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Description of key information

In a Klimisch 1 (reliable without restrictions) in vitro study with the EpiDerm Reconstructed Human Epidermis model, the mean relative absorbance value decreased to 99.3% after treatment with the test item, compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

In a Klimisch 2 (reliable with restrictions) in vivo study in the rabbit eye, no symptoms of eye irritation occurred within 7 days when applied to the left eye of three male rabbits. During the 7-day post-administration assessment period evaluation of the eyes according to Draize (FDA Appraisal of the safety of chemicals in foods, drugs and cosmetics, 1959) revealed no difference between the treated eye and the untreated eye in the same animal or any difference from separate controls.

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 November 2017 to 15 December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
In an international prevalidation study performed by ECVAM, the in vitro skin irritation test using the human skin model EpiDerm™ and EpiSkin™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for skin irritancy potential.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Reconstructed Human Epidermis (MatTek)
- Tissue batch number(s): 25865
- Delivery date: 12 December 2017
- Date of initiation of testing: 13 December 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C for the first 35 minutes and room temperature for 25 minutes
- Temperature of post-treatment incubation: 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: After the end of the treatment interval the inserts were removed immediately from the 6-well plate and tissues were gently rinsed with DPBS at least 15 times in order to remove any residual test material. After the rinsing the inserts were submerged in DPBS at least three times. Afterwards the inserts were once again rinsed with sterile DPBS from the inside and the outside.
- Observable damage in the tissue due to washing: None observed
- Modifications to validated SOP: None

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Microplate reader (Versamax® Molecular Devices)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Mean 4.37%, range 2.20 to 6.78% for positive control
- Barrier function: 5.13 h (acceptance criteria: ET-50 is 4.77 to 8.72 hours)
- Contamination: No contamination with HIV-1 virus, hepatitis B virus, hepatitis C virus, bacteria, yeast or other fungi
- Reproducibility: Relative standard deviation 21.60% for positive control, 9.40% for negative control

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE: The test item passed the MTT- and the colour interference pre-tests.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: One experiment performed.

PREDICTION MODEL / DECISION CRITERIA
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439: An irritation potential of a test item leading to H315 (according to regulation (EC) 1272/2008), and GHS category 2 according to UN GHS (published 2003, 7th revision 2017) is recommended if the mean relative tissue viability of three individual tissues is reduced ≤ 50% of the negative control.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): neat

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): 5%
Duration of treatment / exposure:
60 minutes (incubated for 35 minutes at 37 ± 1.5 °C, 5 ± 0.5 % CO2 and moved to a sterile bench at room temperature until the end of treatment)
Duration of post-treatment incubation (if applicable):
42 hours (24 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2 after first rinse and another 18 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2 after second rinse)
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
99.3
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No
- Colour interference with MTT: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Technical proficiency was proven with 10 reference substances, testing performed in March 2014.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, mean OD was 1.830
- Acceptance criteria met for positive control: Yes, mean relative tissue viability was 4.3%
- Acceptance criteria met for variability between replicate measurements: Yes, the highest relative standard deviation was 15.3%
- Range of historical values if different from the ones specified in the test guideline: Negative control mean OD was 1.830 (historical 1.34 - 2.00) and positive control mean OD was 0.079 (historical 0.03 - 0.11)

Table 1. Skin irritation results

 Treatment group  Replicate  OD(570nm) Well 1  OD(570nm) Well 2  OD(570nm) Well 3  Mean OD of 3 Wells  Mean OD of 3 Wells, blank corrected  Mean OD of 3 tissues, blank corrected  Relative viability (%)  Relative standard deviation (%)  Mean relative viability (%)
 Blank  -  0.035  0.035  0.036  0.035  -  -  -  -  -
 Negative control  1  1.989  1.966  1.951  1.969  1.933  1.830  105.6  5.4  100.0
   2  1.863  1.845  1.855  1.854  1.819    99.4    
   3  1.782  1.765  1.772  1.773  1.738    95.0    
 Positive control  1  0.109  0.112  0.113  0.111  0.076  0.079  4.2  9.5  4.3
   2  0.103  0.104  0.116  0.108  0.073    4.0    
   3  0.121  0.118  0.127  0.122  0.087    4.8    
 Test item  1  1.538  1.529  1.538  1.535  1.500  1.818  82.0  15.3  99.3
   2  1.974  1.977  1.964  1.971  1.936    105.8    
   3  2.051  2.044  2.060  2.052  2.016    110.2    
Interpretation of results:
GHS criteria not met
Conclusions:
After treatment with the test item the mean relative absorbance value decreased irrelevantly to 99.3% compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.
Executive summary:

An in vitro study was performed to assess the irritation potential of the test item by means of the Human Skin Model Test using the EpiDerm Reconstructed Human Epidermis model. The test item passed the MTT- and the Colour Interference pre-tests. The test item, the negative control (DPBS), and the positive control (5% SLS) were applied to triplicate tissue each. The test item and the positive and negative controls were washed off the skin tissues after 60 minutes treatment. After further incubation for about 42 hours the tissues were treated with the MTT solution for 3 hours following 2 hours and 45 minutes extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm. Compared to the relative absorbance value of the negative control the mean relative absorbance value was reduced to 99.3% after exposure of the skin tissues to the test item. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential. The acceptability criteria for the negative control, positive control, variablity and historical results were met. This study is considered to be reliable without restrictions (Klimisch 1) as it was GLP-compliant and was performed according to OECD Guideline 439 (2015) and EU Method B.46 (2008).

