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Description of key information

Results obtained from an in vitro skin irritation test, according to OECD 439, indicated no skin irritation potential of the test item under the utilized testing conditions. Thus, the test item is considered as non-irritant to skin and is therefore not classified.

In an in vitro eye corrosives and severe irritants study, using the Isolated Chicken Eye model with the test item, no ocular corrosion or severe irritation potential was observed. However, the test item was considered as eye irritant in the EpiOcularTM Eye Irritation Test, according to OECD 492.

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:
2015-10-07 to 2016-02-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2012
Deviations:
no
Qualifier:
according to
Guideline:
other: EpiSkin™ SOP, Version 1.8 (February 2009), ECVAM Skin Irritation Validation Study: Validation of the EpiSkin™ test method 15 min - 42 hours for the prediction of acute skin irritation of chemicals.
Version / remarks:
2009
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
other: reconstituted human epidermis
Cell type:
non-transformed keratinocytes
Justification for test system used:
The EPISKIN model has been validated for irritation testing in an international trial. After a review of scientific reports and peer reviewed publications on the EPISKIN method, it showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purposes of distinguishing between skin irritating and no skin irritating test substances (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTMSM, EPISKIN SNC Lyon, France,
- Supplier: SKINETHIC Laboratories; 4, rue Alexander Fleming, 69366 Lyon Cedex 07 - France
- Tissue batch number(s): 15-EKIN-050
- Expiry date: 21 December 2015
- Date of initiation of testing: 7 October 2015

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: approximately 25 mL PBS 1 x solution. The rest of the PBS was removed from the epidermal surface with a suitable pipette tip linked to a vacuum source
- Observable damage in the tissue due to washing: care was taken to avoid damaging to the epidermis.
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 mL of 0.3 mg/mL MTT per well
- Incubation time: 3 hours at 37 °C in an incubator with 5 % CO2, ≥95% humidified atmosphere and protected from light
- Spectrophotometer: Thermo Scientific; Multiscan FC
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

IN CASE OF MTT DIRECT INTERFERENCE:
Optical properties of the test item or its chemical action on MTT may interfere with the assay and lead to a false estimate of viability. This may occur when the test item is not completely removed from the tissue by rinsing or when it penetrates the epidermis. If the test item acts directly on MTT (MTT-reducer), is naturally coloured, or becomes coloured during tissue treatment, additional controls are used to detect and correct for test item interference with the viability measurement.

Check-method for possible direct MTT reduction with test item:
Approximately 10 μL test item was added to 2 mL MTT 0.3 mg/mL (diluted with assay medium) solution and mixed. The mixture was incubated for three hours at 37 °C protected from light. Subsequently, any observed colour changes of the solution was recorded:
- Test items which do not interact with MTT: yellow
- Test items interacting with MTT: blue or purple
If the MTT solution colour becomes blue or purple, the test item interacts with the MTT. It is then necessary to evaluate the part of optical density (OD) due to the non-specific reduction of the MTT (i.e. by using killed epidermis).
The test item showed no direct interaction with MTT. Using of additional control was not necessary.

Check-method to detect the colouring potential of test item:
Prior to treatment, the test item was evaluated for its intrinsic colour or ability to become coloured in contact with water (simulating a tissue humid environment). Approximately 10 μL test item was added to 90 μL of water. The mixture was shaken for 15 minutes at room temperature and any development of colouring was monitored (unaided eye assessment). The test item showed no ability to become coloured in contact with water. Using of additional control was not necessary.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irrtating to skin if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 10 µL per test skin unit (area: 0.38 cm2 per unit)
- Concentration: unchanged

NEGATIVE CONTROL
- Amount applied: 10 µL per test skin unit (area: 0.38 cm2 per unit)
- Concentration: 1x PBS

POSITIVE CONTROL
- Amount applied: 10 µL per test skin unit (area: 0.38 cm2 per unit)
- Concentration: SDS 5% aq.
Duration of treatment / exposure:
15 min
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Value:
71
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:
- Direct-MTT reduction: No colour change was observed after three hours of incubation. The test material did not interact with the MTT, therefore additional controls and data calculations were not necessary. A false estimation of viability can be excluded.
- Colour interference with MTT: The test item showed no ability to become coloured in contact with water therefore additional controls and data calculations were not necessary. A false estimation of viability can be excluded.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prior to routine use of the method Toxi-Coop ZRT. demonstrated the technical proficiency in a separate study (study no.: 392.554.2938) using the ten Proficiency Chemicals according to OECD Test Guideline No. 439.

