Registration Dossier

Administrative data

Description of key information

In two in vitro EPISKIN model tests with the test item the results indicated that the test item is not corrosive or irritating to skin.
In an Isolated Chicken Eye model test the test item was determined to be not severely eye damaging. In an OECD 492 compliant in vitro study the test item was considered to be either eye irritating or severely eye irritating. However, in an in vivo study the test item caused no irritating effects on the ocular mucosa of rabbits.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2014-06-20 to 2014-06-18
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:
26 July 2013
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
06 July 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.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
other: reconstructed 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
- Tissue batch number: 14-EKIN-022
- Expiry date: 23 June 2014
- Date of initiation of testing: 18 June 2014

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: After the incubation time, the EPISKINTMSM units were removed and rinsed thoroughly with PBS 1 x solution to remove all of the test material from the epidermal surface. The rest of the PBS was removed from the epidermal surface with a suitable pipette tip linked to a vacuum source (care was taken to avoid damaging to the epidermis).
- Observable damage in the tissue due to washing: 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 h
- Spectrophotometer: Thermo Scientific; Multiscan FC
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
According to the result of the In Vitro Skin Corrosion Test in the EPISKIN Model, the test item showed no direct interaction with MTT. 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 irritant to skin if the viability after 15 minutes exposure and 42 hours post incubation is less than or equal to 50% of the negative control.
- The test substance is considered to be non-irritant to skin if the viability after 15 minutes exposure and 42 hours post incubation is greater than 50% of the negative control.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 10 µL

NEGATIVE CONTROL
- Amount applied: 10 µL
- Concentration: 1 x PBS

POSITIVE CONTROL
- Amount applied: 10 µL
- Concentration: 5 % aq. Solution
Duration of treatment / exposure:
15 min
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Test item
Value:
87
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
- Direct-MTT reduction: According to the results of the previously performed “In Vitro Skin Corrosion Test with SIKA Hardener AI (SIKA Härter AI) in the EPISKIN Model (Study No.: 644-431-4850), 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 precluded.
- Colour interference with MTT: According to the results of the previously performed “In Vitro Skin Corrosion Test with SIKA Hardener AI (SIKA Härter AI) in the EPISKIN Model (Study No.: 644-431-4850), 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 precluded.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Prior to routine use of the method TOXI-COOP ZRT. demonstrated the technical proficiency, using the ten Proficiency Chemicals according to OECD Test Guideline No. 439.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD value of the three negative control tissues was 0.819.
- Acceptance criteria met for positive control: The mean OD value obtained for the positive control was 0.143 and this result corresponds to 18 % viability when compared to the results obtained from the negative controls.
- Acceptance criteria met for variability between replicate measurements: 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 in vitro skin irritation study revealed that the test item is not irritating to the skin.
Executive summary:

In the OECD 439 compliant in vitro skin irritation study, the purpose was to determine the skin irritation potential of the test item on reconstituted human epidermis in the EPISKIN model.

Disks of epidermal units (three units / chemical) 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 PBS 1 x 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 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 item is considered to be a skin irritant, if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less than or equal to (≤) 50 % when compared to the viability values obtained from 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. 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:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2014-04-25 to 2014-04-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
adopted 26 July 2013
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Version / remarks:
dated May 31st, 2008
Deviations:
no
Qualifier:
according to
Guideline:
other: INVITTOX Protocol No. 118; “EPISKINTM Skin Corrosivity Test” updated December 2011 / February 2012 (ECVAM Database Service on Alternative Methods to Animal Experimentation).
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
other: reconstructed human epidermis
Cell type:
non-transformed keratinocytes
Justification for test system used:
The EPISKIN model has been validated for corrosivity testing in an international trial; it is considered to be suitable for this study (STATEMENT ON THE SCIENTIFIC VALIDITY OF THE EPISKINTM TEST (AN IN VITRO TEST FOR SKIN CORROSIVITY); ECVAM JRC Environment Institute, European Commission; Ispra; 03 April 1998).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTMSM, EPISKIN SNC Lyon, France
- Tissue batch number: 14-EKIN-014
- Expiry date: 28 April 2014
- Date of initiation of testing: 26 March 2014

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After the incubation time, the EPISKIN-SM units were removed and rinsed thoroughly with PBS 1 x solution to remove all of the test material from the epidermal surface. The rest of the PBS was removed from the epidermal surface with a suitable pipette tip linked to a vacuum source (care was taken to avoid damaging to the epidermis).
- Observable damage in the tissue due to washing: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 mL of 0.3 mg/mL
- Incubation time: 3 h
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2


