 seconds) and 240 (± 5) minutes. At the end of the designated incubation period, each tissue insert was rinsed with D-PBS and then incubated with 0.3 mg/ml MTT solution for 180 minutes at room temperature. At the end of the incubation period, the tissues were removed from their plastic inserts. Tissue discolouration was evaluated with the naked eye (discoloured surface area and intensity). The epidermis was then separated from the collagen matrix and both parts were put into acidified isopropanol overnight to extract the formazan (reduced MTT) out of the MTT‑loaded tissues. At the end of the extraction period, the optical density of each extract was measured at 570 nm. Relative viability values were calculated for each tissue and expressed as percentages of the negative control tissue viability which was set at 100% (reference viability).

The relative mean viabilities of the test substance-treated tissues were as follows:

-        3-minute exposure: 86% with a standard deviation of 1%,

-        60-minute exposure: 79% with a standard deviation of 4%,

-        240-minute exposure: 104% with a standard deviation of 1%.

The blue discolouration of the test substance-treated tissues following the 3, 60 and 240 minute exposure periods was representative of viable tissue. Hence, the acceptance criteria were fulfilled and the study was therefore considered to be valid. Under the conditions of the in vitro study, the test substance was considered to be non-corrosive to skin.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
From 02 October 2012 to 16 October 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Read across study hence maximum reliability rating of 2 assigned according to ECHA guidance.
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
unchanged (no vehicle)
Details on test system:
EpiskinTM Model Kit (0.38 cm2 tissues) supplied by SkinEthic Laboratories, Nice, France.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
As the test substance was a solid, 5 µL of water for injection was first applied to each of the three tissues and then, 10 mg ± 2 mg of the test substance were applied evenly to each tissue, taking care to spread it over the whole tissue surface without damaging the tissue sample.
Duration of treatment / exposure:
Exposure period of 15 minutes followed by rinsing and a 42-h recovery period.
Duration of post-treatment incubation (if applicable):
MTT-loading after a 42h-incubation period following rinsing. Observation of MTT-> formazan transformation by viable cells.
Number of replicates:
Triplicate tissues for each tested substance (test substance, negative control, positive control).
Details on study design:
POSITIVE CONTROL
Sodium Dodecyl Sulphate (SDS) at a 5% (w/v) aqueous solution.

NEGATIVE CONTROL
Dulbecco’s Phosphate-Buffered Saline (D-PBS).

METHOD
Preliminary tests were performed to detect the ability of the test substance to directly reduce MMT as well as its coloring potential.
Following the preliminary tests, the skin irritation potential of the test substance was tested in the main test. The test substance and both the negative and positive controls were applied topically on triplicate tissues and incubated at room temperature for 15 (± 1) minutes. At the end of the treatment period, each tissue was rinsed with D-PBS and incubated for 42 (± 1) h at 37°C, 5% CO2 in a humidified incubator. The cell viability was then assessed by means of the colorimetric MTT reduction assay.
Also, the concentration of the inflammatory mediator IL-1α was evaluated in the culture medium retained following the 42-h recovery period. This quantification, based on an ELISA assay, was performed since the mean relative viability of the test substance-treated tissues was > 50% following the MTT reduction assay.

SCORING SYSTEM:
- Optical density (OD) was measured at 540 nm.
Relative viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (reference viability).
Relative mean viability (%) = 100 x mean OD(test item) / mean OD(negative control)

Interpretation: See below
Irritation / corrosion parameter:
% tissue viability
Value:
109
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 15 minutes exposure + 42h recovery period. Remarks: 100% = control. (migrated information)
Other effects / acceptance of results:
PRELIMINARY TESTS
- Test for direct MTT reduction with the test substance: The MTT solution containing the test substance did not turn blue/purple when compared with the negative control. The test substance was therefore considered not to have direct MTT reducing properties. As a result, no additional controls were performed on water-killed tissues in parallel to the main test.

