Registration Dossier
Registration Dossier
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EC number: 224-166-0 | CAS number: 4221-80-1
Skin irritation / corrosion
Administrative data
- Endpoint:
- skin corrosion: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 30 Sep 2014 - 21 Oct 2014
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 014
- Report date:
- 2014
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
- Qualifier:
- according to guideline
- Guideline:
- other: Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006, Part B: Methods for the determination of toxicity and other health effects: In Vitro Skin Corrosion: Human Skin Model Test
- GLP compliance:
- yes (incl. QA statement)
Test material
- Reference substance name:
- 2,4-di-tert-butylphenyl 3,5-di-tert-butyl-4-hydroxybenzoate
- EC Number:
- 224-166-0
- EC Name:
- 2,4-di-tert-butylphenyl 3,5-di-tert-butyl-4-hydroxybenzoate
- Cas Number:
- 4221-80-1
- Molecular formula:
- C29H42O3
- IUPAC Name:
- 2,4-di-tert-butylphenyl 3,5-di-tert-butyl-4-hydroxybenzoate
- Test material form:
- solid: bulk
Constituent 1
- Specific details on test material used for the study:
- STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
In vitro test system
- Test system:
- human skin model
- Cell type:
- non-transformed keratinocytes
- Details on animal used as source of test system:
- The EpiDermTM model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDermTM tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs, 10 mm ∅) and commercially available as kits (EpiDerm™ 200), containing 24 tissues on shipping agarose.
Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia - Vehicle:
- unchanged (no vehicle)
- Details on test system:
- The EpiDermTM model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDermTM tissues (surface 0.6 cm²) are cultured on specially
prepared cell culture inserts (MILLICELLs, 10 mm ∅) and commercially available as kits (EpiDerm™ 200), containing 24 tissues on shipping agarose.
Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia - Control samples:
- other: Control tissues were concurrently treated with 50 μL of de-ionized water (negative control, NC) or with 50 μL of 8 N potassium hydroxide (positive control, PC).
- Amount/concentration applied:
- 25 μL de-ionized water was applied first. Thereafter, a bulk volume of 25 μL of the solid test material was applied with a sharp spoon and homogeneously distributed with the water.
- Duration of treatment / exposure:
- 3 minutes at room temperature or 1 hour in the incubator
- Number of replicates:
- Two tissues per exposure time.
Test system
- Details on study design:
- The tissues were washed with PBS to remove residual test material 3 minutes or 1 hour after start of the application treatment.
Rinsed tissues were kept in 24-well plates (holding plates) at room temperature on assay medium until all tissues per application time were dosed and rinsed. The assay medium was then replaced by MTT solution and tissues were incubated for 3 hours.
After incubation, the tissues were washed with PBS to stop the MTT-incubation. The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in isopropanol. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 6 microtiter wells filled with isopropanol for each microtiter plate.
Results and discussion
In vitro
Resultsopen allclose all
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- 3 min exposure; tissue 1
- Value:
- 107.4
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- 3 min exposure; tissue 2
- Value:
- 107.9
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- 3 min exposure; tissue 3
- Value:
- 108
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- 1 h exposure; tissue 1
- Value:
- 96.5
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- 1 h exposure; tissue 2
- Value:
- 106.1
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- 1 h exposure; tissue 3
- Value:
- 101
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Other effects / acceptance of results:
- The test substance is not able to reduce MTT directly, as determined in a pretest.
Any other information on results incl. tables
Individual and mean OD570 values, individual and mean viability values
| Exposure: 3 min | Exposure: 1 hour | ||||||
| Test substance |
tissue 1 | tissue 2 | mean | tissue 1 | tissue 2 | mean | |
| NC | mean OD570 | 1.689 | 1.716 | 1.703 | 1.987 | 1.880 | 1.934 |
| viability [% of NC] | 99.2 | 100.8 | 100 | 102.8 | 97.2 | 100 | |
| test article | mean OD570 | 1.828 | 1.838 | 1.833 | 1.866 | 2.052 | 1.959 |
| viability [% of NC] | 107.4 | 107.9 | 108 | 96.5 | 106.1 | 101 | |
| PC | mean OD570 | 0.341 | 0.383 | 0.362 | 0.190 | 0.224 | 0.207 |
| viability [% of NC] | 20.0 | 22.5 | 21 | 9.8 | 11.6 | 11 | |
Applicant's summary and conclusion
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- The test article does not show a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy under the test conditions chosen.
- Executive summary:
The potential of the test article to cause dermal corrosion/irritation was assessed by a single topical application of 25 μL bulk volume (about 16 mg) of the undiluted test substance to a reconstructed three dimensional human epidermis model (EpiDerm™). For the corrosion test two EpiDerm™ tissue samples were incubated with the test substance for 3 minutes and 1 hour, respectively. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation 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 testsubstance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability. The EpiDerm™ skin corrosion/irritation test showed the following results: The test substance is not able to reduce MTT directly. Corrosion test: The mean viability of the test-substance treated tissues determined after an exposure period of 3 minutes was 108%,and it was 101% after an exposure period of 1 hour. Based on the observed results it was concluded, that the test article 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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