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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin (Cat. 1A) if the viability after 3 minutes exposure is less than 35%.
- The test substance is considered to be corrosive to skin (Cat 1B and 1C) if the viability after 3 minutes exposure is greater than or equal to 35% AND after 60 minutes exposure smaller than 35%; or if the viability after 60 minutes exposure is greater than or equal to 35% AND after 240 minutes exposure smaller than 35%.
- The test substance is considered to be non-corrosive to skin if the viability after 4 hours exposure is greater than or equal to 35%.
- Justification for the selection of the cut-off point: The cut-off value of 35 % and classification method was validated in an international validation of this kit (Fentem, 1998). The prediction model corresponds to the methods agreed by EU regulatory agencies in line with OECD 431 (OECD, 2015).
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 50 µL

NEGATIVE CONTROL
- Amount applied: 50 µL
- Concentration: 9 g/L

POSITIVE CONTROL
- Amount applied: 50 µL
Duration of treatment / exposure:
4 h
Number of replicates:
2
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Test Item
Value:
110
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
- 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 precluded.
- 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 precluded.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control results of the first experiment did not meet all validity criteria. The one out of three validity criteria was out of range due to technical reason. This criterion requires “In the range 20-100% viability and for ODs ≥ 0.3, difference of viability between the two tissue replicates should not exceed 30%”. The difference of viability of two replicates of NaCl control exceeded this value. According to the OECD 431 (Adopted: 26 July 2013) the experiment was repeated. The data of the first experiment will be archived but not reported. In the additional experiment the mean OD value of the two negative control tissues was 0.726.
- Acceptance criteria met for positive control: The positive control result showed 2 % viability.
- Acceptance criteria met for variability between replicate measurements: The difference of viability between the two tissue replicates:
Negative control: 8%
Positive control: 1%
Test item: 14%
All validity criteria were within acceptable limits in the additional experiment therefore the study can be considered as valid.
Interpretation of results:
other: not corrosive to skin
Conclusions:
In this in vitro EPISKIN model test with SIKA Hardener AI the results indicated that the test item is not corrosive to skin.
Executive summary:

The purpose of this study was to determine the skin corrosion potential of the test item SIKA Hardener AI on reconstituted human epidermis in the EPISKIN model in vitro.

Disks of EPISKIN (two units / chemical / incubation time) were treated with test item and incubated for 4 hours at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in 5% CO2 protected from light. The formazan precipitated was then extracted using acidified isopropanol and quantified spectrophotometrically.

NaCl (9 g/L saline) and glacial acetic acid treated epidermis were used as negative and positive controls respectively.

For each treated tissue, viability was expressed as a % relative to negative control. The test item is considered to be non-corrosive to skin, if the mean relative viability after 4 hours of exposure is above or equal 35 % of the negative control.

The negative control results of the first experiment did not meet one out of three validity criteria. According to the OECD 431 (Adopted: 26 July 2013) the experiment was repeated.

The test item did not show significantly reduced cell viability in comparison to the negative control after four hours of exposure. Both individual tissue viabilities were above 35 % of the mean negative control value. The average test item treated tissue viability was 110 %.

Positive and negative controls showed the expected cell viability values within acceptable limits. The additional experiment was considered to be valid.

In conclusion, in this in vitro EPISKIN model test with SIKA Hardener AI the results indicated that the test item is not corrosive to skin.

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:
2014-08-14 to 2014-08-14
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 Ocular Corrosives and Severe Irritants)
Version / remarks:
26 July 2013
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
chicken
Strain:
other: COBB 500
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 (20.3 ºC to 21.4ºC) during the transport. All eyes used in the assay were from the same groups of eyes collected on one specific day. 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).
- Time interval prior to initiating testing: 2 h
- indication of any existing defects or lesions in ocular tissue samples: none
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 30 μL/eye

Duration of treatment / exposure:
10 seconds
Duration of post- treatment incubation (in vitro):
The control and test eyes were evaluated 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:
The treatment group and the concurrent positive control consisted of three eyes. The negative control group consisted of one eye.
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
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. 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. 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 5 drops/minutes. 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, 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 acclimatisation period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a base line (t=0) for each individual eye. The cornea thickness of the eyes should not increase or reduce by more than ± 5-7 % between the acclimatisation time (-45 to -60 minutes) and the zero time. Slight thinning and changes in thickness (0% to 3%) were observed in the eyes. This is considered normal when maintaining enucleated eyes. Following the equilibration period, the fluorescein retention was measured. Base line values were required to evaluate any potential test item related effects after treatment. The locations of any minor findings were marked on the record sheet as a drawing. All eyes were considered to be suitable for the assay.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: NaCl 9 g/L solution

POSITIVE CONTROL USED: acetic acid 10 % (v/v) solution

APPLICATION DOSE AND EXPOSURE TIME: 30 µL for 10 sec

OBSERVATION PERIOD: 30, 75, 120, 180 and 240 minutes after the post-treatment

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.

