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EC number: 926-195-0 | CAS number: 1176284-65-3
- 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
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- Guideline study under GLP
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 015
- Report date:
- 2016
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
- GLP compliance:
- yes
Test material
- Reference substance name:
- Addition product of maleic anhydride, tall oil fatty acids, linseed oil and methanol
- IUPAC Name:
- Addition product of maleic anhydride, tall oil fatty acids, linseed oil and methanol
- Test material form:
- liquid
- Details on test material:
- UVCB, Purity 100%, brown liquid, homogenous, Batch no.210150084, Expiry date 18 May 2016
Constituent 1
- Specific details on test material used for the study:
- Being the test item a viscous/not dispensable substance, a preliminary trial was performed in order to verify the best way to carry out the treatment. The following trials were performed:
– An aliquot of the test item was frozen in order to be reduced into powder. No relevant change in test item physical state was noted after 3 days at -80 °C.
– Two aliquots of the test item were warmed for approximately 1 hour. No relevant change in test item texture was observed after warming at 37 or 45 °C.
– An aliquot of test item was withdrawn with a 1 mL syringe and 25 mg was weighed directly on the surface of the plate. A sufficiently accurate weight was obtained. Moreover, the test item could be mechanically removed from the well with a cotton stick.
Based on these results, in the preliminary trial the test item was weighed directly on the surface of the tube (Colouring potential test) or plate (Direct MTT reduction test), while in the Main Assay the test item was weighed directly on the surface of each tissue.
In vitro test system
- Test system:
- human skin model
- Source species:
- human
- Cell type:
- non-transformed keratinocytes
- Cell source:
- other:
- Source strain:
- not specified
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin, SkinEthic Laboratories, Lyon, France
- Tissue batch number(s):15-EKIN-040
- Production date:
- Shipping date:
- Delivery date:
- Date of arrival at laboratory: 6 Oct 2015
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 deg C
- Temperature of post-treatment incubation (if applicable):
REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 1
- Observable damage in the tissue due to washing:
- Modifications to validated SOP:
FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability:
NUMBER OF REPLICATE TISSUES: 3
CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues
- Procedure used to prepare the killed tissues (if applicable):
- N. of replicates :
- Method of calculation used:
NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439:SKIN DISC PREPARATION
- Procedure used: Not different; cut-off point cell viability = 40% and SD cell viability >/= 18
- Quality control for skin discs:
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 +/- 2 deg C
- Temperature of post-treatment incubation (if applicable): same
REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: 1
- Observable damage in the tissue due to washing:
- Modifications to validated SOP:
DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT [3-(4,5-Dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS RN. 298-93-1]
- Spectrophotometer:
- Wavelength:
- Filter:
- Filter bandwidth: - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- Amount/concentration applied:
- 20.2 mg/epidermal unit
- Duration of treatment / exposure:
- 15 minutes
- Duration of post-treatment incubation (if applicable):
- 42 hours
Test animals
- Details on test animals or test system and environmental conditions:
- A commercial reconstructed human skin product, EPISkin, was used in the study.
Test system
- Preparation of test site:
- not specified
- Vehicle:
- unchanged (no vehicle)
- Controls:
- yes, concurrent positive control
- yes, concurrent negative control
- no
- Amount / concentration applied:
- 20 mg
- Duration of treatment / exposure:
- 15 minutes
- Observation period:
- 43 hours
- Details on study design:
Maintenance Medium SkinEthic; batch: 15-MAIN3-041
Assay Medium SkinEthic; batch: 15-ESSC-041
Preliminary test: Direct MTT reduction test (Step 1):
Non-specific reduction of MTT was evaluated as follows: two mL of MTT Ready-to-use Solution was incubated with 21.25 mg of test item at 37°C, 5% CO2 and saturated humidity for 3 hours protected from light, simulating test conditions. Observation of blue or purple appearance of the solution at the end of the incubation time was carried out.
Colouring potential test (Step 2)
Chemicals’ colouring potential was assessed for potential interaction with the test system. 11.67 mg of the test item was added to 90 µL of distilled water in a transparent tube and the resulting solution/suspension mixed by using a vortex for 15 minutes. Colouring of the solution/suspension at the end of the incubation time was evaluated.
Main test:
A Main Assay was carried out including the test item, positive and negative controls. Results presented in this report were obtained in a repeated assay. In the original experiment the negative control showed a mean Optical Density value (OD = 0.523) out of the acceptability range (OD >/= 0.600 and = 1.5). Data from this experiment are not presented in this report.
