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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
Endpoint summary
Administrative data
Description of key information
The substance is a skin irritant in an in vitro rHSE model (EPISKIN) and is a non-irritant in an ex vivo BCOP model.
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
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- Guideline study under GLP
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
- GLP compliance:
- yes
- 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. - 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
- Details on test animals or test system and environmental conditions:
- A commercial reconstructed human skin product, EPISkin, was used in the study.
- 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- 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.
- 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.
Reference
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 |
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (irritating)
Eye irritation
Link to relevant study records
- Endpoint:
- eye irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2015
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- Guideline study under GLP
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
- Deviations:
- yes
- Remarks:
- minor. The test item was stored at 25.9 deg C for 8.5 h, rather than at 20 deg C as stated in the protocol. Also,the measurement of opacity was done in a photometer rather than an opacitometer, but an appropriate calculation can be made for correction.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
- Qualifier:
- according to guideline
- Guideline:
- EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
- GLP compliance:
- yes
- Species:
- cattle
- Strain:
- other: Bos primagenius Taurus
- Details on test animals or tissues and environmental conditions:
- Bovine corneas were used from previously slaughtered cattle which were between ages 12-60 months. SOURCE OF COLLECTED EYES
- Source:
- Number of animals:
- Characteristics of donor animals (e.g. age, sex, weight): age 12-60 months, M/F,
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions):
- Time interval prior to initiating testing:
- indication of any existing defects or lesions in ocular tissue samples: eyes checked for defects as part of the selection criteria
- Indication of any antibiotics used:
TEST ANIMALS
- Source: Muller Fleisch GmbH,Birkenfeld, Germany. Isolated fresh.
- Age at study initiation:12-60 months
- Weight at study initiation: No data
ENVIRONMENTAL CONDITIONS
- Temperature (°C): After receipt, incubated 1 h at 32 deg C in Hank's Balanced Salt Solution in exposure chambers. Vessels were sterilized glass or sterilizable plastic.
- Humidity (%):
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light):
IN-LIFE DATES: 2 Sept. 2015 - Vehicle:
- other: Minimal Essential Medium (MEM) without penol but supplemented with sodium bicarbinate, L-glutamine and 10% fetal calf serum (FCS).
- Controls:
- yes, concurrent positive control
- yes, concurrent negative control
- no
- Amount / concentration applied:
- 750 μL
- Duration of treatment / exposure:
- 10 minutes
- Duration of post- treatment incubation (in vitro):
- 2 hours
- Number of animals or in vitro replicates:
- Bovine eyes ex vivo were used in the study.
- Details on study design:
- Light transmission was recorded through each cornea in a spectrophotomer at 570 nm, prior to exposure. This comprised the baseline opacity.
Three replicate measurements were obtained.
The closed chamber method was used. The respective substance (negative control solution, test item or positive control) was applied by pipetting 750 μL of the appropriate liquid through the refill hole in the holder of the cornea. The test item was applied to the epithelium in such a manner that as much as possible of the cornea was covered with the test item.
Exposure time was 10 min at 32 ± 1 °C. After rinsing with cMEM with phenol red and final rinsing with cMEM without phenol red, the anterior chamber was filled with cMEM without phenol red, and the corneas were stored for additional 2 h at 32 ± 1 °C (post-incubation). The cMEM without phenol red was renewed in both chambers of the apparatus. Then, the final opacity value of each cornea was recorded (again by measurement at 570 nm).
The cMEM without phenol red was removed from the front chamber, and 1 mL sodium fluorescein solution (concentration: 4 mg/mL) was added to the front chamber. The chambers were then closed and incubated for 90 min at 32 ± 1 °C. After incubation, the content of the posterior chamber was thoroughly mixed. Then, the permeability of the cornea was measured by reading the optical density at 490 nm of the liquid in the posterior chamber. - Irritation parameter:
- in vitro irritation score
- Run / experiment:
- Mean
- Value:
- 1.83
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Other effects / acceptance of results:
- Mean opacity difference of the negative control is 0.1220.