- Test for the detection of the coloring potential of the test substance: During this test, the water solution containing the test substance did not change color. Therefore, the test substance was found not to have a coloring potential. As a result, no additional controls were used in parallel to the main test.

MAIN TEST

- All of the acceptance criteria for the negative and positive controls was fulfilled, therefore the study was considered to be valid.

- Following the treatment with the test substance, all treated tissues appeared blue which was considered indicative for viable tissues.

- Following a 15 minutes exposure and a 42-h recovery period, the relative mean viability of the tissues treated with the test substance was 109% with a standard deviation of 5% as assessed by the MTT assay (see Table1).

- As the mean viability was >50% after the MTT reduction, the IL-1α concentrations in culture media samples retained from the three negative controls and the three test substance-treated tissues were analyzed by ELISA. The mean IL-1α concentration for treated tissues was 53.5 pg/mL. Due to this value being below 60 pg/mL, the results met the criteria for an in vitro classification as non-irritant to skin.

(For Tables please refer to the PDF under 'attached background material': The individual and mean OD values, standard deviations and tissue viabilities for the test substance, vehicle and positive controls has been presented in Table 1. The qualitative evaluation of the tissue viability have been presented in Table 2.)

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the study, the test substance was considered to be non-irritant to skin.
Executive summary:

An in vitro study was conducted to evaluate the skin irritation potential of the test substance using the EpiskinTMreconstituted human epidermis model according to EU Method B.46 and OECD Guideline 439 in compliance with GLP.

The test substance and both the negative and positive controls were applied topically on triplicate tissues and incubated at room temperature for 15 (± 1) minutes. At the end of the treatment period, each tissue was rinsed with Dulbecco’s phosphate buffered saline (D-PBS) and incubated for 42 (± 1) h at 37°C / 5% CO2in a humidified incubator. Cell viability was then assessed by means of the colorimetric MTT ((3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) reduction assay. Relative viability values were calculated for each tissue and expressed as a percentage of the mean viability of negative control tissues which was set at 100% (reference viability). Also, the concentration of the inflammatory mediator IL-1α was assessed in the culture medium retained following the 42 h recovery period.All acceptance criteria for the negative and positive controls were fulfilled and hence the study was considered to be valid. Following a 15 minutes exposure and a 42 h recovery period, the relative mean viability of the tissues treated with the test substance was 109%, with a standard deviation of 5%. As the mean relative viability was > 50% after MTT reduction, culture media samples retained from the three negative controls and the three test substance-treated tissues were analysed by ELISA. The mean concentration of IL-1α within the retained medium was 53.5 pg/mL. Due to this value being below 60 pg/mL, the test substance was considered to be non-irritant to skin.

Under the conditions of the study, the test substance was therefore considered to be non-irritant to skin.

Endpoint:
skin irritation / corrosion, other
Remarks:
in vivo
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From 22 October 2012 to 05 November 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Read across study hence maximum reliability rating of 2 assigned according to ECHA guidance.
Qualifier:
according to guideline
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: CEGAV, Argenvilliers, France
- Age at study initiation: 2 to 4 months old on the day of treatment
- Mean body weight at study initiation: 2788 g (range: 2700 g to 2945 g)
- Fasting period before study: No
- Housing: The animals were individually housed in noryl cages
- Diet: 110 pelleted diet (free access)
- Water: Tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: At least 5 d before the beginning of the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 18 ± 3°C
- Humidity: 50 ± 20%
- Air changes: approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: From 23 October 2012 to 05 November 2012.
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: a quantity of 0.5 g/flank was used.


