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EC number: 218-664-7 | CAS number: 2212-81-9
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Skin irritation / corrosion
Administrative data
- Endpoint:
- skin irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2012-03-06 to 2012-03-12
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Well-conducted and documented study performed under GLP according to current guideline.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 012
- Report date:
- 2012
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
Test material
- Reference substance name:
- [1,3-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide
- EC Number:
- 218-664-7
- EC Name:
- [1,3-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide
- Cas Number:
- 2212-81-9
- Molecular formula:
- C20H34O4
- IUPAC Name:
- 1,3-bis[2-(tert-butylperoxy)propan-2-yl]benzene
- Test material form:
- solid: flakes
- Details on test material:
- - Substance type: organic peroxide
- Physical state: flaked solid
- Analytical purity: >99.0%
- Expiration date of the lot/batch: 2012-10-01
- Storage condition of test material: -20 °C in dark
Constituent 1
Test animals
- Species:
- human
- Details on test animals or test system and environmental conditions:
- The EPISKIN(TM) model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
Test system
- Type of coverage:
- other:
- Vehicle:
- unchanged (no vehicle)
- Controls:
- not required
- Amount / concentration applied:
- Approximately 10 mg of the test item was applied to the epidermis surface. The epidermis surface had previously been moistened wtih 5 µl of sterile distilled water to improve contact between the solid test item and the epidermis.
- Duration of treatment / exposure:
- Triplicate tissues were treated wtih the test item for an exposure period of 15 minutes. Culbecco's Phosphate Buffered Saline (DPBS) with Ca++ and Mg++ was used as the negative control. Sodium Dodecyl Sulphate (SDS) 5% w/v was used as the positive control.
- Observation period:
- Rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.
- Number of animals:
- not applicable
- Details on study design:
- Pre-incubation: 2 ml maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre-labeled 12-well plate. Each epidermis unit was transferred into the maintenance medium felled wells (3 units per plate). A different 12-well plate wasused for the test item and each control item. The tissues were incubated at 37 °C, 5% CO2 in air overnight.
Application of test item and rinsing (Day 1): 2 ml maintenance medium, warmed to approximately 37 °C, was pipettted into the second column of 3 wells of the 12-well plate. Triplicate tissues were treated with the test item for an exposure period of 15 minutes. the test item was applied topically to the corresponsding tissues ensuring uniform covering. Approximately 10 mg of the test item ws applied to the epidermis surface. The epidermis surface had previously been moistened with 5 µl of sterile distilled water to improve contact between the solid test item and theepidermis. Triplicate tissues treated with 10 µl of DPBS served as the negative controls and triplicate tisues treated with 10 µl of SDS 5% w/v served as the positive controls. The plates were kept in the biological safety cabinet at romo temperature for 15 minutes.
At the end of the exposure period, each tissue was removed from teh well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved uby filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 ml of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.
Following the 42-hour post-exposure incubation period, each 12-well plate was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the medium. 1.6 ml of the maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer at -14 to -30 °C for possible inflammatory mediator determination.
2 ml of a 0.3 mg/ml MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12 well plate(s). The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 °C, 5% CO2 in air. At the end of the 3-hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKIN(TM) biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labelled 1.5 ml micro tubes containing 500 µl acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed throughly on a vortex mixer. The tubes we3re refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of the formazan crystals out of the MTT-loaded tissues.
At the end of the formazan extration period each tube was mixed throughly on a vortex mixer to produce a homogenous coloured solution.
For each tissue, duplicate 200 µl samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 µl of acidified isopropanol alone was added to the two wells designated as "blanks". The optical density was measured (quantitative viability analysis) at 540 nm (without a reference filter) using the Anthos 2001 microplate reader.
Results and discussion
In vitro
Results
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- mean
- Value:
- 99.5
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
In vivo
- Irritant / corrosive response data:
- The relative mean viability of the test item treated tissues was 99.5% after a 15-Minute exposure period.
- Other effects:
- The relative mean tissue viability for the positive control treated tissues was 7.8% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 0.7%. The positive control acceptance criterion was tehrefore satisfied.
The mean OD540 for the negative control treated tissues was 0.847 and the standard deviation value of the percentage viability was 5.8%. The negative control acceptance criterion was therefore satisfied.
The standard deviation calculated from individual percentage tissue viabilities of three identically treated tissues was 3.3%. The test item acceptance criterion was therefore satisfied.
Any other information on results incl. tables
The MTT solution containing the test item did not turn blue which indicated that the test item did not directly reduce MTT.
Applicant's summary and conclusion
- Interpretation of results:
- not irritating
- Remarks:
- Migrated information Criteria used for interpretation of results: EU
- Conclusions:
- Tes test item is considered a Non-Irritant (NI).
- Executive summary:
The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKIN(TM) reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-expousre incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures folowing topical exposure to thetest imte by means of the colourimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3 -[4,5 -dimethylthiazol-2 -yl]-2,5 -diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tisssues relative to the negative controls. The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42 -hour post-exposure incubatino period is also determined for test items whicha re found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end-point will be used to either confirm a non-irritant result or will be used to override the non-irritant result.
Triplicate tissues were treatd wtih the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for posssible inflammatory mediator determinatino. AFter MTTT loading a total biopsy of each epidermis wasmade and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µl samples were tranferred to the appropriate wells of a pre-labelled 96 -well plate. The optical density was measured at 540 nm. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).
The relative mean viability of the test item treated tissues was 99.5% after the 15 -minute exposure period. The quality criteria requried fora cceptance of results in the test were satisfied.
The test item was considered to be Non-Irritant (NI).
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