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EC number: 948-072-0 | CAS number: -
- 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:
- 07 February 2018 to 22 August 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 018
- Report date:
- 2018
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
- Deviations:
- no
- GLP compliance:
- yes
Test material
- Reference substance name:
- dimethyl hydrogen phosphorate, compound with 4-tetrapropyleneaniline {SIDSep} Phosphoric acid, monomethyl ester, compd. with 4-tetrapropylenebenzenamine (1:1)
- Molecular formula:
- N/A
- IUPAC Name:
- dimethyl hydrogen phosphorate, compound with 4-tetrapropyleneaniline {SIDSep} Phosphoric acid, monomethyl ester, compd. with 4-tetrapropylenebenzenamine (1:1)
- Test material form:
- liquid: viscous
- Details on test material:
- Physical Appearance: dark amber, viscous paste
Constituent 1
- Specific details on test material used for the study:
- - Purity: >99.5% (nominal); This substance has an Unknown or Variable composition, is a Complex reaction product, or a Biological material (UVCB)
- Description: Tan paste to semi-solid
In vitro test system
- Test system:
- human skin model
- Source species:
- human
- Cell type:
- other: non-transformed keratinocytes
- Cell source:
- other: not specified
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- The EPISKIN™ Kit consisted of one EPISKIN™ plate containing 12 reconstructed human epidermis tissues (0.38 cm^2), maintenance medium (used for tissue pre-conditioning and testing purposes) and assay medium (used for MTT assay). The maintenance medium and assay medium were refrigerated at 1 to 10 ºC.
Triplicate tissues were treated with the test material for an exposure period of 15 minutes. The test material was applied topically to the corresponding tissues ensuring uniform covering. 10 μL (26.3 μL/cm^2) of the test material was applied to the epidermis surface. Triplicate tissues treated with 10 μL of DPBS served as the negative controls and triplicate tissues treated with 10 μL of SDS 5% w/v served as the positive controls. The plates were kept in the biological safety cabinet at room temperature for 15 minutes. At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. 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.
MTT Loading/Formazan Extraction (Day 3)
Following the 42-hour post-exposure incubation period each 12-well plate was placed onto a plate shaker at room temperature for 15 minutes to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer at -14 to -30 ºC for possible inflammatory mediator IL-1α determination.
MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates. The tissues were transferred to the MTT filled wells, incubated for 3 hours at 37 °C, 5% CO2 in air, and then placed onto absorbent paper to dry. A total biopsy of the epidermis was made, the epidermis was carefully separated from the collagen matrix, and both parts (epidermis and collagen matrix) placed into labeled micro tubes containing acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation, mixed thoroughly on a vortex mixer, and then were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals.
Absorbance/Optical Density Measurements (Day 6)
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous colored solution.
For each tissue, duplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 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 570 nm (without a reference filter) using the LabTech LT-4500 microplate reader. - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- Amount/concentration applied:
- 10 µL (26.3 µL/cm^2) of the test material was applied to the epidermis surface.
- Duration of treatment / exposure:
- Triplicate tissues were treated with the test item for an exposure period of 15 minutes.
- Duration of post-treatment incubation (if applicable):
- At the end of the exposure period each tissue was rinsed before incubating for 42 hours.
- Number of replicates:
- Three
Results and discussion
In vitro
Results
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- 15 minute exposure period and 42 hours post exposure (mean value)
- Value:
- ca. 112.4
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
Any other information on results incl. tables
Direct MTT Reduction
The MTT solution containing the test item turned purple which indicated that the test item directly reduced MTT. Therefore, an additional procedure using water-killed tissues was performed. However, the results obtained showed no effects on OD570 values that indicated the test item was totally rinsed off with no residual test item remained on or in the tissues. It was therefore considered unnecessary to use the results of the water-killed tissues for quantitative correction of results.
Assessment of Color Interference with the MTT endpoint
The solution containing the test item was a brown color, therefore additional color correction tissues were incorporated into the testing procedure. However, the results obtained showed that no color interference occurred. It was therefore considered unnecessary to use the results of the color correction tissues for quantitative correction of results.
Test Item, Positive Control Item and Negative Control Item
The relative mean viability of the test item treated tissues was 112.4% (>50%) after a 15-minute exposure period and 42-hour post-exposure incubation period. It was considered unnecessary to perform the inflammatory mediator IL-1α analysis as the results of the MTT test were unequivocal. The maintenance medium that was retained for possible analysis was discarded without evaluation.
Quality Criteria
The relative mean viability for tissues treated with 5% (w/v) SDS aqueous solution (positive control) was 2.7% relative to the negative control treated tissues and the standard deviation value of the viability was 0.6% (≤18%). The positive control acceptance criteria were therefore satisfied.
The mean OD570 for the negative control (DPBS) treated tissues was 0.736, within the range of 0.6 - 1.5 and the standard deviation value of the viability was 1.2% (≤18%). The negative control acceptance criteria were therefore satisfied.
The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 17.0% (≤18%). The test item acceptance criterion was therefore satisfied.
Individual and Mean OD570 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item
Item |
OD570 of tissues |
Mean OD570 of triplicate tissues |
± SD of OD570 |
Relative individual tissue viability (%) |
Relative mean viability (%) |
± SD of Relative mean viability (%) |
Negative Control Item |
0.743 |
0.736 |
0.009 |
100.9 |
100* |
1.2 |
0.726 |
98.6 |
|||||
0.74 |
100.5 |
|||||
Positive Control Item |
0.019 |
0.02 |
0.004 |
2.6 |
2.7 |
0.6 |
0.016 |
2.2 |
|||||
0.024 |
3.3 |
|||||
Test Item |
0.707 |
0.828 |
0.125 |
96 |
112.4 |
17 |
0.957 |
130 |
|||||
0.819 |
111.2 |
OD = Optical Density
SD = Standard deviation
* = The mean viability of the negative control tissues is set at 100%
Applicant's summary and conclusion
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- The relative mean tissue viability of tissues treated withfor the test item, was 112.4%, greater than 50%, and therefore, the test item was classified as non-irritant to skin. The test item was not classified as a skin irritant according to Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures (EU CLP).
- Executive summary:
The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKINTMreconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric 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/purple formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. The study was performed to the standardised guidelines OECD 439 and EU Test Method B. 46., under GLP conditions.
Triplicate tissues were treated with 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. The test item was found to directly reduce MTT in the pre-test for direct MTT reduction and therefore additional non-viable tissues were incorporated into the testing for correction purposes. The test item was found to have the possibility to cause color interference with the MTT endpoint therefore, as a precaution, additional tissues were incorporated into the testing to correct for this. A third set of controls was included, comprising non-viable tissues, in order to prevent a double correction from a colored test item that also reduces MTT. 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-labeled micro tubes and stored in a freezer for possible inflammatory mediator IL-1α determination. After MTT-loading a total biopsy of each epidermis was made 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 transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density was measured at 570 nm (OD570)
Data are presented in the form of percentage viability (MTT reduction in the test item or positive control treated tissues relative to negative control tissues).
The relative mean viability of the test item treated tissues was 112.4% after the 15-minute exposure period and 42-hours post-exposure incubation period.
The relative mean viability of the tissues treated with the test item was 112.4%, greater than 50%, and therefore, the test item was classified as non-irritant to skin. The test item was not classified as a skin irritant according to Regulation (EC) No. 1272/2008 Classification, Labelling and Packaging of Substances and Mixtures (EU CLP).
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