DOSE FORMULATION PREPARATION
- The test substance was used in its original form. The pH of the test substance was measured with pH paper in order to confirm that the pH value is > 2 and < 11.5.
- Dose formulations preparations were prepared by the testing laboratory extemporaneously on the day of each administration.
- The dose formulation preparations were stored at room temperature and delivered to the study room in paper bags.
Duration of treatment / exposure:
3 min, 1 h, 4 h
Observation period:
1, 24, 48 and 72 h after removal of the dressing; if relevant, daily until reversibility of reactions
Number of animals:
Three males
Details on study design:
RATIONALE FOR STUDY DESIGN
- The sequential study design was as follows:
The test substance was applied for 3 minutes on the skin of a single animal 1:
- as no full thickness destruction of skin tissue was observed in the first hour after removal of the pad, the test substance was applied on another treatment site for 1 h,
After the 1-h application on animal 1:
- as no full thickness destruction of skin tissue was observed in the first hour after removal of the pad, the test substance was applied on another treatment site for 4 h,
After the 4-h application on animal 1:
- as mean value from grading at 24, 48 and 72 h after patch removal was < 2.3 for erythema and for edema, the test substance was applied on the skin of two other animals for 4 h (in animal 2 and 3).
For each animal, the day of dose application was recorded as Day 1 of its observation period.


TEST SITE
- The test substance was placed on a gauze pad, moistened with drinking water treated by reverse osmosis, which was then applied to a skin area of approximately 6 cm2. A quantity of 0.5 g/flank was used. During each exposure time, a dry gauze pad was applied to the opposite flank, in order to check that no alteration of the skin is induced by the semi-occlusive dressing and restraining bandage. The untreated flank acted as control.
- The gauze pad was held in place by a non-irritating semi-occlusive dressing and a restraining bandage.


REMOVAL OF TEST SUBSTANCE
After required period of contact with the skin, the dressing was removed. After removal of the dressing, any residual test substance was wiped off by means of moistened cotton pad with drinking water treated by reverse osmosis.


CLINICAL EXAMINATIONS
- Morbidity and mortality: Each animal was checked for mortality or morbidity once a day during the treatment and observation periods.
- Clinical signs: Each animal was observed once a day, at approximately the same time, for the recording of clinical signs.
- Body weight: The body weight of each animal was recorded on the day of treatment and at the end of the evaluation of cutaneous reactions.
- Examination of cutaneous reactions: For each exposure period, cutaneous reactions were evaluated approximately 1, 24, 48 and 72 h after removal of the dressing.

SCORING SYSTEM:
Dermal irritation was graded in each animal according to the following scoring scale:
- Erythema and eschar formation:
0 no erythema
1 very slight erythema (barely perceptible)
2 well-defined erythema
3 moderate to severe erythema
4 severe erythema (beet redness) to slight eschar formation (injuries in depth).

- Edema formation:
0 no edema
1 very slight edema (barely perceptible)
2 slight edema (edges of area well-defined by definite raising)
3 moderate edema (raised approximately 1 millimeter)
4 severe edema (raised more than 1 millimeter and extending beyond area of exposure).