SCORING SYSTEM:
- Mean corneal swelling (%)
- Mean maximum opacity score
- Mean fluorescein retention score at 30 minutes post-treatment

DECISION CRITERIA: the decision criteria as indicated in the TG was used.
Irritation parameter:
percent corneal swelling
Run / experiment:
Mean maximum corneal swelling at up to 75 min
Value:
5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
percent corneal swelling
Run / experiment:
Mean maximum corneal swelling at up to 240 min
Value:
9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Value:
3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein retention score
Value:
1
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: Based on the overall ICE Class the negative control NaCl (9g/L saline) had no significant effects on the chicken eye in this study.
- Acceptance criteria met for positive control: 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.
Positive and negative control values were within the corresponding historical control data ranges.

 Test item

 

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

5 %

I

Mean maximum corneal swelling at up to 240 min

9 %

II

Mean maximum corneal opacity

3.0

IV

Mean fluorescein retention

1.0

II

Other Observations

None

Overall ICE Class

2xII, 1xIV

 

 

Positive Control: Acetic acid 10% (v/v) solution

 

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

11 %

II

Mean maximum corneal swelling at up to 240 min

21 %

III

Mean maximum corneal opacity

4.0

IV

Mean fluorescein retention

1.0

II

Other Observations

Cornea opacity score 4 was observed in two eyes at 75 minutes after the post-treatment rinse

Overall ICE Class

1xII, 1xIII, 1xIV

 

 

Negative Control: NaCl (9g/L saline)

 

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

0 %

I

Mean maximum corneal swelling at up to 240 min

0 %

I

Mean maximum corneal opacity

0.0

I

Mean fluorescein retention

0.0

I

Other Observations

None

Overall ICE Class

3xI

 

Interpretation of results:
study cannot be used for classification
Conclusions:
In an Isolated Chicken Eye model test the test item was determined to be not corrosive to eye.
Executive summary:

In the OECD 438 compliant in vitro Isolated Chicken Eye Test (ICET), the purpose 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 onto the cornea of isolated chicken eyes in order to potentially classify the test compound as ocular corrosive and/or severe irritant. 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 a volume of 30 μL/eye, in such a way that the entire surface of the cornea attempting to cover the cornea surface uniformly 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.

In this in vitro eye corrosives and severe irritants study, using the Isolated Chicken Eye model with SIKA Hardener AI, no ocular corrosion or severe irritation potential was observed. According to the guideline OECD 438, the test item has been classified as “No prediction can be made”. However, to obtain a definitive classification in relation to the irritation potential, a further in vitro study (OECD 492) is required.

 

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A weight of evidence approach was conducted to the endpoint skin corrosion/ irritation.

 

Skin corrosion in vitro

The purpose of this study was to determine the skin corrosion potential of the test item on reconstituted human epidermis in the EPISKIN model in vitro.

Disks of EPISKIN (two units / chemical / incubation time) were treated with test item and incubated for 4 hours at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in 5% CO2 protected from light. The formazan precipitated was then extracted using acidified isopropanol and quantified spectrophotometrically.

NaCl (9 g/L saline) and glacial acetic acid treated epidermis were used as negative and positive controls respectively.

For each treated tissue, viability was expressed as a % relative to negative control. The test item is considered to be non-corrosive to skin, if the mean relative viability after 4 hours of exposure is above or equal 35 % of the negative control.

The negative control results of the first experiment did not meet one out of three validity criteria. According to the OECD 431 (Adopted: 26 July 2013) the experiment was repeated.

The test item did not show significantly reduced cell viability in comparison to the negative control after four hours of exposure. Both individual tissue viabilities were above 35 % of the mean negative control value. The average test item treated tissue viability was 110 %.

Positive and negative controls showed the expected cell viability values within acceptable limits. The additional experiment was considered to be valid.

In conclusion, in this in vitro EPISKIN model test with SIKA Hardener AI the results indicated that the test item is not corrosive to skin.

Acute dermal toxiciy

In contrast to in vitro test results, in an acute dermal toxicity study signs of dermal irritation were observed in rats. (please refer to Iuclid section 7.2.3)

 

Skin irritation in vitro

In the OECD 439 compliant in vitro skin irritation study. 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 / chemical) 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 PBS 1 x 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 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 item is considered to be a skin irritant, if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less than or equal to (≤) 50 % when compared to the viability values obtained from 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. 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.