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:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
not applicable
Principles of method if other than guideline:
- Principle of test: A single application of 0.1 g of the test item suspended in peanut oil DAB 7 or 0.1 mL of peanut oil DAB 7 was instilled into the conjunctival sac of the left eye of male New Zealand White-EMD rabbits. The right eye remained untreated and served as the control. Animals were observed for one week.
- Short description of test conditions: Experimental conditions were maintained at 23 to 33 °C with 45 to 73 % relative humidity.
- Parameters analysed / observed: Eye lesions
GLP compliance:
no
Species:
rabbit
Strain:
New Zealand White
Remarks:
EMD
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 2.6 to 3.2 kg (mean 2.9 kg)
- Housing: Individually caged in standard cages on wire grids.
- Diet: Altromin MS, ad libitum.
- Water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 to 33 °C
- Humidity (%): 45 to 73 %
Vehicle:
other: peanut oil DAB 7
Controls:
yes, concurrent no treatment
yes, concurrent vehicle
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 0.1 g test item suspended in vehicle

VEHICLE
- Amount(s) applied: 0.1 mL
Duration of treatment / exposure:
Single application
Observation period (in vivo):
The eyes of the rabbits were examined daily (Monday through Saturday) for one week.
Number of animals or in vitro replicates:
3 male rabbits per treatment and vehicle control
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing: No

SCORING SYSTEM: Draize, FDA-Appraisal (1959)

TOOL USED TO ASSESS SCORE: Hand lamp
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
7 d
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
0
Remarks on result:
not measured/tested
Irritation parameter:
iris score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
0
Remarks on result:
not measured/tested
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
0
Remarks on result:
not measured/tested
Irritation parameter:
chemosis score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
0
Remarks on result:
not measured/tested
Irritant / corrosive response data:
A single application of the test item or vehicle was tolerated by the rabbits without irritation and reaction. The treated eye did not differ from the untreated control eye.
Interpretation of results:
GHS criteria not met
Conclusions:
No symptoms of eye irritation occurred within 7 days.
Executive summary:

Eye irritation of the test item was tested in male New Zealand White-EMD rabbits. A single application of 0.1 g of the test item suspended in peanut oil DAB 7 or 0.1 mL of peanut oil DAB 7 was instilled into the conjunctival sac of the left eye. The right eye remained untreated and served as the control. Animals were examined daily for eye lesions for one week and changes were evaluated according to Draize, FDA-Appraisal (1959). No symptoms of eye irritation occurred within 7 days in animals treated with test item or vehicle. The treated eye did not differ from the untreated control eye. This study is considered to be reliable with restriction (Klimisch 2) as it was conducted similar to OECD Guideline 405, however there were minor limitations in experimental design and reporting.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

An in vitro study was performed to assess the irritation potential of the test item by means of the Human Skin Model Test using the EpiDerm Reconstructed Human Epidermis model (2018). The test item passed the MTT- and the Colour Interference pre-tests. The test item, the negative control (DPBS), and the positive control (5% SLS) were applied to triplicate tissue each. The test item and the positive and negative controls were washed off the skin tissues after 60 minutes treatment. After further incubation for about 42 hours the tissues were treated with the MTT solution for 3 hours following 2 hours and 45 minutes extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm. Compared to the relative absorbance value of the negative control the mean relative absorbance value was reduced to 99.3% after exposure of the skin tissues to the test item. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential. The acceptability criteria for the negative control, positive control, variablity and historical results were met. This study is considered to be reliable without restrictions (Klimisch 1) as it was GLP-compliant and was performed according to OECD Guideline 439 (2015) and EU Method B.46 (2008).

Eye irritation of the test item was tested in male New Zealand White-EMD rabbits (1973). A single application of 0.1 g of the test item suspended in peanut oil DAB 7 or 0.1 mL of peanut oil DAB 7 was instilled into the conjunctival sac of the left eye. The right eye remained untreated and served as the control. Animals were examined daily for eye lesions for one week and changes were evaluated according to Draize, FDA-Appraisal (1959). No symptoms of eye irritation occurred within 7 days in animals treated with test item or vehicle. The treated eye did not differ from the untreated control eye. This study is considered to be reliable with restriction (Klimisch 2) as it was conducted similar to OECD Guideline 405, however there were minor limitations in experimental design and reporting.

Justification for classification or non-classification

Two reliable (Klimisch 1 and 2) studies are available to inform the irritation / corrosion classification of the test item (1973, 2018). In the in vitro skin irritation study, the mean relative absorbance value was above the threshold for irritancy (50%). In the eye irritation study, no effects of the test item were observed in 3 rabbits. Therefore, according to CLP Regulation (EC) No 1272/2008, Guidance on the Application of the CLP Criteria" (ECHA 2017) and the 8th Adaptation to Technical Progress to CLP Regulation, the test item is not classified as a skin irritant or eye irritant.