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EC number: 225-561-0 | CAS number: 4927-36-0
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
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- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
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- Flash point
- Auto flammability
- Flammability
- Explosiveness
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- 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
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- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
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- Nanomaterial crystallite and grain size
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- Nanomaterial specific surface area
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- Endpoint summary
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- Environmental data
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- 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
Skin corrosion based on skin sensitisation and eye irritation: Not corrosive
In vitro skin irritation (OECD TG 439): irritating (Category 2)
In vitro eye irritation (OECD TG 438): not irritating
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:
- 02 May, 2016 - 09 May, 2016
- 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:
- 28 July 2015
- 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:
- other: epidermal keratinocytes
- Cell source:
- other: SkinEthic Laboratories, Lyon, France.
- Source strain:
- other: Not applicable
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModelTM, 0.38 cm^2
- Tissue batch number: 16-EKIN-018
- Twenty five μL of the undiluted test substance was added into 12-well plates on top of the skin tissues.
- The test item was applied topically to the corresponding tissues ensuring uniform covering.
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 36.7 - 37.1°C
PRE-TEST PROCEDURE:
Assessment of Direct Test Item Reduction of MTT
MTT Salt Metabolism, Cell Viability Assay
The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt by mitochondrial succinate dehydrogenase in viable cells.
One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified.
Test for Direct MTT Reduction + colour interference:
Damascol/4 was checked for possible direct MTT reduction and colour interference before the study was started. Some non-coloured test items may change into coloured items in aqueous conditions and thus stain the skin tissues during the exposure. To assess the colour interference, 10 μL of Damascol/4 was added to 90 μL Milli-Q water. The mixture was mixed for approximately 15 minutes. A negative control, 10 μL Milli-Q water was tested concurrently. At the end of the shaking period a colour check was performed.
To assess the ability of the test item to reduce MTT, 25 μL of the test item was added to 2 mL MTT solution (0.3 mg/mL in PBS). The mixture was incubated for 3 hours at 37°C. A negative control, sterile Milli-Q water was tested concurrently. At the end of the incubation period a colour check was performed.
PRE-INCUBATION:
On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for approximately 22 hours at 37°C. Maintenance medium and Assay medium were supplied by Skinethic Laboratories, Lyon, France.
APPLICATION/TREATMENT OF TEST SUBSTANCE:
The test was performed on a total of 3 tissues per test item together with negative and positive controls.
Twenty five μL of the undiluted test item was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μL PBS (negative control) and 3 tissues with 25 μL 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. 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.
CELL VIABILITY MEASUREMENT:
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 prelabeled microtubes and extracted with 500 μL isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for 70 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues
. Skin irritation potential of the test item was classified according to remaining cell viability following
exposure of the test item.
DECISION CRITERIA
- A test substance 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 substance and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
- A test substance 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 substance and 42 hours of post incubation is > 50% of the mean viability of the negative controls. - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- Amount/concentration applied:
- Test material
- Applied volume: 25 μL - Duration of treatment / exposure:
- 15-Minute exposure period and 42 hours post-exposure incubation period.
- Number of replicates:
- A total of 9 tissues were used: Triplicate tissues were treated with: test substance, positive control or negative control.
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- Relative mean tissue viability compared to the negative control tissues (100%)
- Value:
- 22
- Vehicle controls validity:
- not examined
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of irritation
- Other effects / acceptance of results:
- Direct MTT Reduction:
Damascol 4 was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because no colour changes were observed it was concluded that Damascol/4 did not interact with the MTT endpoint.
Test Item, Positive Control Item and Negative Control Item
The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with Damascol/4 compared to the negative control tissues was 22%. Since the mean relative tissue viability for Damascol 4 was below 50% it is considered to be irritant.
