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EC number: 843-143-1 | CAS number: 709647-81-4
- 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:
- 10 February 2012 - 03 March 2012
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Remarks:
- The study was conducted according to international guideline and line with ECVAM recommendation under GLP albeit with slight deviation i.e. the optical density measurement was 540 nm instead of 570 nm.
- Justification for type of information:
- Refer to the read-across justification attached in Section 13.2.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 012
- Report date:
- 2012
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
- Version / remarks:
- 24 August 2009
- Deviations:
- yes
- Remarks:
- The optical density measurement was 540 nm instead of 570 nm
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
- Version / remarks:
- 22 July 2010
- Deviations:
- yes
- Remarks:
- The optical density measurement was 540 nm instead of 570 nm
- GLP compliance:
- yes (incl. QA statement)
Test material
- Reference substance name:
- (+)-L-arginine hydrochloride
- EC Number:
- 214-275-1
- EC Name:
- (+)-L-arginine hydrochloride
- Cas Number:
- 1119-34-2
- IUPAC Name:
- (+)- L-arginine hydrochloride
- Test material form:
- solid: crystalline
- Details on test material:
- CAS no. 1119-34-2
EC no. 214-275-1
Batch no. 10110839
Receipt no. 50080
Date of receipt: February 09, 2012
Characteristics: White crystalline powder
Storage conditions: +10°C - +25°C
Stability (retest date): December 15, 2013
Assay (dry basis): 100.0%
Constituent 1
In vitro test system
- Test system:
- human skin model
- Remarks:
- EST1000
- Source species:
- human
- Cell type:
- non-transformed keratinocytes
- Cell source:
- other: human derived epidermis keratinocytes
- Source strain:
- not specified
- Details on animal used as source of test system:
- N/A
- Justification for test system used:
- The principle of the reconstructed human epidermis model test is based on the premise that irritant substances are able to penetrate the stratum corneum by diffusion and are cytotoxic to the cells in the underlying layers. Cell viability was measured by dehydrogenase conversion of the vital dye MTT (3-[4,5- Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Thiazolyl blue1 ; EINECS number 206-069-5, CAS number: 298-93-1), into a blue formazan salt that was quantitatively measured after extraction from tissues. Irritant substances were identified by their ability to decrease cell viability below defined threshold levels (i.e.≤ 50% for UN GHS category 2 irritants). Substances that produce cell viabilities above the defined threshold level will not be classified (i.e.> 50%, no category).
The reconstructed human epidermis model systems may be used to test solids, liquids, semi-solids and waxes. Solids may be soluble or insoluble in water. Whenever possible, solids should be tested as a fine powder. The test item is a solid white powder and therefore the test system was deemed suitable. - Vehicle:
- unchanged (no vehicle)
- Remarks:
- However, the epidermis surface was moistened with Dulbecco's phosphate buffered saline (D-PBS) before application to ensure good contact with the skin.
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Skin model EST1000 (CellSytems® Biotechnology GmbH, 53562 Katharinen, Germany), insert size 0.6 cm^2
- Tissue batch number(s): Batch no. EST-120213-001 (Catalogue no. CS-1001)
- Production date: Not specified
- Shipping date: Not specified
- Delivery date: Not specified
- Date of initiation of testing: 10 February 2012
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: The models were cultivated at 21°C for 20 minutes according to the instructions of the EST1000 supplier CellSystems®.
- Temperature of post-treatment incubation (if applicable): 37 ºC
REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: At the end of the exposure period, the test item was carefully washed from the skin surface with Dulbecco's phosphate buffered saline (D-PBS).
- Observable damage in the tissue due to washing: None reported
- Modifications to validated SOP: N/A
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: Yes
- Wavelength: 540 nm
- Filter: Not specified
- Filter bandwidth: Not specified
- Linear OD range of spectrophotometer: Not specified
FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: MTT conversion assay. The optical density (OD) of the extracted (solubilised) dye from the tissue treated with the negative control (NC) should be at least 20-fold greater than the OD of the extraction solvent alone. The tissue treated with NC should exhibit stability in culture (provide similar viability measurements) for the duration of the test exposure period. Viability was performed after a post-treatment incubation period of the rinsed tissues in fresh medium of 42 h. This period allows both for recovery from weakly irritant effects and for appearance of clear cytotoxic effects. Each skin sample was placed in a MTT solution of 1 mg/mL (37 °C incubation temperature, 5 % CO2, 95 % humidity) for 3 h. The precipitated blue formazan product was extracted using the solvent propanol-2 , and the concentration of the formazan was measured by determining the optical density (OD) at a wavelength of 540 nm in a spectrophotometer. The measurements were made for each of the three tissues in duplicate.
- Barrier function: Stratum corneum was multilayered containing the essential lipid profile to produce a functional barrier with robustness to resist rapid penetration of the cytotoxic marker substance sodium dodecyl sulphate (SDS). The barrier function is assessed either by determination of the concentration at which a marker substance reduces the viability of the tissues by 50% (IC50) after a fixed exposure time, or by determination of the exposure time required to reduce cell viability by 50% (ET50) upon application of the marker substance at a specified, fixed concentration. Each batch of the epidermal model used meets defined production release criteria, set by the supplier, among which those for viability and for barrier function are the most relevant (MTT, 2 hours Triton X-100: target > 50%). The barrier properties of the tissues were verified by the supplier.
