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EC number: 208-706-2 | CAS number: 538-93-2
Skin irritation / corrosion
Administrative data
- Endpoint:
- skin irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- Aug 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Cross-reference
- Reason / purpose for cross-reference:
- other: part of in vitro skin irritation and corrosion test strategy
Reference
- Endpoint:
- skin corrosion: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- Aug 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- other: part of in vitro skin irritation and corrosion test strategy
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
- Version / remarks:
- 29 July 2016
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: EXP-18/11/346
- pH value: ca. 5 (undiluted test substance, determined in the lab prior to start of the GLP study)
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor. - Test system:
- human skin model
- Source species:
- human
- Cell type:
- non-transformed keratinocytes
- Cell source:
- other: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ 200 kit, EPI-200
- Tissue batch number: 25837
FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Acceptance criteria: OD (540 - 570 nm) = 1.0 - 3.0; result: 1.476 ± 0.098
- Barrier function: Acceptance criteria: ET-50 = 4.77 - 8.72 hrs; result: 5.23 hrs
- Contamination: no
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (for 3 min exposure); incubator at 37°C (for 1 h exposure)
REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: The tissues were washed with PBS to remove residual test material 3 minutes or 1 hour after start of the application treatment.
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg / mL MTT diluent
- Incubation time: 3 hours
- Spectrophotometer: SunriseTM Absorbance Reader
- Wavelength: 570 nm
CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
no MTT interference observed
NUMBER OF REPLICATE TISSUES: 2
DECISION CRITERIA (see table 1) - Control samples:
- yes, concurrent negative control
- other: Positive control
- Amount/concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume): 50 µL
- Concentration (if solution): undiluted
NEGATIVE CONTROL
- Amount(s) applied (volume): 50 µL
POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): 8 N - Duration of treatment / exposure:
- 3 minutes and 1 hour
- Number of replicates:
- 2
- Irritation / corrosion parameter:
- % tissue viability
- Remarks:
- 3 minutes exposure
- Value:
- 101.3
- Vehicle controls validity:
- other: = negative control
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: no indication of corrosion
- Irritation / corrosion parameter:
- % tissue viability
- Remarks:
- 1 hour exposure
- Value:
- 114.1
- Vehicle controls validity:
- other: = negative control
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- 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
DEMONSTRATION OF TECHNICAL PROFICIENCY: yes
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: see table 2 - Interpretation of results:
- GHS criteria not met
- Conclusions:
- Based on the results observed, it was concluded that the test substance does not show a skin corrosive potential in the EpiDerm™ in vitro corrosion test under the test conditions chosen.
- Executive summary:
The objective was to assess the skin irritation and corrosion potential of the test substance. By using the currently available methods a single in vitro assay is not sufficient to cover the full range of skin irritating/corrosion potential.
Therefore, two in vitro assays were part of this in vitro skin irritation and corrosion test strategy:
The Skin Corrosion Test (SCT) and Skin Irritation Test (SIT).
The potential of the test substance to cause dermal corrosion/irritation was assessed by a single topical application of 50 μL (corrosion test) or 30 μL (irritation test) of the undiluted test substance to a reconstructed three-dimensional human epidermis model (EpiDerm™).
For the corrosion test, two EpiDerm™ tissues were incubated with the test substance for 3 minutes and 1 hour, each.
Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation by using a colorimetric test. The reduction of mitochondrial dehydrogenase activity measured by reduced formazan production after incubation with a
tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the epidermal tissues treated with the test substance is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability.
The following results were obtained in the EpiDerm™ skin corrosion/irritation test:
The test substance is not able to directly reduce MTT.
Results of the Corrosion Test (SCT):
The final mean viability of the tissues treated with the test substance determined after an exposure period of 3 minutes was 101.3% and it was 114.1% after an exposure period of 1 hour.
Based on the results observed, it was concluded that the test substance does not show a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy under the test conditions chosen.