For the test item, the calculated IVIS (In vitro irritancy scoe) is 1.83. The experiment is considered as sufficient for the classification of the test item because two of the three replicates of the test item lead to the same assessment for the test item. - Interpretation of results:
- GHS criteria not met
- Conclusions:
- The calculated IVIS (in vitro irritancy score) of the undiluted test substance in the BCOP is 1.83. The criteria for a non-irritant (IVIS of 3 or less) is met and the substance is evaluated as not irritating to the eye.
- Executive summary:
less
Reference
Optical Density of the test substance:
Replicate |
Negative Control |
Test Item ZWA 5496/100 |
Positive Control |
||||||
Measured values |
-0.0004 |
0.0007 |
0.0031 |
0.0060 |
0.0413 |
0.0240 |
0.1997 |
0.2629 |
0.2637 |
*Corrected values |
-0.0020 |
0.0035 |
0.0155 |
0.0300 |
0.2065 |
0.1200 |
0.9985 |
1.3145 |
1.3185 |
Mean |
0.0057 |
Absorbance and Opacity Values of the Negative Control:
Parameter |
Negative Control |
||
Absorbance before exposition |
0.1888 |
0.1907 |
0.1792 |
Absorbance after exposition |
0.2395 |
0.2033 |
0.2148 |
Opacity before exposition |
1.5445 |
1.5513 |
1.5108 |
Opacity after exposition |
1.7358 |
1.5970 |
1.6398 |
Opacity Difference |
0.1913 |
0.0457 |
0.1291 |
Absorbance and Opacity Values for the Test Item and the Positive Control:
Parameter |
Test Item ZWA 5496/100 |
Positive Control |
||||
Absorbance before exposition |
0.1297 |
0.2813 |
0.1462 |
0.1425 |
0.1296 |
0.1766 |
Absorbance af- ter exposition |
0.1560 |
0.3495 |
0.2432 |
1.9991 |
1.8398 |
1.9957 |
Opacity before exposition |
1.3480 |
1.9112 |
1.4002 |
1.3884 |
1.3477 |
1.5018 |
Opacity after exposition |
1.4322 |
2.2361 |
1.7507 |
99.7930 |
69.1512 |
99.0148 |
Opacity Difference |
0.0842 |
0.3250 |
0.3504 |
98.4046 |
67.8035 |
97.5130 |
In Vitro Irritancy Scores:
Test Group |
IVIS |
Mean IVIS |
Relative Standard Deviation IVIS |
Negative Control 0.9% NaCl |
0.16 |
0.21 |
66.4% |
0.10 |
|||
0.36 |
|||
Test Item ZWA5496/100 |
0.33 |
1.83 |
79.2% |
3.22 |
|||
1.94 |
|||
Positive Control DMF undiluted |
113.18 |
105.86 |
15.3% |
87.31 |
|||
117.08 |
Note: the high relative standard deviations of the IVIS of test item and negative control are due to mathematical reasons, as the respective means are very small.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not irritating)
Respiratory irritation
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
In the OECD 439 in vitro skin irritation test, two of three values for cell viability suggested that the substance is not a skin irritant, but the mean value of three replicates resulted in a value below the threshold of 40% viability. Additional study of the test material in other models may be indicated. However, since the material is found to be a dermal sensitiser in the LLNA, efforts should be made to minimise dermal exposure of workers.
In the OECD 437 BCOP ex vivo model of eye irritation, the substance is found to be non-irritating, with an irritancy score below 3 (threshold for non-irritant).
An IATA for skin irritation was undertaken with conclusions from a Weight-of-Evidence analysis (Element 7) that the substance is not a skin corrosive. This includes an absence of physico-chemical alerts, available information from an in vivo skin sensitisation study in mice (LLNA) indicating irritation, in vitro data indicating irritation, and absence of reports of adverse skin reactions among human workers.
Justification for classification or non-classification
The substance was found to be a skin irritant in an in vitro (EPISKIN) model. This data, combined with data from a LLNA, indicate classification of the substance as a skin irritant, Category 2, according to Regulation EC No. 1272/2008.
The substance is not an eye irritant, according to the BCOP ex vivo assay, and does not meet the criteria for classification for eye damage or irritation.
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