ACCEPTANCE OF RESULTS:
The mean OD value of the three negative control tissues was 0.861. The mean OD value obtained for the positive control was 0.078 and this result corresponds to 9 % viability when compared to the results obtained from the negative controls. Each calculated standard deviation value (SD) for the % viability was below 18. All validity criteria were within acceptable limits and therefore the study can be considered as valid.
Interpretation of results:
GHS criteria not met
Conclusions:
The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilized testing conditions. According to the current OECD Guideline No. 439, the test item is considered as non-irritant to skin and is therefore not classified.
Executive summary:

The purpose of this study was to determine the skin irritation potential of the test item on reconstituted human epidermis in the EPISKIN model in vitro. Disks of epidermal units (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test material was terminated by rinsing the epidermal units with 1x PBS solution. Epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5 % CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in 5 % CO2 and protected from light. The resulting formazan chrystals were extracted with acidified isopropanol and quantified with the optical densities (OD) recorded spectrophotometrically. SDS 5 % aq. and 1 x PBS treated (three units / positive and negative control) epidermis units were used as positive and negative controls, respectively. For each treated tissue, viability was expressed as a percentage relative to negative control. The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control. In this in vitro skin irritation test using the EPISKIN model, the test item did not show significantly reduced cell viability in comparison to the negative control (mean relative viability: 71 %). All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

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:
weight of evidence
Study period:
2016-02-03 to 2016-03-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
2013
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
Version / remarks:
2010
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
chicken
Strain:
other: ROSS 308
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: TARAVIS KFT. 9600 Sárvár, Rábasömjéni út 129. Hungary
- Storage, temperature and transport conditions of ocular tissue: Head collection was performed by a slaughter house technician. Heads were removed immediately after sedation of the chickens (sedation was happened by electric current). The heads were transported to Toxi-Coop ZRT at the earliest convenience for use approximately within 2 hours from collection. The ambient temperature was optimal (19.5ºC to 20.5 ºC) during the transport. All eyes used in the assay were from the same groups of eyes collected on one specific day.
- indication of any existing defects or lesions in ocular tissue samples: After collection, the heads were inspected for appropriate quality and wrapped with paper moistened with saline, then placed in a plastic box that can be closed (4-5 heads/box).
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: The undiluted test item was applied at a volume of 30 μL from a micropipette, in such a way that the entire surface of the cornea was covered with test substance.
Duration of treatment / exposure:
The time of application was monitored. After an exposure period of 10 seconds the cornea surface was rinsed thoroughly with 20 mL saline solution at ambient temperature, while taking care not to damage the cornea but attempting to remove the entire residual test item, if possible. The eye in the holder was then returned to its chamber. The time while the eye was out of the chamber was limited to a minimum.
Duration of post- treatment incubation (in vitro):
The control and test item treated eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within ± 5 minutes were considered acceptable.
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
Eyes selection:
After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of fluorescein solution 2 % (w/v) was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL saline solution. Then the fluorescein-treated cornea was examined with a slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged (i.e., fluorescein retention ≤ 0.5). If the cornea was in good condition, the eyeball was carefully removed from the orbit.
Preparation of eyes:
The eye ball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper. The nictitating membrane and other connective tissue were cut away. The prepared eyes were kept on wet papers in a closed box to maintain an appropriate humidity. The treatment group and the concurrent positive control consisted of three eyes. The negative control group consisted of one eye.
Eyes examination and acclimatisation time:
The enucleated eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head). Again, too much pressure on the eye by the clamp was avoided. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was needed to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with saline solution dripping from a stainless steel tube, at a rate of approximately 3 or 4 drops/minute. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.
The appropriate number of eyes was selected and, after being placed in the superfusion apparatus, the eyes were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the saline solution which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or a high corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured using the depth measuring device on the slit lamp microscope (Haag-Streit BQ 900) with the slit-width set at 9½, equaling 0.095 mm. Any eye with cornea thickness deviating more than 10 % from the mean value for the eyes, or eyes that showed any other signs of damage, were rejected and replaced. If the selected eyes were appropriate for the test, acclimatisation started and was conducted for approximately 45 to 60 minutes. The temperature of the circulating water was verified to ensure that the temperature in all chambers was in the range of 32 ± 1.5 °C during the acclimatisation and treatment periods.

