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Description of key information

The test substane is not irritant to skin and eyes.

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:
22/01/2016-31/05/2016
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)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Details on test system:
PRE-TEST
1) Assessment of direct test Item reduction of MTT
- 25 +/- 2 mg of test item were added to 1 mL of MTT solution
- Incubation temperature: 37°C +/- 1.5 °C
- Incubation duration: 1h
- Assessment: blue/purple coloration of the MTT solution indicates chemical interference of the test chemical with MTT reduction.

2) Assessment of colored or staining materials
- 25 mg of test item were mixed with 300 µL of deionised water
- Incubation temperature water mixture: 37 +/- 1.5°C
- Incubation time water mixture: 1h
- Assessment: coloration of the test mixture indicates possible interation of the MTT measurement.

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200-SIT Kit
- Kit Lot number(s): 23314

PRE-WARMING OF EPIDERM TISSUES
- Quality control EpiDermTM tissues:
1. air bubbles between agarose and insert are not > 30% of the total surface,
2. liquid on top of the insert is removed with steriles cotton tips,
3. if again moisture is observed on top of the inserts after the pre-incubation or in case of visible defects the respective skin models were discarded.
- Pre-incubation in assay medium for 60 minutes in the incubator (37 ± 1.5 °C, 5 ± 1% CO2, 95 ± 5% RH). Transfer inserts from upper wells into the lower wells and continue pre-incubation for 23 hours (37 ± 1.5 °C, 5 ± 1% CO2, 95 ± 5% RH).

MAIN EXPERIMENT

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 +/- 1.5°C
- Temperature of post-treatment incubation (if applicable): 37 +/- 1.5°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: at least 15 rinsings with DPBS.
- After the rinsigs, the inserts were submerged in DPBS at least 3 times and once again rinsed with DPBS from the inside and outside.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL in the MTT diluent
- Incubation time: 3h
- Extraction: with isopropanol solution for 67.3h at 2 - 8 °C
- Spectrophotometer: microplate reader Versamax Molecular Devices, Softmax Pro version 4.7.1
- Wavelength: 570 +/- 1 nm

NUMBER OF REPLICATE TISSUES: 3

DATA EVALUATION
- Negative control: Mean OD of 3 negative control tissues was calculated after blank correction. This value corresponds to 100% tissue viability.
- Test item or the positive control: individual relative tissue viability is calculated according to: relative viability (%) = {mean OD (test item/positive control) / mean OD negative control} * 100
The mean relative viability ± rel. standard deviation of 3 individual tissues was calculated and used for classification

PREDICTION MODEL / DECISION CRITERIA
For the current test, an irritation potential of a test item according to EU classification H315 (according to regulation (EC) 1272/2008) is recommended if the mean relative tissue viability of three individual tissues is reduced ≤ 50% of the negative control.
- In vitro result: mean tissue viability =< 50% ; classification Irritant Category 2 (H315)
- In vitro result: mean tissue viability > 50% ; classification Non Irritant

VALIDATION
- Negative control: absolute OD negative control is indicator of tissue viability and should be ≥ 0.8 and ≤ 2.8.
- Positive control: An assay is meeting the acceptance criterion if mean relative tissue viability of the positive control is ≤ 20%.
- Standard deviation: SD of 3 identical replicates should be < 18%.
- OD values should not be below historically established boundaries.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL: 25mg (+- 39 mg/cm2 according to guideline) in 25µL DPBS
Duration of treatment / exposure:
60 minutes: 35 minutes in incubator + 25 minutes on sterile bench at room temperature
Duration of post-treatment incubation (if applicable):
24 hours
Number of replicates:
3
Irritation / corrosion parameter:
other: cell viability (%)
Run / experiment:
Mean of all runs
Value:
112.4
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The mean absorbance of the 3 tissues treated with the test item was 1.757. The mean relative absorbance value is calculated from this absorbance and corresponds to the cell viability. The cell viability was 112.4% for the test item with the negative control set to 100%. The threshold for classification for irritancy is ≤ 50%, consequently the test item was not irritant to skin.

