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EC number: 654-333-7 | CAS number: 5471-90-9
- 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
Endpoint summary
Administrative data
Description of key information
Key value for chemical safety assessment
Skin irritation / corrosion
Link to relevant study records
- Endpoint:
- skin irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 15/10/2019 - 11/11/2019
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
- Version / remarks:
- adopted 18 June 2019
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
- Version / remarks:
- 20 July 2012
- Deviations:
- no
- GLP compliance:
- yes
- Test system:
- human skin model
- Source species:
- human
- Cell type:
- non-transformed keratinocytes
- Cell source:
- other: adult donor
- Source strain:
- other:
- Justification for test system used:
- In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModelTM
- Tissue batch number(s): 19-EKIN-043 & 19-EKIN-045
- Production date:
- Shipping date: October 22 2019 & November 5 2019
- Delivery date: October 22 2019 & November 5 2019
- Date of initiation of testing: October 22 2019 & November 5 2019
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37.0 ± 1.0°C (actual range 35.8 - 37.2°C)
- Temperature of post-treatment incubation (if applicable): 37.0 ± 1.0°C (actual range 35.8 - 37.2°C)
REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: /
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT concentrate (Sigma Aldrich, Zwijndrecht, The Netherlands; 3 mg/ml in PBS) diluted (10x) in Assay medium (final concentration 0.3 mg/ml).
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm
- Filter: /
- Filter bandwidth: /
- Linear OD range of spectrophotometer: /
After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 ml MTT-solution (0.3 mg/ml in PBS). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in pre-labeled microtubes and extracted with 500 µl isopropanol (Merck, Darmstadt, Germany).
Tubes were stored refrigerated and protected from light for approximately 67 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: /
- Barrier function: /
- Morphology: /
- Contamination: /
- Reproducibility: /
NUMBER OF REPLICATE TISSUES: 3
CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Since the test item reacted with the MTT medium, in addition two killed tissues treated with test item and two killed untreated tissues were used for the cytotoxicity evaluation with MTT.
After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item.
After rinsing, the cell culture inserts were each dried carefully and moved to a new well on
2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.
NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1
PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
A test item is considered irritant in the skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
A test item is considered non-irritant in the in vitro skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls. - Control samples:
- yes, concurrent negative control
- yes, concurrent no treatment
- yes, concurrent positive control
- other: two killed tissues treated with test item
- Amount/concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 14.1 to 20.2 mg
NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25 µl PBS
POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 µl 5% SDS - Duration of treatment / exposure:
- 15 minutes
- Duration of post-treatment incubation (if applicable):
- After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item.
After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C. - Number of replicates:
- 3 tissues per test item together with negative and positive controls
- Irritation / corrosion parameter:
- % tissue viability
- Value:
- ca. 101
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Remarks:
- SD = 6.8%
- Other effects / acceptance of results:
- The test item was checked for color interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because a color change was observed by adding MTT-medium it was concluded that the test item did interact with the MTT endpoint.
In addition to the normal procedure, two killed tissues treated with test item and two killed non treated tissues were used for the cytotoxicity evaluation with MTT.
The non-specific reduction of MTT (% NSMTT) by the test item was 7.6% of the negative control tissues. The net OD of the treated killed tissues was subtracted from the ODs of the test item treated viable tissues.
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range
- Acceptance criteria met for positive control: The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 22 ± 25 %.
- Acceptance criteria met for variability between replicate measurements: The standard deviation value of the percentage viability of three tissues treated identically (6.8%) was less than 18%
The standard deviation of the viability of the positive control was 25% which is above the acceptability criteria (18%).
Evaluation: The high standard deviation is caused by one of three skin tissues treated with the positive control that showed a higher value. The OD value of this deviating skin tissue (0.309) is well within the historical data range for the positive control (0.023 – 0.449), and outside the range for the negative control (0.422 – 1.547). Additionally, standard deviations of the negative control (of which the OD values are used for the calculations) and test item treated tissues are all acceptable. Therefore, this deviation had no influence on the integrity of the study. - Interpretation of results:
- GHS criteria not met
- Conclusions:
- In conclusion, CH03951 is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.
- Executive summary:
The objective of this study was to evaluate CH03951 for its ability to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small model (EPISKIN-SMTM). The possible skin irritation potential of the test item was tested through topical application for
15 minutes. The study procedures described in this report were based on the most recent OECD 439 (2019) and EC 440 (2008) guidelines.Batch CH03951/ME of the test item was an off white powder. Skin tissue was moistened with 5 μL of Milli-Q water and at least 10 mg of the test item was applied directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.