- Any other lesions were noted, if applicable.
Irritation parameter:
erythema score
Basis:
animal #1
Time point:
other: 24, 48, 72 h (mean)
Score:
1.7
Max. score:
2
Reversibility:
fully reversible within: Day 7
Irritation parameter:
erythema score
Basis:
animal #2
Time point:
other: 24, 48, 72 h (mean)
Score:
1
Max. score:
2
Reversibility:
fully reversible within: Day 3
Irritation parameter:
erythema score
Basis:
animal #3
Time point:
other: 24, 48, 72 h (mean)
Score:
1.3
Max. score:
2
Reversibility:
fully reversible within: Day 4
Irritation parameter:
edema score
Basis:
animal #1
Time point:
other: 24, 48, 72 h (mean)
Score:
0
Max. score:
0
Irritation parameter:
edema score
Basis:
animal #2
Time point:
other: 24, 48, 72 h (mean)
Score:
0
Max. score:
0
Irritation parameter:
edema score
Basis:
animal #3
Time point:
other: 24, 48, 72 h (mean)
Score:
0
Max. score:
0
Irritant / corrosive response data:
CUTANEOUS REACTIONS:
- After a 3-minute exposure in animal 1, no cutaneous reactions were observed.
- After a 1-h exposure, a well-defined erythema (grade 2) was noted in animal 1 from Day 1 to Day 3, followed by a very slight erythema (grade 1) on Days 4 and 5. In addition, a very slight edema (grade 1) was observed on day 1.
- After a 4-h exposure, a well-defined erythema (grade 2) was noted from Day 1 until Day 2 in 2/3 animals (i.e., in animal 2 and 3) and until Day 3 in 1/3 animals (i.e., in animal 1), followed by a very slight erythema (grade 1) up to Day 3 in animal 2, Day 4 in animal 3 or Day 7 in animal 1.
- Mean scores calculated for each animal over 24, 48 and 72 h were as follows:
- erythema: 1.7, 1.0, 1.3; showing no significant inflammation,
- edema: 0.0, 0.0; 0.0; showing no significant inflammation.
Other effects:
MORTALITY: No unscheduled deaths occurred during the study.
CLINICAL SIGNS: No clinical signs indicative of systemic toxicity were observed in any animals.
BODY WEIGHT: The body weight of the animals was unaffected by the test substance treatment.
Interpretation of results:
GHS criteria not met
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the study conditions, the test substance was considered to be slightly irritating when applied topically to rabbits.
Executive summary:

An in vivo study in New Zealand White rabbits was run according to OECD Guideline 404 and EU Method B.4 in compliance with GLP.

Here, 0.5 g of the undiluted test substance (moistened with drinking water treated by reverses osmosis) was applied to a skin area of approximately 6 cm2under semi-occlusive conditions for 4 h. Each animal was observed once a day for mortality and clinical signs. The cutaneous reactions were evaluated approximately 1, 24, 48 and 72 h after removal of the dressing and then daily until the reversibility of cutaneous reactions. The mean values of the scores for erythema and edema were calculated for each animal. Body weight was recorded at the beginning and the end of the observation period. No unscheduled deaths occurred during the study and no clinical signs were noted in any animal. The body weight of the animals was unaffected by treatment. After a 4 h exposure, a well-defined erythema was noted from Day 1 until Day 2 in 2/3 animals or until Day 3 in 1/3 animals, followed by a very slight erythema up to Days 3, 4 or 7. Mean scores calculated for each animal over 24, 48 and 72 h were as follows:

-        Erythema: 1.7, 1.0, 1.3; showing no significant inflammation;

-        Edema: 0.0, 0.0; 0.0; showing no significant inflammation.

Under the study conditions, the test substance produced slight skin irritation in rabbits which was reversible within 7 d

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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
May 21, 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Read across study hence maximum reliability rating of 2 assigned according to ECHA guidance.
Principles of method if other than guideline:
The rabbit enucleated eye test was designed to assess the ocular irritancy potential of the test substance in the rabbit following application onto the cornea of the enucleated eye. The assay has undergone inter-laboratory validation and has been shown to reliably detect test substances that are negligible, or moderate to severe ocular irritants.
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Not applicable
Vehicle:
unchanged (no vehicle)
Controls:
other: yes, two enucleated eyes treated with saline (0.9% Sodium chloride)
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL of the test substance (which was found to weigh approximately 48 mg)
- Concentration (if solution):