EQUILIBRATION AND BASELINE RECORDINGS
At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness, opacity, and fluorescein retention to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than ±5-7 % within approximately 45 to 60 minutes before the start of application. No changes in thickness were observed in the eyes. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effects after treatment. If any eye was considered to be unsuitable following baseline assessment, it was discarded.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: Saline

POSITIVE CONTROL USED: Acetic acid 10% (v/v)

APPLICATION DOSE AND EXPOSURE TIME: 30 µL for 10 sec

OBSERVATION PERIOD: The control and test item treated eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within ± 5 minutes were considered acceptable.
The cornea thickness and cornea opacity were measured at all time points. Fluorescein retention was measured on two occasions, at base line (t=0) and 30 minutes after the post-treatment rinse.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: The time of application was monitored. After an exposure period of 10 seconds the cornea surface was rinsed thoroughly with 20 mL saline solution at ambient temperature, while taking care not to damage the cornea but attempting to remove the entire residual test item, if possible. The eye in the holder was then returned to its chamber. The time while the eye was out of the chamber was limited to a minimum.

METHODS FOR MEASURED ENDPOINTS:
The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects (e.g., pitting or loosening of the epithelium).
Results from corneal opacity, swelling, and fluorescein retention were evaluated separately to generate an Isolated Chicken Eye (ICE) class for each endpoint. The ICE classes for each endpoint were then combined to generate an Irritancy Classification for each test substance.

SCORING SYSTEM:
The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects (e.g., pitting or loosening of the epithelium).
Results from corneal opacity, swelling, and fluorescein retention were evaluated separately to generate an Isolated Chicken Eye (ICE) class for each endpoint. The ICE classes for each endpoint were then combined to generate an Irritancy Classification for each test substance.

DECISION CRITERIA: The conclusion on eye irritancy was based on the OECD guideline on quantitative assessments.
Irritation parameter:
percent corneal swelling
Run / experiment:
at up to 75 min
Value:
7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
percent corneal swelling
Run / experiment:
at up to 240 min
Value:
9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein leakage
Run / experiment:
mean
Value:
1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Run / experiment:
mean maximum
Value:
0.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: None.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes. Based on the overall ICE Class the negative control NaCl (9 g/L saline) had no significant effects on the chicken eye in this study.
- Acceptance criteria met for positive control: yes. Based on the overall ICE Class the positive control Acetic acid 10 % (v/v) solution was classed as corrosive/severely irritating, UN GHS Classification: Category 1.
- Range of historical values if different from the ones specified in the test guideline: Positive and negative control values were within the corresponding historical control data ranges.
Conclusions:
In this in vitro eye corrosives and severe irritants study, using the Isolated Chicken Eye model with the test item, no ocular corrosion or severe irritation potential was observed. The overall ICE score was 3xII.
Executive summary:

The purpose of this Isolated Chicken Eye Test (ICET) was to evaluate the potential ocular corrosivity or severe irritancy of the test item by its ability to induce toxicity in enucleated chicken eyes. The test compound was applied in a single dose (30 μL /eye) onto the cornea of isolated chicken eyes in order to potentially classify the test compound as either 1: causing "serious eye damage" [category 1 of the Globally Harmonised System for the Classification and Labelling of chemicals (GHS)], or 2: not requiring classification for eye irritation or serious eye damage according to the GHS. The ICET does not fully replace the in vivo rabbit eye test (OECD 405); however, the ICET is used as part of a tiered testing strategy for regulatory purposes.