Quality Criteria
The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 24%. The absolute mean OD570 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 less than 16%, indicating that the test system functioned properly. - Interpretation of results:
- other: Skin irritant Category 2
- Remarks:
- Based on CLP criteria (EC 1272/2008 and its updates)
- Conclusions:
- The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the substance compared to the negative control tissues was 22%. Since the mean relative tissue viability for the substance was below 50%, the substance is considered to be an irritant. Based on this result, Damascol 4 should be classified for skin irritation (Skin Irrit. 2) in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
- Executive summary:
The possible skin irritation potential of the substance was tested in vitro using a human skin model through topical application for 15 minutes. The study procedures described in this report were according to OECD TG 439 guideline and GLP principles. Skin tissue was treated by topical application of 25 μL undiluted test substance. After 42 hours 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 (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) MTT at the end of treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. Reliable negative and positive controls were included. The positive control had a mean cell viability of 24% after 15 minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically was less than 5%, indicating that the test system functioned properly. The relative mean tissue viability obtained after 15 minutes treatment with the substance compared to the negative control tissue was 22%. Since the mean relative tissue viability for the substance was below 50% after 15 minutes treatment the substance is considered to be an irritant. Based on this result, Damascol 4 should be classified for skin irritation (Skin Irrit. 2 / H315: Causes skin irritation) in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
Reference
Mean OD570 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item:
Item |
OD570 of tissues |
Mean OD562 of triplicate tissues |
± SD of OD570 |
Relative individual tissue viability (%) |
Relative mean viability (%) |
Negative Control Item |
0.890 |
0.832 |
0.132 |
100 |
|
0.925 |
|||||
0.680 |
|||||
Positive Control Item |
0.190 |
0.199 |
0.010 |
21 |
24 |
0.210 |
23 |
||||
0.195 |
29 |
||||
Test Item |
0.181 |
0.179 |
0.007 |
20 |
22 |
0.171 |
18 |
||||
0.186 |
27 |
SD = Standard deviation
*The mean viability of the negative control tissues is set at 100 %
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:
- 22-03-2016 to 04-04-2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Triskelion, Utrechtseweg 48, 3700 AV, Zeist
- Species:
- other: eyes of male or female chickens (ROSS, spring chickens)
- Details on test animals or tissues and environmental conditions:
- TEST ANIMALS
- Source: Slaughterhouse v.d. Bor, Nijkerkerveen, The Netherlands
- Age at study initiation: approximately 7 weeks
- Weight at study initiation: approximately 1.5 - 2.5 kg
-Heads of the animals were cut off immediately after sedation of the animals by electric shock and incision of the neck for bleeding, and before they reached the next station on the process line. The heads were placed in small plastic boxes on a bedding of paper tissues moistened with isotonic saline. Next, they were transported to the testing facility. During transportation, the heads were kept at ambient temperature.
- The preparation and validation of the eyes prior to the ICE-test were all according to OECD guideline 438. - Vehicle:
- unchanged (no vehicle)
- Controls:
- other: Positive controls: Benzalkonium Chloride. Negative control: Phosphate buffered saline (PBS).
- Amount / concentration applied:
- TEST MATERIAL
- Amount applied: 30 µL - Duration of treatment / exposure:
- 10 seconds
- Duration of post- treatment incubation (in vitro):
- 240 minutes
- Number of animals or in vitro replicates:
- 3 eyes
- Details on study design:
- REMOVAL OF TEST SUBSTANCE
- Washing: The eyes were rinsed with 20 mL saline
- Time after start of exposure: 10 seconds
SCORING SYSTEM: According to OECD 438 guideline. Examination of the eyes after 0, 30, 75, 120, 180, and 240 minutes
TOOL USED TO ASSESS SCORE: All examinations were carried out with the hand-slit lamp microscope. Fluorescein retention was only scored at approximately 30 minutes after treatment.
CONTROLS: A negative control (30 µL physiological saline) and 3 positive controls (30 µL Benzalkonium Chloride 5%) were included. - Irritation parameter:
- percent corneal swelling
- Run / experiment:
- Slit-lamp examination
- Value:
- 2
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Maximum mean score
- Irritation parameter:
- cornea opacity score
- Run / experiment:
- Slit-lamp examination
- Value:
- 0.5
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Maximum mean score
- Irritation parameter:
- fluorescein retention score
- Run / experiment:
- slit-lamp examination
- Value:
- 0.2
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Other effects / acceptance of results:
- Slit-lamp examination: The test substance caused corneal effects consisting of very slight corneal swelling (mean of 2%), very slight opacity (mean score of 0.5) and no or very slight fluorescein retention (mean score of 0.2). The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control BAC 5% caused moderate or severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants.
