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Administrative data

Description of key information

Skin irritation

The test item was determined to be not irritant to skin in the in vitro Human Skin Model Test.

 

Eye irritation

The test substance is not irritating to the eye of rabbit.

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:
2016-05-18 to 2016-06-20
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:
July 28, 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
REACH; 1st ATP 2009: EC Regulation No 761/2009 of 23 July 2009 amending, for the purpose of its ATP, EC Regulation No 440/2008 laying down test methods
Deviations:
no
Qualifier:
according to
Guideline:
other: UN GHS (published 2003, last (6th) revision 2015)
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: human skin model EpiDerm™
- Tissue batch number: 23341
- Delivery date: 14 June 2016
- Date of initiation of testing: 14 June 2016 (preincubation phase)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C, 5 ± 0.5% CO2
- Temperature of post-treatment incubation: 37 ± 1.5 °C, 5 ± 0.5 % CO2

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: Rinsed with DPBS at least 15 times, 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: No
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: (1 mg/mL)
- Incubation time: 3 hours
- Spectrophotometer: Versamax® Molecular Devices, Softmax Pro, version 4.7.1
- Wavelength: 570 nm
- Filter: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Positive: 4.64%, range 4.00 - 5.90 %

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Since the MTT solution did not turn blue/purple, the test item was not considered to reduce MTT and an additional test with freeze-killed tissues did not have to be performed.

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 mean tissue viability is ≤ 50 %.
- The test substance is considered to be non-irritant to skin if the mean tissue viability is > 50 %

Acceptability of the Assay
Criterion 1
Negative control: The absolute OD 570 nm of the negative control tissues in the MTT test is an indicator of tissue viability obtained after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is meeting the acceptance criterion if the mean OD570 of the negative control tissues is ≥ 0.8 and ≤ 2.8.

Criterion 2
Positive control: An assay is meeting the acceptance criterion if mean relative tissue viability of the positive control is ≤ 20%.

Criterion 3
Standard deviation: The 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
- Amount applied: 30 μL (47 μL/cm2)
- Concentration: undiluted

NEGATIVE CONTROL
- Amount applied : 30 μL DPBS (MatTek)

POSITIVE CONTROL
- Amount applied: 30 μL
- Concentration: 5% SLS solution in deionised water
Duration of treatment / exposure:
1 hour
Duration of post-treatment incubation (if applicable):
42.75 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean of 3 tissues
Value:
94.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100 % viability
Positive controls validity:
valid
Remarks:
4.5 % viability
Other effects / acceptance of results:
- Direct-MTT reduction: test item did not reduce MTT
- Colour interference with MTT: test item did not change colour in the colour interference test
- After treatment with the negative control the absorbance values were well in the required range of the acceptability criterion of mean OD = 0.8 and = 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues.
- Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 60 minutes treatment interval, and thus assuring the validity of the test system; the mean relative absorbance value of the positive control was 4.5% compared to the negative control.
Interpretation of results:
GHS criteria not met
Conclusions:
The test item was determined to be not irritant to skin in the in vitro Human Skin Model Test.
Executive summary:

This in vitro study was performed to assess the irritation potential of the test item by means of the Human Skin Model Test.

The test item did not reduce MTT (test for direct MTT reduction), and it did not change colour when mixed with deionised water (test for colour interference). Also its intrinsic colour was not intensive.

Consequently, additional tests with freeze-killed or viable tissues were not necessary. Each three tissues of the human skin model EpiDerm™ were treated with the test item, the negative or the positive control for 60 minutes.

30 μL of the test item were applied to each tissue, and spread to match the surface of the tissue. Furthermore, 30 μL of either the negative control (DPBS) or the positive control (5% SLS) were applied to each tissue. After treatment with the negative control the absorbance values were well within the required range of the acceptability criterion of mean OD0.8 and2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues.

Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 60 minutes treatment interval, and thus assuring the validity of the test system.

After treatment with the test item the mean relative absorbance value decreased irrelevantly to 94.2% compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of50%. Therefore, the test item is not considered to possess an irritant potential.

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is not irritant to skin.

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
Study period:
2000-02-22 to 2000-05-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
Feb. 1987
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Version / remarks:
Jan. 1997
Deviations:
no
GLP compliance:
yes
Species:
rabbit
Strain:
other: SPF albino
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: BI Pharma KG, 88397 Biberach
- Weight at study initiation: 2.7 - 3.3 kg
- Housing: Individually in PPO cages (floor area: 2576 sq.cm) with perforated floor
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature: 20°C +/- 3°C
- Humidity: 55% +/- 15%
- Air changes: 10 times/hour
- Photoperiod: 12 hours light/12 hours dark

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Amount applied : 0.1 mL

Duration of treatment / exposure:
24 hours
Observation period (in vivo):
7 days
Number of animals or in vitro replicates:
4
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing: 20 mL 0.9% sodium chloride solution
- Time after start of exposure: 24 hours (Fluorescein was instilled)