 

Conclusion

Based on the available in vitro skin corrosion and irritation test, the test item was considered to be non-irritant to skin.

 

 

Eye irritation/corrosion

A Weight of Evidence approach was conducted to the endpoint of eye irritation/corrosion

OECD 405 Study, in vivo

The acute eye irritation study of the test item was performed in three New Zealand White rabbits.The irritation effect of the test item was evaluated according to the Draize method (OECD No.: 405, 2012). The test item was placed into the conjunctival sac of the left eye of each animal. The untreated right eye served as control. A volume of 0.1 mL of the test item was used, as a single dose. The eyes of the test animals were not washed out after the application of test item.The eyes were examined at 1, 24, 48 and 72 hours after the application. One hour after the treatment, slight conjunctival redness, chemosis and discharge were observed in animals. 24 hours after the treatment, all animals became free of symptoms. 72 hours after the treatment all animals were free of symptoms and the study was terminated. The animals’ individual mean scores (considering readings at 24, 48 and 72 hours after the treatment) were as follows:

cornea opacity : 0.00, 0.00, 0.00

iris : 0.00, 0.00, 0.00

erythema : 0.00, 0.00, 0.00

chemosis : 0.00, 0.00, 0.00

discharge : 0.00, 0.00, 0.00

No systemic toxicity was observed on the day of the treatment and during the 72-hour observation period. The body weight of animals corresponded to their species and age. Sign of pain and distress as discharge was observed in one animal on the treatment day at first observation. In conclusion, the test item applied to the rabbits’ eye mucosa, caused slight conjunctival irritant effects which were fully reversible within 24 hours. According to Regulation (EC) No 1272/2008, the test item has not been classified into any category.

OECD 438 study, in vitro

In the OECD 438 compliant in vitro Isolated Chicken Eye Test (ICET), the purpose 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 onto the cornea of isolated chicken eyes in order to potentially classify the test compound as ocular corrosive and/or severe irritant. 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 a volume of 30 μL/eye, in such a way that the entire surface of the cornea attempting to cover the cornea surface uniformly 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.

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. According to the guideline OECD 438, the test item has been classified as “No prediction can be made”. However, to obtain a definitive classification in relation to the irritation potential, a further in vitro study (OECD 492) is required.

OECD 492, in vitro

The purpose of this study was to determine the acute ocular irritation potential of the test item on three-dimensional RhCE tissue in the EpiOcular™ model in vitro. Before treatment the tissues were pre-wetted with approximately 20 μL of Ca++Mg++Free-DPBS and were incubated at standard culture conditions for 30 ± 2 minutes. Disks of EpiOcular™ (two units) were treated with (50 μL/units) test item and incubated for 30 minutes (± 2 min) at standard culture conditions (37°C in an incubator with 5 % CO2, 90±10% humidified atmosphere).

Exposure of test material was terminated by rinsing with Ca++Mg++ Free-DPBS solution. After rinsing, the tissues were incubated for a 12 ± 2 minutes immersion incubation (Post-Soak) at room temperature. At the end of the Post-Soak immersion test items treated tissues were incubated for 120 ± 15 minutes at standard culture conditions (Post-treatment Incubation). Fresh Assay Medium was used during the Post-Soak and Post-incubation. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5 % CO2 protected from light, 90±10% humidified atmosphere. The formazan precipitated was then extracted using isopropanol and quantified spectrophotometrically.

Sterile deionized water and methyl acetate treated tissues were used as negative and positive controls respectively. The Disks of EpiOcular™ (two units / control) were treated with positive and negative control and incubated 30 ± 2 minutes.

The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean percent tissue viability after exposure and post-exposure incubation is less than or equal (≤) to 60% of the negative control. Depending on the regulatory framework in member countries, the test chemical is identified as not requiring classification and labelling according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60%.

The test item showed significantly reduced cell viability in comparison to the negative control after 30 minutes of exposure. Both individual tissue viabilities were below 60 % of the mean negative control value. The average test item treated tissue viability was 57 %.

Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

 

Conclusion
Based on the in vitro data available the test item would be considered as eye irritant Cat 2, according to Regulation (EC) No1272/2008. However, based on in vivo data available, the test item is not irritating to ocular tissue and is thus not classified.

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 skin and eye irritation or corrosion, the test item is not classified as skin irritant or corrosive according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighth time in Regulation (EU) No 2016/918. In addition, based on in vivo data available on eye irritation, the substance is not classified as eye irritant according to Regulation (EC) No1272/2008 (CLP), as amendet for eighth time in Regulation (EU) No 2016/218.