Microscopic examination: Microscopic examination of the corneas treated with Damascol did not reveal any abnormalities. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities. Microscopic examination of the corneas treated with the positive control BAC 5% revealed slight, moderate or severe erosion and very slight or slight vacuolation of the epithelium, the epithelium partly detached from the basement membrane (one cornea), and endothelial necrosis (one cornea). - Interpretation of results:
- other: not an eye irritant
- Remarks:
- based on CLP criteria (EC 1272/2008 and its updates)
- Conclusions:
- Under the test conditions chosen (OECD 438 and GLP), the test substance is not considered to be an eye irritant and does not need to be classified in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
- Executive summary:
In accordance with OECD guideline 438 and GLP, the test substance was examined for its in vitro eye irritating potential using the Isolated Chicken Eye (ICE) Test. In the ICE test, 3 eyes were exposed to 30 µL test substance for 10 seconds. In addition, one negative control eye (30 µL saline) and three positive control eyes (30 µL Benzalkonium Chloride (BAC)) were tested. After the exposure the eyes were rinsed with 20 mL saline and were examined at approximately 0, 30, 75, 120, 180, and 240 minutes after treatment. The test substance caused corneal effects consisting of very slight corneal swelling (mean of 2%), very slight opacity (mean score of 0.5) and no or very slight fluorescein retention (mean score of 0.2). Microscopic examination of the corneas did not reveal any abnormalities. The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. Microscopic examination did not reveal any abnormalities. The positive control BAC 5% caused moderate or severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination revealed slight, moderate or severe erosion and very slight or slight vacuolation of the epithelium, the epithelium partly detached from the basement membrane (one cornea), and endothelial necrosis (one cornea). Based on these results, the test substance is considered to be not irritating to the eye and does not need to be classified in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
Reference
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not irritating)
Additional information
Skin corrosion: The substance is not considered corrosive based on absence of irritation and corrosion effects in the skin sensitisation test and eye irritation test applied up to 100%.
In vitro skin irritation
The possible skin irritation potential of the substance was tested in vitro using a human skin model through topical application for 15 minutes. The study procedures described in this report were according to OECD TG 439 guideline and GLP principles. Skin tissue was treated by topical application of 25 μL undiluted test substance. After 42 hours 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 (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) MTT at the end of treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. Reliable negative and positive controls were included. The positive control had a mean cell viability of 24% after 15 minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically was less than 5%, indicating that the test system functioned properly. The relative mean tissue viability obtained after 15 minutes treatment with the substance compared to the negative control tissue was 22%. Since the mean relative tissue viability for the substance was below 50% after 15 minutes treatment the substance is considered to be an irritant.
In vitro eye irritation
In accordance with OECD guideline 438 and GLP, the test substance was examined for its in vitro eye irritating potential using the Isolated Chicken Eye (ICE) Test. In the ICE test, 3 eyes were exposed to 30 µL test substance for 10 seconds. In addition, one negative control eye (30 µL saline) and three positive control eyes (30 µL Benzalkonium Chloride (BAC)) were tested. After the exposure the eyes were rinsed with 20 mL saline and were examined at approximately 0, 30, 75, 120, 180, and 240 minutes after treatment. The test substance caused corneal effects consisting of very slight corneal swelling (mean of 2%), very slight opacity (mean score of 0.5) and no or very slight fluorescein retention (mean score of 0.2). Microscopic examination of the corneas did not reveal any abnormalities. The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. Microscopic examination did not reveal any abnormalities. The positive control BAC 5% caused moderate or severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination revealed slight, moderate or severe erosion and very slight or slight vacuolation of the epithelium, the epithelium partly detached from the basement membrane (one cornea), and endothelial necrosis (one cornea). Based on these results, the test substance is considered to be not irritating to the eye.
Justification for classification or non-classification
Based on absence of corrosive indications in the skin sensitisation and eye irritation the substance is not considered corrosive.
Based on the positive results found in the in vitro skin irritation test the substance is considered a skin irritant. In accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC and its updates), this results in a Category 2 classification for this endpoint (H315: Causes skin irritation).
Based on the negative results found in the in vitro eye irritation test, the substance does not need to be classified as an eye irritant in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC and its updates).
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