SCORING SYSTEM:

Cornea
Opacity-degree of density (area most dense taken for reading)
No ulceration or opacity: 0
Scattered or diffuse area of opacity (other than slight dulling of normal luster), details of iris clearly visible: 1
Easily discernible translucent areas, details of iris slightly obscured: 2
Nacreous areas, no details of iris visible, size of pupil barely discernible: 3
Complete corneal opacity, iris not discernible: 4

Iris
Normal: 0
Markedly deepened folds, congestion, swelling, moderate circumcorneal injection (any of these or combination of any thereof)
iris still reacting to light (sluggish reaction is positive): 1
No reaction to light, haemorrhage, gross destruction (any or all of these): 2

Conjunctivae
Redness (refers to palpebral and bulbar conjunctivae excluding cornea and iris)

Vessels normal: 0
Some vessels definitely injected: 1
More diffuse, deeper crimson red, individual vessels not easily discernible: 2
Diffuse beefy red: 3

Chemosis
No swelling: 0
Any swelling above normal (includes nictitating membrane): 1
Obvious swelling with partial eversion of lids: 2
Swelling with lids about half closed: 3
Swelling with lids about half closed to completely closed: 4

TOOL USED TO ASSESS SCORE: hand-slit lamp with white and UV-light/ magnifying glass with 2 x magnification (biomicroscope) / fluorescein
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:
cornea opacity score
Basis:
animal #4
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:
iris score
Basis:
animal #4
Time point:
24/48/72 h
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #1
Time point:
24/48/72 h
Score:
1
Max. score:
3
Reversibility:
fully reversible within: 7 days
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.67
Max. score:
3
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #3
Time point:
24/48/72 h
Score:
0.67
Max. score:
3
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #4
Time point:
24/48/72 h
Score:
1
Max. score:
3
Reversibility:
fully reversible within: 7 days
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.67
Max. score:
4
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
chemosis score
Basis:
animal #4
Time point:
24/48/72 h
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 48 hours
Interpretation of results:
GHS criteria not met
Conclusions:
The test substance is not irritating to the eye of rabbit.
Executive summary:

The acute eye irritation/corrosion study was conducted according to OECD guideline no. 405 and GLP.

The test substance was applied in one eye of four female albino rabbits. The eyes were examined and the changes were graded according to a numerical scale one hour, 24, 48 and 72 hours as well as 7 days after dosing.

Slight to well-defined signs of irritation were observed on the treated eyes.

All effects were fully reversible within 7 days.

For the four animals the mean scores after 24/48 and 72 hours was for cornea opacity: 0, 0, 0, 0; iris:0, 0, 0, 0; conjunctival redness: 1.0, 0.67, 0.67, 1.0; chemosis: 0.67, 0.33, 0.33, 0.33, all signs were fully reversible at 7 days.

Based on these results the test substance is not irritating to the eye of rabbit.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation

An in vitro study was performed to assess the irritation potential of the test item by means of the Human Skin Model Test according to OECD 439 and GLP.

The test item did not reduce MTT (test for direct MTT reduction), and it did not change colour when mixed with deionised water (test for colour interference). Also its intrinsic colour was not intensive.Consequently, additional tests with freeze-killed or viable tissues were not necessary.

Each three tissues of the human skin model EpiDerm™ were treated with the test item, the negative or the positive control for 60 minutes.

30 μL of the test item were applied to each tissue, and spread to match the surface of the tissue. Furthermore, 30 μL of either the negative control (DPBS) or the positive control (5% SLS) were applied to each tissue. After treatment with the negative control the absorbance values were well within the required range of the acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues.

Treatment with the positive control induced a sufficient decrease in the relative absorbance as compared to the negative control for the 60 minutes treatment interval, and thus assuring the validity of the test system.

After treatment with the test item the mean relative absorbance value decreased irrelevantly to 94.2% 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 conclusion, it can be stated that in this study and under the experimental conditions reported, the test item is not irritant to skin.

Eye irritation

The acute eye irritation/corrosion study was conducted according to OECD guideline no. 405 and GLP.

The test substance was applied in one eye of four female albino rabbits. The eyes were examined and the changes were graded according to a numerical scale of one hour, 24, 48 and 72 hours as well as 7 days after dosing.

Slight to well-defined signs of irritation were observed on the treated eyes.

All effects were fully reversible within 7 days.

For the four animals the mean scores after 24/ 48and 72 hours was for cornea opacity: 0, 0, 0, 0; iris:0, 0, 0, 0; conjunctival redness: 1.0, 0.67, 0.67, 1.0; chemosis: 0.67, 0.33, 0.33, 0.33, all signs were fully reversible at 7 days.

Based on these results the test substance is not irritating to the eye of rabbit.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

Based on the available experimental test data, the test substance is not considered to be classified for skin irritation/corrosion or eye irritation under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.