Results

The test item, Acetic acid 10 % (v/v) solution (positive control) and NaCl (9 g/L saline) (negative control) were applied in such a way that the entire surface of the cornea was uniformly covered with the test substance or positive or negative control. Three test item treated eyes and three positive control eyes and one negative control eye were used in this study. After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with ~20 mL saline solution at ambient temperature and this procedure was repeated for each eye. In this ICET, the test item did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. Positive and negative controls showed the expected results. The experiments were considered to be valid.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation:

The purpose of this study was to determine the skin irritation potential of the test item on reconstituted human epidermis in the EPISKIN model in vitro. Disks of epidermal units (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test material was terminated by rinsing the epidermal units with 1x PBS solution. Epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5 % CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in 5 % CO2 and protected from light. The resulting formazan chrystals were extracted with acidified isopropanol and quantified with the optical densities (OD) recorded spectrophotometrically. SDS 5 % aq. and 1 x PBS treated (three units / positive and negative control) epidermis units were used as positive and negative controls, respectively. For each treated tissue, viability was expressed as a percentage relative to negative control. The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control. In this in vitro skin irritation test using the EPISKIN model, the test item did not show significantly reduced cell viability in comparison to the negative control (mean relative viability: 71 %). All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

Eye irritation:

ICET:

The purpose of the Isolated Chicken Eye Test (ICET) was to evaluate the potential ocular corrosivity or severe irritancy of the test item by its ability to induce toxicity in enucleated chicken eyes. The test compound was applied in a single dose (30 μL /eye) onto the cornea of isolated chicken eyes in order to potentially classify the test compound as either 1: causing "serious eye damage" [category 1 of the Globally Harmonised System for the Classification and Labelling of chemicals (GHS)], or 2: not requiring classification for eye irritation or serious eye damage according to the GHS. The ICET does not fully replace the in vivo rabbit eye test (OECD 405); however, the ICET is used as part of a tiered testing strategy for regulatory purposes. The test item, Acetic acid 10 % (v/v) solution (positive control) and NaCl (9 g/L saline) (negative control) were applied in such a way that the entire surface of the cornea was uniformly covered with the test substance or positive or negative control. Three test item treated eyes and three positive control eyes and one negative control eye were used in this study. After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with ~20 mL saline solution at ambient temperature and this procedure was repeated for each eye. In this ICET, the test item did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. Positive and negative controls showed the expected results. The experiments were considered to be valid.

EIT

The test item was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 30 min. 50 µL of the liquid test substance was applied to each tissue. After treatment, the respective substance was rinsed from the tissue; then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to formazan. The formazan production was evaluated by measuring the optical density (OD) of the resulting solution. Demineralised water was used as negative control, Methyl acetate was used as positive control. The controls showed the following results: After treatment with the negative control, the absorbance values were within the required acceptability criterion of 0.8< mean OD< 2.5,OD was 2.2. The positive control showed clear eye irritating effects, the relative absorbance value was reduced to 22.9 % (< 50 %). Variation within tissure replicates was acceptable (< 20 %). After treatment with the test item, the relative absorbance values were reduced to 33.4 %. This value is below the threshold for eye irritation potential (< 60%).

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on irritation/corrosive potential, the test item is classified and labelled as Eye Irritant Category 2 (H319: “Causes severe eye irritation”) according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighth time in Regulation (EU) No 2016/918. The test item is not classified as Skin Irritant/Corrosive according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighth time in Regulation (EU) No 2016/918.