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EC number: 211-581-7 | CAS number: 670-96-2
Skin irritation / corrosion
Administrative data
- Endpoint:
- skin irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 25 - 27 Oct 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 017
- 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:
- adopted Jul 2015
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit
Test material
- Reference substance name:
- 2-phenylimidazole
- EC Number:
- 211-581-7
- EC Name:
- 2-phenylimidazole
- Cas Number:
- 670-96-2
- Molecular formula:
- C9H8N2
- IUPAC Name:
- 2-phenyl-1H-imidazole
- Test material form:
- solid: flakes
Constituent 1
In vitro test system
- Test system:
- human skin model
- Source species:
- human
- Cell type:
- non-transformed keratinocytes
- Cell source:
- other: EpiDerm™ (EPI-200-SIT)
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ reconstructed human epidermis model (MatTek)
- Tissue batch number(s): 25850
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C
- Temperature of post-treatment incubation: 24 ± 2 °C
REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: The tissues were washed by filling and emptying the inserts 15 times with DPBS in one-minute intervals.
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h ± 5 min
- Wavelength: 570 nm
- Filter bandwidth: ± 30 nm
FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the RHE model was assessed by undertaking a MTT cell viability test.
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 µL of 1% Triton X-100. The ET-50 value was determined to be 6.16 h.
- Contamination: A sterility test was performed and revealed no contamination of the cells.
NUMBER OF REPLICATE TISSUES: The test item, the positive and negative controls were tested in batch-triplicates. Therefore, a total number of nine tissues was used in this study.
CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- MTT reducing capacity: The test item did not show reduction of MTT, since the mixture did not turn blue/purple.
- Colouring potential: The mixture with the test substane in water and/or isopropanol did not show a colouring potential.
PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the viability is less than 50%.
- The test substance is considered to be non-irritant to skin the viability is greater than or equal to 50%.
ACCEPTABILITY CRITERIA
The test meets acceptance criteria if:
- mean absolute OD(570 nm) of the three negative control tissues is ≥ 0.8 and ≤ 2.8
- mean relative tissue viability of the three positive control tissues is ≤ 20%
- standard deviation (SD) of relative tissue viability obtained from each three concurrently tested tissues is ≤ 18%. - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- Amount/concentration applied:
- TEST MATERIAL
- Amount(s) applied: 25 mg
NEGATIVE CONTROL
- Amount(s) applied: 30 µL
POSITIVE CONTROL
- Amount(s) applied: 30 µL
- Concentration: 5% - Duration of treatment / exposure:
- 35 ± 1 min at 37 ± 1 °C, thereafter at room temperature until 60 ± 1 min incubation time was over.
- Duration of post-treatment incubation (if applicable):
- 24 ± 2 h at 37 ± 1 °C
- Number of replicates:
- The test item, the positive and negative controls were tested in batch-triplicates. Therefore, a total number of nine tissues was used in this study.
Results and discussion
In vitro
Results
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- Mean of 3 tissued
- Value:
- 110.9
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Other effects / acceptance of results:
- - Direct-MTT reduction: The test item did not show reduction of MTT, since the mixture did not turn blue/purple.
- Colour interference with MTT: The mixture with the test substane in water and/or isopropanol did not show a colouring potential.
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean absolute OD 570 of the three negative control tissues was ≥ 0.8 and ≤ 2.8 (1.581).
- Acceptance criteria met for positive control: The mean relative tissues viability (% negative control) of the positive control was ≤ 20% (3.3%).
- Acceptance criteria met for variability between replicate measurements: Standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (1.0 - 5.3%).
Any other information on results incl. tables
Table 2: Summary of results
| Name | Negative control | Positive control | Test item | ||||||
| Tissue | 1 | 2 | 3 | 1 | 2 | 3 | 1 | 2 | 3 |
| Absolute OD 570 | 1.625 | 1.601 | 1.495 | 0.114 | 0.084 | 0.088 | 1.750 | 1.847 | 1.712 |
| 1.644 | 1.592 | 1.528 | 0.113 | 0.084 | 0.088 | 1.640 | 1.837 | 1.707 | |
| OD 570 (Blank corrected) | 1.581 | 1.557 | 1.451 | 0.070 | 0.040 | 0.044 | 1.706 | 1.803 | 1.668 |
| 1.600 | 1.548 | 1.484 | 0.069 | 0.040 | 0.044 | 1.596 | 1.793 | 1.663 | |
| Mean OD 570 of the Duplicates (Blank corrected) | 1.591 | 1.552 | 1.467 | 0.070 | 0.040 | 0.044 | 1.651 | 1.798 | 1.666 |
| Total Mean OD 570 of 3 Replicate tissues (Blank corrected) | 1.537 * | 0.051 | 1.705 | ||||||
| Standard Deviation OD 570 | 0.063 | 0.016 | 0.081 | ||||||
| Relative tissue viability (%) | 103.5 | 101.0 | 95.5 | 4.5 | 2.6 | 2.9 | 107.5 | 117.0 | 108.4 |
| Mean relative tissue viability (%) | 100.0 | 3.3** | 110.9 | ||||||
| Standard Deviation Tissue Viability (%) *** | 4.1 | 1.0 | 5.3 | ||||||
| Coefficient of variation (%) | 4.1 | 31.3 | 4.7 | ||||||
* Blank-corrected mean OD 570 of the negative control corresponds to 100% absolute tissue viability.
** Mean relative tissue viability of the three positive control tissues is ≤ 20%.
*** Standard deviation obtained from the three concurrently tested tissues is ≤ 18%.
Applicant's summary and conclusion
- Interpretation of results:
- other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008
- Conclusions:
- CLP: not classified
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