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Diss Factsheets

Administrative data

Description of key information

In vitro Skin Irritation: By applying the EPISKIN model, the test item is considered to be not irritating to skin.


In vitro Eye Irritation:
By applying the Isolated Chicken Eye Model, the test item was determined to be not severely eye damaging.

A further in vivo eye irritation study showed that the substance is neither irritating nor corrosive.

 

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:
2015-04-22 and 2015-06-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2013
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2012
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EpiSkin SOP, Version 1.8 (February 2009)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
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-018
- Expiry date: 11 May 2015
- Date of initiation of testing: 6 May 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: 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.
- 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 ONTROL
- 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
Details on test animals or test system and environmental conditions:
not applicable
Irritation / corrosion parameter:
% tissue viability
Value:
82
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: During the check-method for possible direct MTT reduction, colour change was observed after three hours of incubation. The test item interacted with the MTT, therefore additional controls and data calculations were necessary.
The non-specific MTT reduction (NSMTT) was determined to be 1.473 %. As the NSMTT was below 30 % the true MTT metabolic conversion and the correction of viability percentages were undertaken.
- Colour interference with MTT: The test item showed no ability to become coloured in contact with water. The intrinsic colour of test item is brownish orange, however in low amount (such as treatment amount) it was observed as a very light coloured liquid and therefore considered to be not able to significantly stain the tissues and lead false estimate of viability. Additional controls and data calculations were not
necessary. A false estimation of viability can be precluded.


ACCEPTANCE OF RESULTS:
The mean OD value of the three negative control tissues was 0.836. The mean OD value obtained for the positive control was 0.033 and this result corresponds to 4 % 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:
not irritating
Conclusions:
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 / 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 1x 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. The test item acted directly on MTT (MTT-reducer), therefore additional controls (test item treated killed tissues and untreated killed tissues) were used to detect and correct for test substance interference with the viability measurement. 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 (mean relative viability: 81 %). 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. The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item is considered as non-irritant to skin and is therefore not classified.

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

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2016-03-08 to 2016-04-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
2nd October 2012
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2400 (Acute Eye Irritation)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: S & K LAP Kft. 2173 Kartal, Császár út 135, HUNGARY
- Age at study initiation: Young adult rabbits, 11 weeks old
- Weight at study initiation: 2540-2900 g
- Housing: Animals were housed individually in metal cages.
- Diet: The animals received C.HYF rabbit mixed diet produced by Cargill Takarmány Zrt., 5300 Karcag, Madarasi út 0399., Hungary, ad libitum.
- Water: Animals received tap water from watering bottles ad libitum. The drinking water is periodically analysed and is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
- Acclimation period: 5 days in first animal and 6 days in second and third animal

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3 °C
- Humidity (%): 30 - 70 %
- Air changes: above 10 changes/hour
- Photoperiod: Artificial light, from 6 a.m. to 6 p.m.
Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: A volume of 0.1 mL of the test item solution was used for the study, in a single dose.
Duration of treatment / exposure:
The eyes of the test animals were not washed out 24 hours after the application of test item, because the test substance was removed from the eye of the test animal by physiological mechanisms and the test item did not cause immediately severe irritation or corrosion after test item application.
Observation period (in vivo):
72 h
Duration of post- treatment incubation (in vitro):
not applicable
Number of animals or in vitro replicates:
Three healthy male animals were selected for the test.
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing: Not performed