Colour interference pre-experiment: No color changes observed; the test substance did not cause optical interference.

Pre-experiment assessing direct reduction of MTT by the test chemical: No color changes observed; the test substance did not cause direct reduction of MTT.

Detailed results:

Dose Group Exposure interval Tissue number Mean absorbance of 3 Wells Mean Absorbance of three wells blank corrected Mean Absorbance of 3 wells after blank correction* Rel. Absorbance (%) Tissue 1,2 + 3** Rel. Standard Deviation (%) Mean Rel. Absorbance (% of Negative Control)***
Blank     0.000          
Negative Control 60 min 1 1.532 1.495 1.563 95.6 4.1 100.0
2 1.644 1.591 100.6
3 1.717 1.631 103.8
Positive Control 60 min 1 0.118 0.081 0.080 5.2 4.4 5.1
2 0.118 0.107 4.8
3 0.104 0.132 5.3
Test item 60 min 1 1.773 1.735 1.757 111.0 4.9 112.4
2 1.904 1.875 118.4
3 1.706 1.731 107.8

* Mean of three replicate wells after blank correction

** Relative absorbance per tissue (rounded value): 100*(absorbance tissue)/(mean absorbance negative control)

*** Relative absorbance per treatment group (rounded values): 100* (mean absorbance test item/positive control)/mean absorbance negative control)

Validity criteria: the validity criteria are met

- Absorbance negative control: within the acceptability criterion (mean OD ≥0.8 and ≤2.8)

- Positive control: decrease in the relative absorbance compared to the negative control

- Relative standard deviations: were below 5% (threshold < 18%)

Interpretation of results:
not irritating
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
The test substance is not irritant to skin according to the OECD 439 in vitro test.
Executive summary:

In the current study the irritant effects of the test item were asessed in an in vitro test performed according to OECD 439 (in vitro Skin Irritation), EU B.46 (In vitro skin irritation: reconstructed human epidermis model test) and GLP. The test uses a Human Skin Model test and MTT as a read-out system. The Human Skin Model test can be used to identify chemicals that do not require classification for skin irritation or skin corrosion according to the UN GHS classification system. Consequently, a negative result in this test can also be used to conclude that no classification for Skin irritation Cat 2 or Skin corrosion Cat 1 is required under CLP. However, a positive result in this test does not allow to differentiate between Skin irritation and Skin corrosion.

In this in vitro test, the test chemical was topically applied to a 3 -dimensional reconstructed human epidermis (RhE) tissue. The tissue cell viability was measured as the degree of enzymatic conversion of the vital dye MTT by the viable cells to the corresponding blue MTT formazan salt, which is quantified after extraction from the tissue. The test item, positive and negative controls were tested in triplicate.

A pre-test is performed to ensure that the colour of the test chemical does not interfere with the MTT formazan measurement (optical interference), and that the test chemical does not cause (non-enzymatic) reduction of MTT into MTT formazan (chemical interference).

In the pre-test, buccoxime did not cause optical or chemical interference.

In the main study, the cell viability was found to be 112.4% with the negative control set to 100%. The threshold for classification for irritancy is ≤ 50%. Treatment with the positive control induced a decrease in the mean relative absorbance compared with the negative control to 5.1%. The validity criteria of the study were met.

In conclusion, the test item was found to be not irritant to the skin under the experimental conditions.

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:
key study
Study period:
29.01-19.02.2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD 492: Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage (July, 2015)
Deviations:
no
Qualifier:
according to
Guideline:
other: MatTek Corporation Protocol: EpiOcular TM Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; for use with MatTek Corporation's Reconstructed Human EpiOcular TM Model; 14 July 2014
Deviations:
no
Principles of method if other than guideline:
The EpiOcular Eye Irritation Test (EIT) was validated by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) and cosmetics Europe between 2008 and 2013. The test consists of a topical exposure of the neat test item to human reconstructed cornea model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT (3-4,5-dimethyl thiazole 2-yl)2,5-diphenyl-tetrazoliumbromide), present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. Reduction of cell viability in comparison to untreated negative controls is used to predict eye irritation potential.
GLP compliance:
yes (incl. certificate)
Species:
human
Strain:
other: epidermal keratinocytes
Details on test animals or tissues and environmental conditions:
CELL CULTURE
- Source: MatTek Corporation (Ashland, MA 01721, USA)
- Model: human-derived epidermal keratinocytes forming a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium.
- Surface: 0.6 cm2
- Storage conditions: 2 - 8 °C on medium-supplemented agarose gels