VEHICLE
- Amount(s) applied (volume or weight with unit):
- Concentration (if solution):
- Lot/batch no. (if required):
- Purity:
Duration of treatment / exposure:
10 sec
Observation period (in vivo):
60, 120, 180 and 240 minutes following treatment.
Number of animals or in vitro replicates:
Three enucleated eyes for test and two enucleated eyes for control group.
Details on study design:
PRE-TEST PROCEDURES
Superfusion chamber:
The water heating circulator, was adjusted so that the temperature of the water flowing through the water jacket of the superfusion apparatus, gave a stable temperature, of 32 ±1.5°C, within the chambers of the apparatus. A peristaltic pump was used to supply saline solution at a flow rate of 0.15 to 0.4 mL/minute (at approximately 32°C) into the rear of each chamber of the apparatus in order to irrigate the surface of the cornea.
Selection of eyes:
Prior to enucleation, the eyes of the provisionally selected rabbits were examined for evidence of ocular irritation or defect, following application of Fluorescein Sodium drops BP (1 % w/v). Examination was aided with the Kowa SL-5 slit-lamp biomicroscope. Corneal thickness values were also recorded using the DGH-1000 Ultrasonic pachymeter. Only animals whose eyes showed no evidence of ocular irritation or defect were used for testing purposes.
Enucleation of eyes:
The donor rabbits were sacrificed by intravenous administration of an overdose of sodium 'pentobarbitone. Immediately afterwards, two to three drops of saline solution (approximately 32°C) were applied to the cornea to prevent desiccation during excision. The eye was then carefully removed, positioned in a perspex clamp and placed within the chamber of the superfusion apparatus, with the saline drip at the rear of the chamber adjusted so that saline solution was allowed to irrigate the surface of the cornea. The eyes were then allowed to equilibrate for approximately thirty minutes. Following the equilibration period, the eyes were re-examined to ensure they had not been damaged during excision. Corneal thickness was also measured using the ultrasonic pachymeter. Any eyes in which the corneal swelling was greater than 100/0 relative to the preenucleation measurement, or in which the cornea was stained with fluorescein, were rejected. The post equilibration corneal thickness values for each eye were recorded.

PROCEDURE
Test substance administration:
Three eyes were treated with test substance, two additional eyes remained untreated for control purposes. The treatment eye was removed from the superfusion apparatus whilst still being held in the perspex clamp. The clamp/eye was then placed horizontally
into a petri dish.
A volume of 0.1 mL of the test substance, which was found to weigh approximately 48 mg (as measured by gently compacting the required volume into an adapted syringe) was sprinkled as evenly as possible over the surface of the cornea. After ten seconds the test substance was washed off the cornea using a minimum of 20 mL of saline solution (approximately 32°C).
Immediately following washing of the corneal surface, the treated eye was returned to the superfusion chamber and the saline drip repositioned to irrigate the eye.
The untreated eyes were similarly washed and used for control purposes.

Examination:
Examination of the eye was facilitated by use of a slit-lamp biomicroscope. The thickness of the cornea was measured using an ultrasonic pachymeter. For each enucleated eye a measurement was made at the optical centre, and at a further four locations at the apex of the cornea. A mean value for corneal thickness was then calculated. Measurements for corneal thickness were carried out pre-enucleation, post equilibration and approximately 60, 120, 180 and 240 minutes following treatment.
The condition of the corneal epithelium was assessed approximately 60, 120, 180 and 240 minutes following treatment. Assessment was facilitated by the use of the slit-lamp biomicroscope.
The uptake of fluorescein by the corneal epithelium was assessed pre-enucleation, post equilibration and approximately 240 minutes following treatment, according to the numerical evaluation (i.e., McDonald - Shadduck Score System). This was carried out using the cobalt blue filter of the slit-lamp biomicroscope, following application of Fluorescein Sodium drops.
Irritation parameter:
cornea opacity score
Remarks:
mean
Run / experiment:
240 min
Value:
0
Negative controls validity:
valid
Remarks:
similar results
Irritation parameter:
fluorescein retention score
Remarks:
mean
Run / experiment:
240 min
Value:
0
Negative controls validity:
valid
Remarks:
similar results in control
Irritation parameter:
other: corneal thickening
Remarks:
mean
Run / experiment:
240 min
Value:
395.6
Negative controls validity:
valid
Remarks:
388.7 micro m in control
Irritation parameter:
percent corneal swelling
Remarks:
mean
Run / experiment:
240 min
Value:
4
Negative controls validity:
valid
Remarks:
2.5% in control
Other effects / acceptance of results:
- Corneal Opacity: No corneal effects were noted in the test eyes or control eyes during the study period.
- Corneal Thickness: Corneal swelling of the test eyes during the study period was comparable to that observed in the control eyes over the same period.
- Corneal Condition: The condition of the corneal epithelium of the test eyes and control eyes appeared normal during the study period.
- Fluorescein Uptake: No fluorescein uptake was noted in the test eyes or control eyes 240 minutes after treatment.

