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Description of key information

In the key in vitro skin irritation study, the test item was found to be irritating to the skin in the in vitro human epidermis model assay (OECD 439).

In the key in vitro skin corrosion study, the test item was found to be non corrosive in the Human skin model test (OECD 431).

Based on the key in vivo eye irritation study derived from the read-across substance isoamyl isovalerate, the test item was found to be not irritating to the eyes of rabbit (OECD 405).

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:
key study
Study period:
2016-04-28 to 2016-10-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
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)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Specific details on test material used for the study:




STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Epi-200 SIT kit purchased from MatTek Corporation (Bratislava, Slovakia) containing EpiDerm™ tissues on agarose
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm tissues (surface 0.6 cm2)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C / 60 minutes
- Temperature of post-treatment incubation (if applicable): 37°C / 60 minutes

CONTROL
- Negative Control: 30 µL DPBS (Gibco) was used as negative control per tissue;
- Positive Control: 30 µL of a 5% SLS solution in deionised water (MatTek) was used a positive control per tissue, freshly prepared prior to the start of the experiment.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: tissues were gently rinsed with DPBS at least 15 times

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

NUMBER OF REPLICATE TISSUES: triplicate

PREDICTION MODEL / DECISION CRITERIA
For the current test, an irritation potential of a test item is determined and classification is recommended if the mean relative tissue viability of three individual tissues is reduced to less than or equal to 50% of the negative control.
Mean tissue viability =50% = irritant (I)
Mean tissue viability >50% = non-irritant (NI)

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 at or below 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:
30 µL
Duration of treatment / exposure:
60 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Remarks:
after 60 min
Value:
8.8
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: mean relative absorbance
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 4.3% compared to the negative control.
Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
Under the experimental conditions reported, the test substance is irritant to skin.
Executive summary:

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

The test item did not reduce MTT (test for direct MTT reduction), and it did not changen 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. 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 ethyl isovalerate the mean relative absorbance value decreased to 8.8% compared to the relative absorbance value of the negative control. This value is below the threshold for irritancy of = 50%. Therefore, the test item is considered to possess an irritant potential.

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08-08-2016 to 2016-10-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
(2015)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Test system:
human skin model
Source species:
human
Cell type:
other: keratinocytes
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ tissues (Epi-200 kit, MatTek)
- Tissue batch number(s) : Lot Not: 23351
- Date of initiation of testing: 08 August 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 minutes or 60 minutes at 37±1.5°C
- Temperature of post-treatment incubation (if applicable): 37±1.5°C

CONTROL
- Negative Control: 50 µL deionised water was applied to each set of duplicate tissues for the 3 minutes and 1 hour exposure periods
- Positive Control: 50 µL 8.0 N Potassium Hydroxide (Sigma) was applied to each set of duplicate tissues for the 3 minutes and 1 hour exposure periods

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: tissues were gently rinsed using a wash bottle / multipipette containing DPBS (20 times)

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

NUMBER OF REPLICATE TISSUES: duplicate

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%, 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%
- 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
Amount/concentration applied:
50 µL
Duration of treatment / exposure:
3 minutes and 60 minutes
Duration of post-treatment incubation (if applicable):
3 hours
Number of replicates:
2
Irritation / corrosion parameter:
% tissue viability
Remarks:
after 3 min
Value:
101.7
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: mean relative absorbance
Irritation / corrosion parameter:
% tissue viability
Remarks:
after 60 min
Value:
38.5
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: mean relative absorbance
Interpretation of results:
GHS criteria not met
Conclusions:
Under the reported experimental conditions, the test substance was non corrosive to skin according to EU CLP and UN GHS.
Executive summary:

This in vitro study was performed to assess the corrosive potential of the test substance by means of the Human Skin Model Test with EpiDerm™ tissues models.

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. Independent duplicate tissues of EpiDerm™ were exposed to either the test item, the negative control (deionised water) or the positive control (8.0 N KOH) for 3 minutes and 1 hour,

respectively. 50 µL of the test item were dispensed directly onto duplicate EpiDermTM tissue surface, and spread to match the surface of the tissue. After exposure to the negative control the absorbance values met the required acceptability criterion of a mean OD570 = 0.8 and = 2.8 for both treatment intervals thereby confirming the acceptable quality of the tissues. Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 minutes exposure period and for the 1 hour exposure period. The 1 hour exposure caused a decrease of the cell viability < 15% of the negative control. The CV in the range 20 – 100% viability between the tissue replicates is = 30%, thus the validity of the test system and the specific batch of the tissue models is confirmed.