IN-LIFE DATES: From: 16.02.2016
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 mg
- Concentration (if solution): 100 %
Duration of treatment / exposure:
6 hours
Observation period (in vivo):
not applicable
Duration of post- treatment incubation (in vitro):
Ca. 18 hours
Number of animals or in vitro replicates:
duplicate
Details on study design:
PRE-TEST
1) Assessment of direct test Item reduction by MTT
- 50 mg of test item were added to 1 mL of a 1.0 mg/mL MTT solution in DMEM in a 6-well plate.
- Incubation temperature: 37°C +/- 1.5 °C
- Incubation duration: approx. 3 h
- Assessment: blue/purple coloration of the MTT solution indicates chemical interference of the test chemical with MTT reduction.

2) Assessment of colored or staining materials
- 50 mg of test item were added to 1.0 mL of water and to 2 mL of isopropanol in 6-well plates.
- Incubation temperature water mixture: 37 +/- 1.5°C
- Incubation time water mixture: at least 1h
- Incubation temperature isopropanol mixture: room temperature
- Incubation time isopropanol mixture: 2-3 hours
- Assessment: coloration of the test mixtures indicates possible interation of the MTT measurement.

TEST KIT INFORMATION
- EpiOcular and MTT-100 kits are purchased from MatTek Corporation (Ashland, MA 01721, USA)
- Test Kit Lot No: 21597


EXPERIMENTAL DESIGN
- Incubation: overnight at standard culture conditions for 30 minutes.
- Exposure conditions: standard culture conditions (37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH)
- Removal test item: extensively rinsing with Ca++Mg++-free DPBS
- Post- soak immersion: incubation in assay medium was intended to remove any test item or control absorbed into the tissue.
- Post-treatment: blotting on absorbent material and incubation for 18 hours in assay medium at standard culture conditions.

CONTROLS
- Negative: deionised water
- Positive: methyl acetate

MTT
- Concentration solution: 1.0 mg/mL in DMEM
- Assessment of Direct Test Item Reduction by MTT: 3h incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air
- Assessment of Colored or Staining Materials: 1h incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air
- Assay: incubation for 180 ± 10 minutes at standard culture conditions and rinsed 3x with DPBS afterwards.

DATA EVALUATION
1) Mean OD value of the blank control wells (ODBlk) for each experiment was calculated.
2) Mean ODBlk from each OD value of the same experiment (blank corrected values) was subtracted.
3) Mean value of 2 aliquots for each tissue (= corrected test item OD) was calculated.
4) Mean value of 2 relating tissues for each control and test item (= corrected mean OD) was calculated. For further calculations only the corrected mean negative control OD value was needed.
5) The corrected OD value of the negative control corresponds to 100% viability. Corrected negative control OD = Negative Control OD - ODBlk = 100% Viability

CALCULATIONS
1) % viability of the 2 relating tissues for each control and test item relative to the negative control (100% control) was calculated.
Viability [%]= 100 x (corrected test item OD / corrected mean negative control OD)
2) The difference of the viability between duplicate tissues Is calculated.
3) The mean test item viability (TI viability) was calculated and the test item is classified according to the prediction model.

VALIDITY CRITERIA
The results are acceptable according to MatTek Protocol, if:
1) The negative control OD is > 0.8 and < 2.5,
2) The mean relative viability of the positive control is below 50% of the negative control viability.
3) The difference of viability between the two relating tissues of a single test item is < 20% in the same run (for positive and negative control tissues and tissues of test items). This applies also to the killed controls (items and negative killed control) and the colorant controls which are calculated as percent values related to the viability of the relating negative control.
Irritation parameter:
other: Cell viability (%)
Run / experiment:
Mean of all runs
Value:
82.9
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The mean absorbance of the 2 tissues exposed to the test item was 1.292. From this the relative absorbance value is calculated and this corresponds to the cell viability. For the test item the cell viability was calculated to be 82%, when the negative control was set to 100 %. The threshold for irritancy is ≤ 60%, consequently the test item was not irritant to eye.