Table 1: Maximal ocular irritation observations

Corneal opacity

 Fluorescein uptake 

 Corneal Swelling (%) 

Condition of Corneal Epithelium 

 Test Eyesa 

 Control Eyesb 

Cldy x Area

Int x Area

60 mins

120 mins

240 mins

60 mins

120 mins

240 mins

0

0

10.4

8.6

4.0

7.7

5.6

2.5

normal

a = For each time point the swelling recorded is the mean of three eyes

b = For each time point the swelling recorded is the mean of two eyes

Cldy = Corneal cloudiness

Int = Intensity of fluorescein uptake

mins = Minutes following treatment

Interpretation of results:
GHS criteria not met
Conclusions:
Under the study conditions, the test substance was considered unlikely to have the potential to cause severe ocular irritancy in vivo.
Executive summary:

An in vitro study was performed to assess the ocular irritancy potential of the test substance in rabbit following application onto the cornea of enucleated eyes, in compliance with GLP.

A volume of 0.1 mL of test substance, which was found to weigh approximately 48 mg, was applied onto the cornea of each of three enucleated rabbit eyes which had been maintained at a temperature of 32 ± 1.5°C within a superfusion chamber. A further two enucleated eyes were treated, for control purposes, with saline solution (0.9% sodium chloride). Effects on corneal opacity, condition of the corneal epithelium, fluorescein uptake (240 minutes following treatment) and the percentage change in corneal thickness (corneal swelling) were observed 60, 120, 180 and 240 minutes after treatment. No corneal effects (opacity) were noted in the test eyes or control eyes during the study period (i.e. 240 minutes after treatment). Corneal swelling of the test eyes during the study period was comparable to that observed in the control eyes over the same period. No fluorescein uptake was seen in test and control eyes and the condition of the corneal epithelium in the test eyes or controls appeared normal during the study period.

Under the study conditions, the test substance was therefore considered unlikely to have the potential to cause severe ocular irritancy in vivo.

Endpoint:
eye irritation, other
Remarks:
In vivo
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From 24 May 2012 to 01 June 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Read across study hence maximum reliability rating of 2 assigned according to ECHA guidance.
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
Two New Zealand White (Hsdlf:NZW) strain rabbits were supplied by Harlan Laboratories UK Ltd., Leicestershire, UK. At the start of the study the animals weighed 2.62 or 3.01 kg and were 12 to 20 weeks old. After an acclimatisation period of at least 5 d each animal was given a number unique within the study which was written with a black indelible marker-pen on the inner surface of the ear and on the cage label.
The animals were individually housed in suspended cages. Free access to mains drinking water and food (2930C Teklad Global Rabbit diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study. The diet and drinking water were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
The temperature and relative humidity were set to achieve limits of 17 to 23°C and 30 to 70% respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was at least 15 changes/h and the lighting was controlled by a time switch to give 12 h continuous light (06:00 to 18:00) and 12 h darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL of the test substance (which was found to weigh approximately 66 mg)
- Concentration: Undiluted

Duration of treatment / exposure:
Duration of treatment same as observation period.
Observation period (in vivo):
The eyes were examined for any ocular reaction approximately 1, 24, 48, and 72 h after the treatment.