SCORING SYSTEM: The eye irritation scores were evaluated according to the scoring system by Draize (1977) and OECD 405 (2nd October 2012).
Irritation parameter:
chemosis score
Basis:
mean
Remarks:
All animals
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
cornea opacity score
Basis:
mean
Remarks:
All animals
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
mean
Remarks:
All animals
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
animal 1
Time point:
24/48/72 h
Score:
0
Max. score:
3
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
animal 2
Time point:
24/48/72 h
Score:
0.33
Max. score:
3
Reversibility:
fully reversible within: 72 h
Irritation parameter:
conjunctivae score
Basis:
mean
Remarks:
animal 3
Time point:
24/48/72 h
Score:
0
Max. score:
3
Irritant / corrosive response data:
One hour after treatment, some conjunctival hyperaemic blood vessels (score 1) were observed in two animals (No.: 376, 375).
24 hours after treatment, all animals were free of symptoms.
48 hours after treatment, some conjunctival hyperaemic blood vessels (score 1) were observed in animal No.: 376. Two animals (No.: 379, 375) were free of symptoms.
72 hours after treatment, all animals were free of symptoms.
72 hours after treatment, the study was terminated, since no primary irritation symptoms occurred.
During the study the control eyes of the animals were symptom-free.
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. Signs of pain and distress as discharge were not observed.
Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, the test item SIKA Hardener LPP, applied to the rabbits’ eye mucosa, caused slight conjunctival irritant effect which was fully reversible within 72 hours. According to Regulation (EC) No 1272/2008, the test item has not been classified into any category.
Executive summary:

The acute eye irritation study of the test item SIKA Hardener LPP 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 solution 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 was observed in two animals. 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, 0.33, 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. Signs of pain and distress as discharge were not observed. In conclusion, the test item SIKA Hardener LPP, applied to the rabbits’ eye mucosa, caused slight conjunctival irritant effect which was fully reversible within 72 hours. According to Regulation (EC) No 1272/2008, the test item has not been classified into any category.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2015-05-13 and 2015-06-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
2013
Deviations:
no
Qualifier:
according to guideline
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. QA statement)
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 (20.1 ºC to 20.8ºC) 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).
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, taking care not to damage or touch the cornea with the application equipment.
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 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, 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 and opacity 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. Changes in thickness were not 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. The location of any minor findings was marked on the record sheet as a drawing, if applicable. If any eye was considered to be unsuitable following baseline assessment, it was discarded.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: NaCl solution

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.


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 120 min
Value:
2
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:
3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein retention score
Value:
1.2
Vehicle controls validity:
not valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Run / experiment:
at up to 30 min
Value:
0.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Run / experiment:
at up to 240 min
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: 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.
Interpretation of results:
other: not GHS cat. 1
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. According to the OECD guideline 438 the test substance has been classified as "No prediction can be made" not cat. 1.
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 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 LPP (SIKA Härter LPP), no ocular corrosion or severe irritation potential was observed. According to the guideline OECD 438, SIKA Hardener LPP (SIKA Härter LPP) has been classified as “No prediction can be made” not cat. 1.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In Vitro 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 / 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 1x 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. The test item acted directly on MTT (MTT-reducer), therefore additional controls (test item treated killed tissues and untreated killed tissues) were used to detect and correct for test substance interference with the viability measurement. 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 (mean relative viability: 81 %). 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. The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item is considered as non-irritant to the skin and is therefore not classified.

Eye Irritation

One in vitro study according to OECD guideline 438 was performed to determine the eye irritation potential. The test showed that the test substance does not induce severe eye damage. The test item has been classified as "No prediction can be made". Therefore a further in-vivo test was performed to determine the eye irritation potential of the substance. Both tests are described in the following section.

In vitro

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 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. According to the guideline OECD 438, the test item has been classified as “No prediction can be made”.However, the test showed that the test substance does not induce severe eye damage (cat. 1, GHS).

In vivo

The acute eye irritation study of the test item SIKA Hardener LPP 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 solution 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 was observed in two animals. 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, 0.33, 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. Signs of pain and distress as discharge were not observed. In conclusion, the test item SIKA Hardener LPP, applied to the rabbits’ eye mucosa, caused slight conjunctival irritant effect which was fully reversible within 72 hours. According to Regulation (EC) No 1272/2008, the test item has not been classified into any category.

Conclusion:

By applying the Isolated Chicken Eye Model, the test item was determined to be not severely eye damaging. A further in vivo eye irriation study showed that the substance is neither irritating nor corrosive.

Justification for classification or non-classification

Skin irritation

Based on the available experimental data the test item is not classified and labelled for skin irritation/ corrosion according to Regulation (EC) No 1272/2008.

Eye Irritation

Based on the available experimental data the test item is not classified and labelled for eye irritation cat. 2 and eye corrosion cat. 1 according to Regulation (EC) No 1272/2008.