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Description of key information

Skin irritation potential of the test substance was tested in an in vitro study, according to OECD 439. Experimental data indicate no irritation potential to skin. Therefore, an in vitro skin corrosion study does not need to be conducted.

Eye irritation potential of the test item was examined in a tiered approach including an in vitro test, according to OECD 438, and an in vivo study according to OECD 405. Based in the experimental data the test item is considered to not possess an irritating or corrosive potential to the eye.

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:
2015-09-21 to 2015-11-18
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), 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. QA statement)
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: 15-EKIN-040
- Expiry date: 12 October 2015
- Date of initiation of testing: 07 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: 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


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

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 mean value (n = 3)
Value:
60
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 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:
- Acceptance criteria met for negative control: The mean OD value of the three negative control tissues was 0.877.
- Acceptance criteria met for positive control: The mean OD value obtained for the positive control was 0.183 and this result corresponds to 21 % 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 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 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: 60 %). 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 vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2016-03-08 to 2016-05-04
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:
2012
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
- Weight at study initiation: 2670-2850 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. Copy of the Certificate of food are maintained in Toxi-Coop Zrt.’s archive.
- 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. Copies of the relevant Certificates of Analysis are maintained in Toxi-Coop Zrt.’s archive.
- Acclimation period: 5 days in first animal and 6 days in second and third animal

ENVIRONMENTAL CONDITIONS
- Temperature: 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, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 0.1 mL
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):
The eyes were examined at 1, 24, 48 and 72 hours after the application.
Number of animals or in vitro replicates:
3
Details on study design:
REMOVAL OF TEST SUBSTANCE
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.

SCORING SYSTEM: The eye irritation scores were evaluated according to the scoring system by Draize (1977) and OECD 405 (2nd October 2012).
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
iris score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
iris score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
3
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.33
Max. score:
3
Reversibility:
fully reversible within: 48 h
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0.33
Max. score:
3
Reversibility:
fully reversible within: 48 h
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.66
Max. score:
4
Reversibility:
fully reversible within: 72 h
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Irritant / corrosive response data:
The eyes were examined at 1, 24, 48 and 72 hours after the application. 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. Sign of pain and distress as discharge was observed in all animals on the treatment day.
Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, the test item 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 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 and discharge were observed in animals. 24 hours after the treatment, slight conjunctival redness and chemosis were observed in animals. One animal 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, 0.33, 0.33; chemosis: 0.00, 0.66, 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 all animals on the treatment day.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin Irritation / Corrosion:

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 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: 60 %). 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 / Corrosion:

Eye irritating properties of the test item were examined in a tiered approach. First, an in vitro study was conducted, classifying the test substance either as eye corrosive GHS category 1, or as not requiring classification. In a second step an in vivo study was conducted in order to confirm non irritant potential of the test item.

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 (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 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 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 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 and discharge were observed in animals. 24 hours after the treatment, slight conjunctival redness and chemosis were observed in animals. One animal 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, 0.33, 0.33; chemosis: 0.00, 0.66, 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 was observed in all animals on the treatment day.

Conclusion:

In this tiered weight of evidence approach experimental in vitro and in vivo data indicate no eye irritating or corrosive properties of the test item.

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 / corrosion, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighth time in Regulation (EU) No 2016/918.

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