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Administrative data

Description of key information

The test substance was determined to be irritating in the in vitro human skin model test with EpiDerm.

The test substance was determined to be non-corrosive in the in vitro human skin model test with EpiDerm.

The test substance was shown to be not irritating to rabbit eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2015-11-09 to 2015-12-07
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:
2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2008
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:
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™, Epi-200- SIT Kit
- Tissue batch number: 23305
- Delivery date: 2015-12-01
- Date of initiation of testing: 2015-12-01

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C, for 35 min in incubator and for following 25 min at room temperature
- Temperature of post-treatment incubation: 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: at least 15 times, no defined volume
- Modifications to validated SOP: No modifications were made.

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

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test item did not prove to be a MTT reducer and the test item did not dye water.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritating to skin if the mean tissue viability is less than or equal to 50 % (Cat. 2).
- The test substance is considered to be non-irritating to skin if the mean tissue viability is greater than 50 %.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 30 μL (47 μL/cm2)

NEGATIVE CONTROL
- Amount applied: 30 μL

POSITIVE CONTROL
- Amount applied: 30 μL
Duration of treatment / exposure:
60 min
Duration of post-treatment incubation (if applicable):
After rinsing the tissues was incubated for 24 h in fresh medium. After the 24 h the tissues were transfered to new 6 wells with fresh medium and incubated for additional 17.5 h.
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 min exposure
Value:
8.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100 %
Positive controls validity:
valid
Remarks:
4.9
Remarks on result:
positive 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

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The tissue viability met the acceptance criterion as the mean OD570 of the negative control tissues was ≥ 0.8 and ≤ 2.8.
- Acceptance criteria met for positive control: The assay met the acceptance criterion as the mean relative tissue viability of the positive control was ≤ 20 %.
- Acceptance criteria met for variability between replicate measurements: The SD of 3 identical replicates was < 18 %. OD values were below historically established boundaries.
Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
The test substance was determined to be irritating in the in vitro human skin model test with EpiDerm.
Executive summary:

The in vitro Human Skin Model Test with EpiDerm study according to OECD 439 was performed to assess the irritation potential of the test substance. Independent triplicate tissues of EpiDerm were exposed to either the test item, the negative control (DPBS) or the positive control (5 % SDS in water) for 1 hour. 30 μL of the test item were dispensed directly onto triplicate EpiDerm tissue surface, and spread to match the surface of the tissue. After exposure of the tissues to the test substance the mean tissue viability was 8.4 % after 1 h exposure. This value is bwlow the threshold for irritation, which is defined to be < 50 %. Therefore, the test substance was considered to be skin irritating. In conclusion, it can be stated that in this study and under the reported experimental conditions, the test was irritating to skin according to EU CLP and UN GHS (Category 2, H315: Causes skin irritation). No observations were made which might cause doubts concerning the validity of the study outcome.

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2016-03-17 to 2016-03-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
July 28, 2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Version / remarks:
30 May 2008
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:
Validation studies have shown that tests employing human skin models are able to discriminate between known skin corrosives (Optional sub-category 1A, optional subcategory 1B and 1C) and non-corrosives. The test protocol may also enable the distinction between severe and less severe skin corrosives.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Kit
- Tissue batch number: 23324
- Shipping date: 2016-03-15
- Date of initiation of testing: 2016-03-17

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C, for exposure of 3 ± 0.5 min at room temperature
- Temperature of post-treatment incubation: 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 20 times, no defined volume
- Modifications to validated SOP: There was the following alteration from the MatTek test protocol: Concerning exposure conditions (chapter 3.6.2): Only the 6-well plates for the 60 ± 5 minutes exposure period was placed into an incubator (37 ± 1.5 °C, 5 ± 0.5% CO2) during treatment, the wells for the 3 ± 0.5 minutes exposure periods stayed at room temperature in the sterile bench.

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

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test item did not prove to be a MTT reducer and the test item did not dye water.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50% (Cat. 1A), or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15% (Cat. 1B and 1C).
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
other: Controls for MTT assay
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 50 μL (79.4 μL/cm2)

NEGATIVE CONTROL
- Amount applied: 50 μL

POSITIVE CONTROL
- Amount applied: 50 μL
Duration of treatment / exposure:
3 min and 1 h
Duration of post-treatment incubation (if applicable):
3 h MTT incubation, 67.5 h extraction
Number of replicates:
2
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 min exposure
Value:
98.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100.0 %
Positive controls validity:
valid
Remarks:
27.9 %
Remarks on result:
other: no indication of corrosion
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 h exposure
Value:
98.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100.0 %
Positive controls validity:
valid
Remarks:
6.6 %
Remarks on result:
other: no indication of corrosion
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: the mean OD of the tissue replicates treated with the negative control was ≥ 0.8 and ≤ 2.8 for every exposure time
- Acceptance criteria met for positive control: the mean viability of the tissue replicates treated with the positive control for 1 hour, was <15% compared to the negative control
- Acceptance criteria met for variability between replicate measurements: the Coefficient of Variation (CV) in the range 20 – 100% viability between tissue replicates was ≤ 30%
Interpretation of results:
other:
Remarks:
not GHS cat. 1
Conclusions:
The test substance was determined to be non-corrosive in the in vitro human skin model test with EpiDerm.
Executive summary:

The in vitro Human Skin Model Test with EpiDerm™ study according to OECD 431 was performed to assess the corrosion potential of the test substance. Independent duplicate tissues of EpiDerm™ were exposed to either the test item, the negative control (deionised water) or the positive control (8.0 N Potassium Hydroxide) for 3 min or 1 hour. 50 μL of the test item were dispensed directly onto EpiDermTM tissue surface, and spread to match the surface of the tissue. After exposure of the tissues to the test item the relative absorbance value decreased to 98.2 % after 3 minutes exposure. After 1 hour exposure the relative absorbance value was reduced to 98.1 %. Both values did not exceed the threshold for corrosivity which is defined to be 50 % after the 3 minutes exposure and 15% after the 1 hour exposure. Therefore, the test item was not considered to be corrosive. In conclusion, it can be stated that in this study and under the reported experimental conditions, the test substance was non corrosive to skin according to EU CLP and UN GHS. No observations were made which might cause doubts concerning the validity of the study outcome.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (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:
1985-05-30 to 1985-06-02
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
yes
Remarks:
For details on deviations please refer to "Principles of method"
Principles of method if other than guideline:
Deviation:
- The identification and stability of the test material were not determined.
- The oberservation period was 72 h. Afterwards the study was determinated as no effect was observed.
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Age at study initiation: 12-16 weeks
- Weight at study initiation: 2.43-3.05 kg
- Housing: individually, in suspended metal cages
- Diet: ad libitum, Rabbit Diet, A.W. Tindall Limited, Holbeach, Lincolnshire
- Water: ad libitum, tap water
- Acclimation period: min. 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.5 ± 2.0
- Humidity (%): 58-60
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 0.1 mL
- Concentration: 100 %

Duration of treatment / exposure:
Single treatment.
Test substance was not removed from the eye (only self-cleaning of eye).
Observation period (in vivo):
72 h.
Observations done after 1, 24, 48 and 72 h.
Number of animals or in vitro replicates:
3
Details on study design:
SCORING SYSTEM:
- Assessment of damage/irritation: Draize J.H., 1959, Association of Food and Drug Officials of the United States, Austin, Texas, "The Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics"
- classification of ocular irritation potential: modified version of the system described by Kay J.H. and Calandra J.C., J.Soc. Cosmet, Chem. 1962 13 281 to 289

TOOL USED TO ASSESS SCORE: standard opthalmoscope
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
fully reversible
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
fully reversible
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
24/48/72 h
Score:
0.33
Max. score:
3
Reversibility:
fully reversible
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
fully reversible
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
fully reversible
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
fully reversible
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.33
Max. score:
3
Reversibility:
fully reversible
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
fully reversible
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
fully reversible
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
fully reversible
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
fully reversible
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0.33
Max. score:
3
Reversibility:
fully reversible
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
fully reversible
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
fully reversible
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0.33
Max. score:
3
Reversibility:
fully reversible
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
fully reversible
Remarks on result:
no indication of irritation
Interpretation of results:
GHS criteria not met
Conclusions:
A study in rabbits was conducted to evaluate the irritation potential of the test substance in the eye. The results showed a minimal irritation effect of the test substance on rabbit eyes.
Executive summary:

The potential of the test substance to cause eye irritation was tested using rabbits accordin to OECD 405. The animals were observed for 72 hours. The mean score for cornea opacity and iris were 0 for 24/48/72 h. For conjuntivae redness it was 0.33 and for chemosis 0 at 24/48/72 h. For all animals the individual mean scores for cornea opacity and iris were 0 for 24/48/72 h. Animal 1 had a score for conjuntivae redness of 0.33 and 0 for chemosis at 24/48/72 h. Animal 2 had a score for conjuntivae redness of 0.33 and 0 for chemosis at 24/48/72 h. Animal 3 had a score for conjuntivae redness of 0.33 and 0 for chemosis at 24/48/72 h. All effects observed were reversible within 48 h. Due to these results the test substance was found to be slightly irritating for the eye but without classification.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation/corrosion

Weight of evidence

A weight of evidence approach was conducted to evaluate the skin irritation and corrosion potential of the test substance. Therefore two in vitro studies were conducted. In addition a supporting in vivo study was available.