After exposure of the tissues to the test item the relative absorbance value increased to 101.7% after 3 minutes exposure. After 1 hour exposure the relative absorbance value was reduced to 38.5%. 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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1999-09-06 to 1999-09-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes
Test material information:
Composition 1
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Charles River, Deutschland GmbH, Stolzenseeweg 32-36, 88353 Kißlegg
- Weight at study initiation: 1.8 to 1.9 kg
- Housing: individually in PPO cages
- Diet (e.g. ad libitum): pelleted complete rabbit diet ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20+-3°c
- Humidity (%): 55%+-15%
- Air changes (per hr): 10 air changes per hr
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
0.1 ml per application
Duration of treatment / exposure:
24 h
Observation period (in vivo):
1 h, 24 h, 48 h, 72 h and 7 d
Number of animals or in vitro replicates:
4 females
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): rinsing with 20 ml 0.9% sodium chloride solution
- Time after start of exposure: 24 h after exposure

SCORING SYSTEM:
- According to OECD guideline.

TOOL USED TO ASSESS SCORE: fluorescein / UV-lamp
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
fully reversible
Irritation parameter:
iris score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
fully reversible
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
24/48/72 h
Score:
0.6
Max. score:
1
Reversibility:
fully reversible
Irritation parameter:
chemosis score
Basis:
mean
Time point:
24/48/72 h
Score:
0.1
Max. score:
0.33
Reversibility:
fully reversible

Scores for ocular lesions

 Rabbit No.    Individual mean score from readings after 24, 48 and 72 hours
 1623  Cornea opacity, degree  0.00
   Iris  0.00
   Conjunctiva Redness  0.67
   Chemosis  0.00
     
 1624  Cornea opacity, degree  0.00
   Iris  0.00
   Conjunctiva Redness  1.00
   Chemosis  0.33
     
 1625  Cornea opacity, degree  0.00
   Iris  0.00
   Conjunctiva Redness  0.67
   Chemosis  0.00
     
 1626  Cornea opacity, degree  0.00
   Iris  0.00
   Conjunctiva  0.00
   Chemosis  0.00
Interpretation of results:
GHS criteria not met
Conclusions:
The test substance is non irritating to the eye according to GHS criteria.
Executive summary:

The test item was tested for eye irritation in an in-vivo test in rabbits (according to OECD 405 and GLP). Four rabbits had the test item instilled in the left eye. The eyes were examined and the changes were graded according to a numerical scale one hour, 24, 48 and 72 hours as well as day 7 after dosing. Slight signs of irritation were observed on the treated eyes. By 7 days, 4 of 4 treated eyes had returned to normal. It is concluded that the substance is not irritating to the eyes of rabbits under the conditions of the study.

Endpoint:
eye irritation: in vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted using a read across substance
Justification for type of information:
The read across justification is included as an attachment to Iuclid section 13.
Reason / purpose:
read-across source
Related information:
Composition 1
Test material information:
Composition 1
Irritation parameter:
cornea opacity score
Time point:
24/48/72 h
Score:
0
Remarks on result:
other: 3 -methylbutyl isovalerate
Irritation parameter:
iris score
Time point:
24/48/72 h
Score:
0
Remarks on result:
other: 3 -methylbutyl isovalerate
Irritation parameter:
conjunctivae score
Time point:
24/48/72 h
Score:
0.6
Remarks on result:
other: 3 -methylbutyl isovalerate
Irritation parameter:
chemosis score
Time point:
24/48/72 h
Score:
0.1
Remarks on result:
other: 3 -methylbutyl isovalerate
Interpretation of results:
GHS criteria not met
Conclusions:
The source substance ( Read -across substance 3 -methylbutyl isovalerate) is non irritating to the eye according to GHS criteria. Therefore, using a read across approach, it is considered that the target substance is not irritant under CLP.
Executive summary:

The eye irritation of the target substance was predicted from the source substance 3 -methylbutyl isovalerate (see read-across justification).

3 -methylbutyl isovalerate as a read across substance was tested for eye irritation in an in-vivo test in rabbits (according to OECD 405 and GLP). Four rabbits had the test item instilled in the left eye. The eyes were examined and the changes were graded according to a numerical scale one hour, 24, 48 and 72 hours as well as day 7 after dosing. Slight signs of irritation were observed on the treated eyes. By 7 days, 4 of 4 treated eyes had returned to normal. It is concluded that the substance is not irritating to the eyes of rabbits under the conditions of the study. Therefore, using a read across approach, it is considered that the target substance is not irritant under CLP.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Based on the available data, the test substance is classified for skin irritation as Category 2, 'Causes skin irritation', H315, and does not meet CLP classification for eye irritation.