The test item did interact with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). Therefore, in addition to the normal procedure, two killed tissues treated with test item and two killed untreated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT (% NSMTT) by the test item was 7.6% of the negative control tissues. The net OD of the treated killed tissues was then subtracted from the ODs of the test item treated viable tissues.
Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 101%. Since the mean relative tissue viability for the test item was above 50% after 15 ± 0.5 minutes treatment the test item is considered to be non-irritant.
The positive control had a mean cell viability of 22% after 15 ± 0.5 minutes exposure. The absolute mean OD570(optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was < 7% for the negative control and the test item. The standard deviation of the viability of the positive control was 25% (see deviation).
In conclusion, CH03951 was non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.
Reference
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not irritating)
Eye irritation
Link to relevant study records
- Endpoint:
- eye irritation: in vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 405 (Acute Eye Irritation / Corrosion)
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
- Principles of method if other than guideline:
- None.
- GLP compliance:
- yes (incl. QA statement)
- Species:
- rabbit
- Strain:
- New Zealand White
- Details on test animals or tissues and environmental conditions:
- The 2 animals received an acclimatisation period of at least five days. The temperature and relative humidity were set to achieve limits of 17 to 23°C and 30 to 70% respectively. The rate of air exchange wat at least fifteen changes per hour and the lighting was controlled by a time switch to give twelve houds continuous light (06:00 ti 18:00) and twelve hours darkness. the animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
- Vehicle:
- unchanged (no vehicle)
- Controls:
- other: left eye of the animal used as control
- Amount / concentration applied:
- 0.1 mL of test item (94 mg) was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball.
- Duration of treatment / exposure:
- Eyelids were held together for about one second imeediately after treatment, to prevent loss of the test item, and then released.
- Observation period (in vivo):
- 72 hours
- Number of animals or in vitro replicates:
- 2
- Details on study design:
- Assessment of ocular damage/irritation was made approx. 1 hour, 24, 48 and 72 hours following treatment. Any other ocular effects were also noted. Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.
- Irritation parameter:
- overall irritation score
- Basis:
- mean
- Time point:
- 24/48/72 h
- Score:
- 3.3
- Max. score:
- 7
- Reversibility:
- fully reversible within: 72h
- Irritation parameter:
- cornea opacity score
- Basis:
- mean
- Time point:
- 24/48/72 h
- Score:
- 0
- Max. score:
- 0
- Irritation parameter:
- iris score
- Basis:
- mean
- Time point:
- 24/48/72 h
- Score:
- 0
- Max. score:
- 0
- Irritation parameter:
- conjunctivae score
- Basis:
- mean
- Time point:
- 24/48/72 h
- Score:
- 0.833
- Max. score:
- 2
- Reversibility:
- fully reversible within: 72 hours
- Irritation parameter:
- chemosis score
- Basis:
- mean
- Time point:
- 24/48/72 h
- Score:
- 0.5
- Max. score:
- 1
- Reversibility:
- fully reversible within: 72 hours
- Irritation parameter:
- other: conjunctivae: discharge
- Basis:
- mean
- Time point:
- 24/48/72 h
- Score:
- 0.333
- Max. score:
- 2
- Reversibility:
- fully reversible within: 48 hours
- Irritant / corrosive response data:
- No corneal or iridial effects were noted during the study. Moderate conjunctival irritation was noted in both treated eyes 1 hour after treatment. Moderate conjunctival irritation was noted in one treated eye and minimal conjunctival irritation was noted in the other treated eye at the 24h observation with minimal conjunctivak irritation noted in both treated eyes at the 48h observation.
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- The test item produced a maximum group mean score of 8.0 and was classified as a mild irritant (Class 4 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.
The test item does not meet the criteria for classification according to CLP. - Executive summary:
Introduction
The study was performed to assess the irritancy potential of the test item to the eye of the New Zealand White rabbit.
Results
A single application of the test item to the non-irrigated eye of two rabbits produced moderate conjunctival irritation. Both treated eyes appeared normal at the 72 -hour observation.
Conclusion
The test item produced a maximum group mean score of 8.0 and was classified as a mild irritant (Class 4 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.
The test item does not meet the criteria for classification according to CLP.
Reference
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not irritating)
Respiratory irritation
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Justification for classification or non-classification
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
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