Colour change pre-experiments: No change in color was observed.

Pre-experiment assessing direct reduction of MTT by the test chemical: No color changes observed; the test substance did not cause direct reduction of MTT.

Results after treatment for 6 hours with test item and the controls:

Dose group Mean Absorbance*
Tissue 1 and 2
Mean Absorbance*
of 2 tissues
Rel. Absorbance (%) Tissue 1 and 2** Absolute value
 of the difference of the
 Rel. Absorbance (%)
Tissue 1 and 2
Rel. Absorbance
 (% of Negative Control)**
Negative
Control
1.545 1.558 99.2 1.6 100.0
1.610 100.8
Positive
Control
0.635 0.612 40.8 2.9 39.3
0.637 37.8
Test Item 1.197 1.292 76.9 12.2 82.9
1.425 89.0

*Mean of two replicates wells after blank correction

** Relative absorbance (rounded values): 100 * (absorbance (test item/positive control)) / (absorbance negative control)

Validity criteria:

- The negative control OD is > 0.8 and < 2.5 ( 1.564 and 1.612)

- The mean relative viability of the of the positive control is below 50% of the negative control viability (39.3%).

- The difference of viability between the two relating tissues of a single item is < 20% in the same run (value between 1.6 % to 12.2%).

Interpretation of results:
not irritating
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
The test item does not possess eye irritating potential under the test conditions set in an in vitro OECD 492 study.
Executive summary:

In the current study the eye irritation potential of the test item was assessed in an in vitro study according to OECD 492 guideline, MatTek Corporation Protocol EpiOcular Eye Irritation Test and under GLP. The EpiOcular EIT can be used to identify chemicals that do not require classification for eye irritation or serious eye damage according to the UN GHS classification system. Consequently, a negative result in this test can also be used to conclude that no classification for Eye damage Cat 1 or Eye Irritation Cat 2 is required under CLP. However, a positive result in this test does not allow to differentiate between Eye damage Cat 1 and Eye Irritation Cat 2.

In this in vitro test, the test chemical was topically applied to a 3 -dimensional reconstructed human cornea-like eipithelium (RhCE ) tissue. The tissue viability was measured following exposure and a post-treatment incubation period. The RhCE tissue viability is measured as the degree of enzymatic conversion of the vital dye MTT by the viable cells to the corresponding blue MTT formazan salt, which is quantitatied after extraction from the tissue. The test item, positive and negative controls were tested in duplicate. The exposure duration was 6 hours.

A pre-test is performed to ensure that the colour of the test chemical does not interfere with the MTT formazan measurement (optical interference), and that test chemical does not cause chemical (non-enzymatic) reduction of MTT into MTT formazan (chemical interference).

In the pre-tests, buccoxime did not cause optical nor chemical interference.

In the main study, the cell viability was calculated to be 82%, when the negative control was set to 100 %. The threshold for irritancy is ≤ 60%. Treatment with the positive control induced a decrease in the mean relative absorbance compared with the negative control to 39.3%. The validity criteria of the study were met.

In conclusion, the test item was found to be not irritant to the eye under the experimental conditions.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation:

Two studies are available that assess possible irritation or corrosion effects of the test substance towards the skin. The studies were performed according to OECD guideline and GLP.

In the key study (Sinn S. , 2016) the test substance was tested in vitro by means of the Human Skin Model test according to OECD 439 (in vitro Skin Irritation), EU B.46 (In vitro skin irritation: reconstructed human epidermis model test) and GLP. In this test, 25 mg of the test item , 30µL of either the negative control (DPBS) or the positive control (5% Sodium Lauryl Sulphate) was applied to the tissue, each in triplicate.