Table 2: Historic control data
Historic Range of NC |
|
||||
OD570 |
|||||
Exposure Time |
Period |
Mean OD |
SD |
Mean + 2 SD |
Mean - 2 SD |
3 minutes |
Jan 2016 - Jul 2017 |
1.800 |
0.195 |
2.191 |
1.410 |
60 minutes |
Jan 2016 - Jul 2017 |
1.780 |
0.168 |
2.116 |
1.444 |
Historic Range of PC |
|
||||
OD570 |
|||||
Exposure Time |
Period |
Mean OD |
SD |
Mean + 2 SD |
Mean - 2 SD |
3 minutes |
Jan 2016 - Jul 2017 |
0.265 |
0.083 |
0.431 |
0.098 |
60 minutes |
Jan 2016 - Jul 2017 |
0.096 |
0.021 |
0.138 |
0.053 |
Viability (%) |
|
||||
Exposure Time |
Period |
Mean % |
SD |
Mean + 2 SD |
Mean - 2 SD |
3 minutes |
Jan 2016 - Jul 2017 |
14.7 |
4.3 |
23.2 |
6.1 |
60 minutes |
Jan 2016 - Jul 2017 |
5.3 |
0.9 |
7.1 |
3.6 |
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 018
- Report date:
- 2018
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
- Version / remarks:
- 28 July 2015
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
Test material
- Reference substance name:
- Isobutylbenzene
- EC Number:
- 208-706-2
- EC Name:
- Isobutylbenzene
- Cas Number:
- 538-93-2
- Molecular formula:
- C10H14
- IUPAC Name:
- (2-methylpropyl)benzene
- Test material form:
- liquid
- Details on test material:
- - Analytical purity: 99.9%
- Lot/batch No.: EXP-18/11/346
Constituent 1
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: EXP-18/11/346
- pH value: ca. 5 (undiluted test substance, determined in the lab prior to start of the GLP study)
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor.
In vitro test system
- Test system:
- human skin model
- Source species:
- human
- Cell type:
- non-transformed keratinocytes
- Cell source:
- other: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ 200 kit, EPI-200
- Tissue batch number: 25837
FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Acceptance criteria: OD (540 - 570 nm) = 1.0 - 3.0; result: 1.476 ± 0.098
- Barrier function: Acceptance criteria: ET-50 = 4.77 - 8.72 hrs; result: 5.23 hrs
- Contamination: no
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (for 25 min) + incubator at 37°C (for 35 min)
REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: The tissues were washed with sterile PBS to remove residual test material 1 hour after start of application.
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg / mL MTT diluent
- Incubation time: 3 hours
- Spectrophotometer: SunriseTM Absorbance Reader
- Wavelength: 570 nm
CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
no MTT interference observed
NUMBER OF REPLICATE TISSUES: 3
DECISION CRITERIA (see table 1) - Control samples:
- yes, concurrent negative control
- other: Positive control
- Amount/concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume): 30 µL
- Concentration (if solution): undiluted
NEGATIVE CONTROL
- Amount(s) applied (volume): 30 µL
POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5% (w/v) - Duration of treatment / exposure:
- 25 min at room temperature following 35 minutes at 37°C
- Duration of post-treatment incubation (if applicable):
- 42 ± 2 hours at 37°C (after 24 ± 2 hours medium change)
- Number of replicates:
- 3
Results and discussion
In vitro
Results
- Irritation / corrosion parameter:
- % tissue viability
- Value:
- 80.9
- Vehicle controls validity:
- other: = negative control
- 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: yes
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: see table 2
Any other information on results incl. tables
Table 2: Historic control data
Historic Range of NC |
|
|||
OD570
Period |
Mean OD |
SD |
Mean + 2 SD |
Mean - 2 SD |
Jan 2016 - Jul 2017 |
1.887 |
0.175 |
2.237 |
1.537 |
Historic Range of PC |
|
|
|
|
OD570
Period |
Mean OD |
SD |
Mean + 2 SD |
Mean - 2 SD |
Jan 2016 - Jul 2017 |
0.054 |
0.012 |
0.078 |
0.030 |
Viability (%)
Period |
Mean % |
SD |
Mean + 2 SD |
Mean - 2 SD |
Jan 2016 - Jul 2017 |
2.9 |
0.5 |
3.9 |
1.8 |
Applicant's summary and conclusion
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- Based on the results observed, it was concluded that the test substance does not show a skin irritation potential in the EpiDerm™ in vitro skin irritation test under the test conditions chosen.
- Executive summary:
The objective was to assess the skin irritation and corrosion potential of the test substance. By using the currently available methods a single in vitro assay is not sufficient to cover the full range of skin irritating/corrosion potential.
Therefore, two in vitro assays were part of this in vitro skin irritation and corrosion test strategy:
The Skin Corrosion Test (SCT) and Skin Irritation Test (SIT).
The potential of the test substance to cause dermal corrosion/irritation was assessed by a single topical application of 50 μL (corrosion test) or 30 μL (irritation test) of the undiluted test substance to a reconstructed three-dimensional human epidermis model (EpiDerm™).
The irritation test was performed with three EpiDerm™ tissues which were incubated with the test substance for 1 hour followed by a 42-hour post-incubation period.
Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation by using a colorimetric test. The reduction of mitochondrial dehydrogenase activity measured by reduced formazan production after incubation with a
tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the epidermal tissues treated with the test substance is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability.
The following results were obtained in the EpiDerm™ skin corrosion/irritation test:
The test substance is not able to directly reduce MTT.
Results of the Irritation Test (SIT):
The final mean viability of the tissues treated with the test substance determined after an exposure period of 1 hour with an about 42-hour post-incubation was 80.9%.
Based on the results observed, it was concluded that the test substance does not show a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy under the test conditions chosen.
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