At the end of the 15 minute exposure, each tissue was rinsed with approximately 25 mL of sterile D-PBS filling and emptying the tissue insert of the plate. The test item was mechanically removed with cotton sticks and then the remaining substance was washed with sterile D-PBS, filling and empting the tissue insert, until reaching the complete removal of the test item. The excess liquid was carefully removed and the sample transferred in new wells pre-filled with 2 mL/well of maintenance medium.
Post-exposure period
A 43 hour recovery period was allowed by incubation at 37°C, 5% CO2 and saturated humidity.
MTT Assay
Each tissue insert was incubated with 2 mL/well of MTT ready-to-use solution for approximately 3 hours at 37°C, 5% CO2 and saturated humidity. At the end of the incubation period, tissues were placed on absorbent paper to dry. A total biopsy was carried out by means of a biopsy punch to allow biopsies of the same dimensions.
The epidermis was separated from the collagen matrix and both placed in a microtube prefilled with 500 µL of acidic isopropanol. Tubes were preserved for approximately 3 days at 4°C to allow formazan extraction. At the end of the extraction period, debris were eliminated by short centrifugation of the tubes (approximately 11000 rpm for 2 minutes) and aliquots of 200 µL from each sample were read in duplicate for their absorbance at 595 nm. OD values were recorded. Six aliquots (200 µL) of acidic isopropanol were analysed and used as blank.
Sample Test System Treatment Amount per well Number of replicates Sample code
Negative Live D-PBS 20 µL 3 N1 N2 N3
control tissue
Positive Live 5% SDS in water 20 µL 3 P1 P2 P3
control tissue
Test item Live
tissue ZWA 5496/100 20± 2 mg 3 A1 A2 A3
Results and discussion
In vitro
Results
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- main study
- Value:
- 36
- Vehicle controls validity:
- not examined
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Other effects / acceptance of results:
- Of 3 replicates, two showed viability > 40% (44.6 and 45.0%, respectively), while one showed viability of 18.5%. The average was 36.0% viability with a SD viability of 15.2.
Any other information on results incl. tables
PRELIMINARY TEST
Direct MTT reduction test (Step 1)
Test item (mg) |
MTT ready to use solution(mL) |
Container |
Incubation condition |
Colour Observation |
20 |
2.0 |
well |
3 h at 37°C, 100% nominal humidity 5% CO2 |
No colour change (no interaction) |
Colouring potential test (Step 2)
Test item (mg) |
Water (mL) |
Container |
Incubation condition |
Colour Observation |
10 |
90 |
Eppendorf tube |
15’, ambient condition, in agitation |
No colour change (no interaction) |
MAIN ASSAY
BLANK NegativeControl
OD |
|
OD |
OD-blank Viability(%) |
0.079 |
N1-1 |
0.821 |
0.7392 0.7647 113.5 |
0.081 |
N1-2 |
0.872 |
0.7902 |
0.082 |
N2-1 |
0.698 |
0.6162 0.6342 94.1 |
0.083 |
N2-2 |
0.734 |
0.6522 |
0.083 |
N3-1 |
0.675 |
0.5932 0.6222 92.4 |
0.083 |
N3-2 |
0.733 |
0.6512 |
Mean 0.0818 Mean 0.7555 Mean 0.67370 -------> 100.0
SD 0.0016 SD 0.0757 SD 0.07904 11.7
CV(%) 1.96 CV(%) 10.02 CV(%) 11.73 11.70
Positive Control
|
OD OD-blank Viability(%)
0.0772 0.0832 12.3
0.0892
0.0292 0.0897 13.3
0.1502
0.0202 0.0232 3.4
0.0262
Mean 0.1472 Mean 0.06537 -------> 9.7
SD 0.0506 SD 0.03666 5.5
CV(%) 34.38 CV(%) 56.08 56.70
Test Item |
OD |
OD-blank Viability(%) |
|||
A1-1 |
0.183 |
0.1012 0.1247 18.5 |
|||
A1-2 |
0.230 |
0.1482 |
|||
A2-1 |
0.374 |
0.2922 0.3007 44.6 |
|||
A2-2 |
0.391 |
0.3092 |
|||
A3-1 |
0.379 |
0.2972 0.3032 45.0 |
|||
A3-2 |
0.391 |
0.3092 |
|||
Mean |
0.3247 |
Mean |
0.24287 |
-------> |
36.0 |
SD |
0.0930 |
SD |
0.10234 |
|
15.2 |
CV(%) |
28.64 |
CV(%) |
42.14 |
|
42.22 |
Applicant's summary and conclusion
- Interpretation of results:
- Category 2 (irritant) based on GHS criteria
- Conclusions:
- The EPISKIN in vitro model was found to be positive, (considered an irritant), based on the mean cell viability (36.0%) when compared to the negative control. the test em is considered irritant.
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