After 60 minutes the tissues were washed extensively and the amount of extracted colorant was determined photometrically at 570 nm. The validation criteria set for the negative and positive controls were met indicating the quality and validity of the study.

The mean relative absorbance value of the test item, corresponding to the cell viability, was not reduced (112.4%) when compared to the negative control (100%). Also, it was above the treshold for irritancy (50%) indicating the substance is not skin irritant.

In the supporting study (Huygevoort, 1998) the test substance was tested for its potential irritation/corrosion effect by placing a single dose of the test substance as a 2% solution in parrafin oil on the skin of albino rabbits (semi-occlusive method). 3 rabbits were exposed on the clipped skin for 4 hours, followed by observations at 1, 24, 48, and 72 hours.

The exposure resulted in very slight erythema in the treated skin-area of 1 rabbit 1 hour after exposure, however, the effects were fully reversible. No further signs of irritation were observed in any other animal.

The erythema and oedema scores were 0 and therefore the test item does not have to be classified for skin irritation.

Both studies indicate the test item is not irritant to the skin.

Eye irritation:

There are two studies available assessing the eye irritation potential of the test item.

In the key study (Sinn, 2016) an in vitro test was performed using the Human Cornea Model Test according to OECD 492.

The test item, positive and negative controls were tested in duplicate and the exposure duration was 6 hours.

In the pre-tests the test item did not cause optical nor chemical interference.

In the main study, the cell viability was calculated to be 82%, when the negative control was set to 100 %. The threshold for irritancy is ≤ 60%.Treatment with the positive control induced a decrease in the mean relative absorbance compared with the negative control to 39.3%. The validity criteria of the study were met.

In conclusion, the test item was found to be not irritant to the eye under the experimental conditions.

This is in line with the observations made in the supporting study (unknown, 1983). In this study the eye irritant potential of the test item was assessed according to the Draize method, however, without following an OECD or GLP.

6 albino rabbits were used in the study and the right eye served as a control. The eyes were scored 1, 2 and 8 hours and 1, 2, 3, 4, 5, 6 and 7 days after treatment.

The ocular lesions score comprising the effects seen in cornea, iris and conjunctiva was calculated. This score was 0.5 and was the mean for 6 animals at 24, 48 and 72 hours.


Justification for selection of skin irritation / corrosion endpoint:
The study was performed according to internationally accepted guidelines and GLP, without significant deviations.

Justification for selection of eye irritation endpoint:
The in vitro study has been performed according to internationally accepted guidelines and GLP without signicant deviations.

Justification for classification or non-classification

Skin irritation:

As indicated in section 3.2.2.1 of Annex I of the CLP Regulation N° 1272/2008 'In vitro alternatives that have been validated and accepted may also be used to help make classification decisions. In some cases enough information may be available from structurally related compounds to make classification decisions.'

The criteria set out in Table 3.2.2 for skin irritation are not directly applicable to the results from the in vitro study according to OECD 439, therefore the criteria specified for this in vitro test were used for classification purposes. The threshold for irritancy is 50% and the key study revealed a mean relative absorbance value of 112.4% for the test item. The results indicate that the substance should not be classified as skin irritant.

Additionally, the supporting in vivo skin irritation study showed erythema and oedema scores of 0, confirming that the test substance does not fulfil the criteria for classification as skin irritant.

Eye irritation:

As indicated in section 3.3.2.3 of Annex I of the CLP Regulation N° 1272/2008 'In vitro alternatives that have been validated and accepted can be used to make classification decisions'

The criteria set out in Tables 3.3.1 and 3.3.2 for eye irritation are not directly applicable to the results from the in vitro study according to OECD 492, therefore the criteria specifically for this in vitro test, as described in the applicable OECD guideline, were used for classification purposes. The threshold for irritancy is 60% and the key study revealed a mean relative absorbance value of 82% for the test item. The results indicate that the substance should not be classified as eye irritant.

Additionally, the supporting in vivo eye irritation study showed an overal eye irritation score of 0.5, confirming that the test substance does not fulfil the criteria for classification as skin irritant.