The in vitro Human Skin Model Test with EpiDerm study according to OECD 439 was performed to assess the irritation potential of the test substance. Independent triplicate tissues of EpiDerm were exposed to either the test item, the negative control (DPBS) or the positive control (5 % SDS in water) for 1 hour. 30 μL of the test item were dispensed directly onto triplicate EpiDerm tissue surface, and spread to match the surface of the tissue. After exposure of the tissues to the test substance the mean tissue viability was 8.4 % after 1 h exposure. This value is below the threshold for irritation, which is defined to be < 50 %. Therefore, the test substance was considered to be skin irritating. In conclusion, it can be stated that in this study and under the reported experimental conditions, the test was irritating to skin according to EU CLP and UN GHS (Category 2, H315: Causes skin irritation). No observations were made which might cause doubts concerning the validity of the study outcome.

The in vitro Human Skin Model Test with EpiDerm study according to OECD 431 was performed to assess the corrosion potential of the test substance. Independent duplicate tissues of EpiDerm were exposed to either the test item, the negative control (deionised water) or the positive control (8.0 N Potassium Hydroxide) for 3 min or 1 hour. 50 μL of the test item were dispensed directly onto EpiDerm tissue surface, and spread to match the surface of the tissue. After exposure of the tissues to the test item the relative absorbance value decreased to 98.2 % after 3 minutes exposure. After 1 hour exposure the relative absorbance value was reduced to 98.1 %. Both values did not exceed the threshold for corrosivity which is defined to be 50 % after the 3 minutes exposure and 15% after the 1 hour exposure. Therefore, the test item was not considered to be corrosive. In conclusion, it can be stated that in this study and under the reported experimental conditions, the test substance was non corrosive to skin according to EU CLP and UN GHS. No observations were made which might cause doubts concerning the validity of the study outcome.

 

Supporting study

A skin irritation test according to OECD 404 was performed using 3 rabbits with an exposure time of 4 h. The animals were observed for 7 days. The experiment was terminated on day 7 when most effects were not detectable anymore or in clear regression. The test substance caused erythema and edema development that was reversible. The mean Primary Cutaneous Irritation Index for 24/72h was 2.3 with the undiluted (100 %) test substance. A concentration of 25 % resulted in a Primary Cutaneous Irritation Index of 0.3. A concentration of 5 % as well as 1 % resulted in an index of 0.0. The erythema score for animal 1 for 24/48/72h with the undiluted test substance was 2.0 and the edema score was 0.67. The erythema score for animal 2 for 24/48/72h with the undiluted test substance was 1.67 and the edema score was 0.33. The erythema score for animal 3 for 24/48/72h with the undiluted test substance was 2.0 and the edema score was 1.0. Due to these results the test substance was found to be moderately skin irritating but without classification.

 

Conclusion

The in vivo study in rabbits showed moderate skin irritating properties of the test substance. In this study a concentration of 25 % showed no irritation while a concentration of 100 % showed an irritation potential. As the test was terminated after 7 days with not reversible effects, instead of 14 days, no final conclusion on reversibility and consequences for classification was possible. Therefore further tests were conducted to evaluate the skin irritation/corrosion potential. The in vitro test according to OECD 439 showed skin irritating properties of the test substance and the OECD 431 study demonstrated that the test substance does not have skin corrosive potential. As these results are in line with the preliminary terminated in vivo study, this study was considered as supporting. The in vitro studies were considered together as weight of evidence. All studies were performed with the undiluted substance and therefore the classification for skin irritation cat. 2 represents the worst case.

 

Eye irritation/corrosion

The potential of the test substance to cause eye irritation was tested according to OECD 405 using rabbits. The animals were observed for 72 hours. The mean score for cornea opacity and iris were 0 for 24/48/72 h. For conjuntivae redness it was 0.33 and for chemosis 0 at 24/48/72 h. For all animals the individual mean scores for cornea opacity and iris were 0 for 24/48/72 h. Animal 1 had a score for conjuntivae redness of 0.33 and 0 for chemosis at 24/48/72 h. Animal 2 had a score for conjuntivae redness of 0.33 and 0 for chemosis at 24/48/72 h. Animal 3 had a score for conjuntivae redness of 0.33 and 0 for chemosis at 24/48/72 h. All effects observed were reversible within 48 h. Due to these results the test substance was found to be slightly irritating for the eye but without classification.

 

Justification for classification or non-classification

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

The available experimental test data is reliable and suitable for classification purposes under Regulation (EC) No 1272/2008.

Based on available data on skin irritation/corrosion, the test item is classified for skin irritation into category 2 and labeled with H315 (Causes skin irritation) according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighth time in Regulation (EU) No 2016/918.

Based on available data on 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.