Number of animals or in vitro replicates:
Two
Details on study design:
PROCEDURE:
Only animals free of ocular damage were used.
Initially, a single rabbit was treated. A volume of 0.1 mL of the test substance, which was found to weigh approximately 66 mg (as measured by gently compacting the required volume into an adapted syringe) was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of the test substance, and then released. The left eye remained untreated and was used for control purposes. Immediately after administration of the test substance, an assessment of the initial pain reaction was made according to the six point scale.
After consideration of the ocular responses produced in the first treated animal, a second animal was treated.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): No

SCORING SYSTEM: Draize scale for scoring ocular Irritation (Draize J H (1977) "Dermal and Eye Toxicity Tests" In: Principles and Procedures for Evaluating the Toxicity of Household Substances, National Academy of Sciences, Washington DC p.48 to 49).

TOOL USED TO ASSESS SCORE: Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
other: 1 h
Score:
6
Max. score:
110
Reversibility:
fully reversible within: 72 h
Remarks on result:
other: Based on Draize scoring method
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
other: 24 h
Score:
2
Max. score:
110
Reversibility:
fully reversible within: 72 h
Remarks on result:
other: Based on Draize scoring method
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
other: 48 h
Score:
1
Max. score:
110
Reversibility:
fully reversible within: 72 h
Remarks on result:
other: Based on Draize scoring method
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
other: 72 h
Score:
0
Max. score:
110
Reversibility:
fully reversible
Remarks on result:
other: Based on Draize scoring method
Irritant / corrosive response data:
- No corneal or iridial effects were noted during the study.
- Minimal conjunctival irritation was noted in both the animals 1 and 24 h after treatment and in one animal at the 48-h observation. However, these effects were reversible within 48 h in one animal and within 72 h in the other animal.


See below tables for further details.
Other effects:
Only one of the animal showed an expected gain in bodyweight during the study.

Table 1: Individual Scores and Individual Total Scores for Ocular Irritation

Rabbit Number and Sex  72058 Male  72080 Male
Initial pain reaction = 1  Initial pain reaction = 1
Time after treatment  1 h 24 h 48 h 72 h 1 h 24 h 48 h 72 h
CORNEA                 
E = Degree of opacity  0 0 0 0 0 0 0 0
F = Area of cornea involved  0 0 0 0 0 0 0 0
Score (E x F) x 5  0 0 0 0 0 0 0 0
IRIS                 
D  0 0 0 0 0 0 0 0
Score (D x 5)  0 0 0 0 0 0 0 0
CONJUNCTIVAE                 
A = Redness  1 1 0 0 1 1 1 0
B = Chemosis  1 0 0 0 1 0 0 0
C = Discharge  1 0 0 0 1 0 0 0
Score (A + B + C) x 2  6 2 0 0 6 2 2 0
Total Score  6 2 0 0 6 2 2 0

Table 2: Individual Total Scores and Group Mean Scores for Ocular Irritation

 Rabbit Number and Sex   Individual total scores at: 
1 h 24 h 48 h 72 h
 72058 Male   6   2   0   0 

 72080 Male 

 6   2   2   0 
 Group Total   12   4   2   0 
 Group Mean Score   6.0   2.0   1.0   0 
Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the study, the test substance produced minimal conjunctival irritation which was reversible within 72 h.
Executive summary:

An in vivo study was conducted to evaluate the acute eye irritation potential of the test substance in New Zealand White rabbits according to the EU Method B.5 and OECD Guideline 405 in compliance with GLP.

A volume of 0.1 mL of test substance (i. e, 66 mg) was placed into the conjunctival sac of the right eye of two New Zealand White rabbits without irrigation. The left eye remained untreated and was used for control purposes. Assessment of ocular damage/irritation was made approximately 1, 24, 48 and 72 h following treatment according to the Draize scoring method. No corneal or iridial effects were noted during the study. Minimal conjunctival irritation was seen in both animals 1 and 24 h after treatment and in one animal at the 48 h observation. These effects were reversible within 48 h in one animal and within 72 h in the other.

In conclusion, under the conditions of the study, the test substance produced minimal conjunctival irritation in rabbits which was reversible within 72 h

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Based on the findings of reliable studies on irritation and corrosion, classification of the substance is not justified.