- Morphology: The test item was applied topically to a three-dimensional reconstructed human epidermis model, comprised of normal, human-derived epidermal keratinocytes, which had been cultured to form a multilayered, highly differentiated model of the human epidermis. It consisted of organised basal, spinous and granular layers, and a multilayered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The containment properties of the model prevented the passage of material around the stratum corneum to the viable tissue, which would lead to poor modelling of skin exposure.
- Contamination: The skin model was free of contamination by bacteria, viruses, mycoplasma, or fungi.
- Reproducibility: Concurrent negative and positive controls were used, each in triplicate, to demonstrate that viability (NC), barrier function and resulting issue sensitivity (PC) of the tissues are within a defined historical acceptance range. The NC item was Dulbecco's phosphate buffered saline (D-PBS) and the PC item was 5% aqueous sodium dodecyl sulphate (SDS)
NUMBER OF REPLICATE TISSUES: 3 tissues in duplicate
CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues
- Procedure used to prepare the killed tissues (if applicable): N/A
- No. of replicates: 3 tissues in duplicate
- Method of calculation used:
The MTT conversion assay was used to measure cell viability. The OD values obtained for each test sample were used to calculate a mean percentage viability relative to the negative control, which is arbitrarily set at 100%.
NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
PREDICTION MODEL / DECISION CRITERIA
The test item is to be considered to be an irritant to skin in accordance with if the tissue viability after exposure and post-treatment incubation is ≤ 50%.
The test item is to be considered to have no category if the tissue viability after exposure and post-treatment incubation was > 50%.
Assay acceptability criteria: For each assay using valid batches, tissues treated with the NC exhibit OD reflecting the quality of the tissue that followed all shipment and receipt steps and all the irritation protocol processes. The OD values of controls should not be below historical established lower boundaries. Similarly, tissues treated with the PC, i.e. 5% aqueous SDS, should reflect the sensitivity retained by tissues and their ability to respond to an irritant substance in the conditions of each individual assay. - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- Amount/concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Three samples of 30 mg test item were applied to the skin model with a surface area of 0.6 cm^2 to uniformly cover the skin surface.
- Concentration (if solution): N/A
VEHICLE: N/A
NEGATIVE CONTROL
- Amount(s) applied (volume or weight): Dulbecco’s phosphate buffered saline (D-PBS) in triplicate (concentration not specified)
- Concentration (if solution): N/A
POSITIVE CONTROL
- Amount(s) applied (volume or weight): sodium dodecyl sulphate
- Concentration (if solution): 5 % aqueous
Concurrent negative and positive controls were used, each in triplicate, to demonstrate that viability (NC), barrier function and resulting issue sensitivity (PC) of the tissues are within a defined historical acceptance range. - Duration of treatment / exposure:
- 20 m
- Duration of post-treatment incubation (if applicable):
- 42 h
- Number of replicates:
- 3 tissues in duplicate
Results and discussion
In vitro
Resultsopen allclose all
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- Tissue 3 (mean)
- Value:
- 134
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- Tissue 2 (mean)
- Value:
- 76
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- Tissue 1 (mean)
- Value:
- 98
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Other effects / acceptance of results:
- - OTHER EFFECTS:
- Visible damage on test system: None reported
- Direct-MTT reduction: None reported
- Colour interference with MTT: None reported
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: N/A, model variability criteria not provided in OECD Guideline
- Range of historical values if different from the ones specified in the test guideline: N/A
The mean viability of the cells exposed to the test item was 98.0% of the mean negative control value. The OD540 values were well above the cut-off percentage cell viability value that distinguishes irritant from non-irritant test items of >50 % for a 20 minute exposure.
The test item was considered to be non-cytotoxic and predicted to be not irritant to skin.
The viability of cells treated with the positive reference item, 5 % SDS, was 6.9 % of the negative controls and was below the cut-off value. Hence, 5 % SDS is predicted to cause pronounced skin irritation.
Any other information on results incl. tables
Table 1. Summary results of in vitro skin irritation optical density (OD)
Formazan extraction solution | Extinction# (n= 3 tissues) | SD | % extinction compared to control |
Negative control D-PBS |
0.548 | 0.027 |
- |
Test item | 0.537 | 0.152 | 98.0 |
Positive control 5 % SDS |
0.038 | 0.002 | 6.9 |
# : at λ = 540 nm
D-PBS: Dulbecco's phosphate buffered saline
SD: standard deviation
SDS: 5 % aqueous deviation sodium dodecyl sulphate
Table 2. Individual values optical density (OD)
Extinction at λ = 540nm
Replicates (duplicate measurements per tissue) | Negative control (D-PBS) | Test item | Postive control 5 % SDS |
Tissue 1 | 0.592 | 0.412 | 0.035 |
0.566 | 0.464 | 0.036 | |
Tissue 2 |
0.537 | 0.447 | 0.042 |
0.534 | 0.472 | 0.034 | |
Tissue 3 |
0.533 | 0.727 | 0.040 |
0.526 | 0.697 | 0.038 | |
Mean | 0.548 | 0.537 | 0.038 |
SD | 0.027 | 0.152 | 0.002 |
D-PBS: Dulbecco's phosphate buffered saline
SD: standard deviation
SDS: 5 % aqueous deviation sodium dodecyl sulphate
Applicant's summary and conclusion
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- Based on the conditions of the study, the test item was concluded not to be irritating to the skin.
- Executive summary:
An in vitro study was performed in accordance with OECD 439 (2010), in order to assess the skin irritation potential of the test item.
The EST1000 three-dimensional reconstructed human epidermis skin model was utilised. Cell viability was determined by measuring the optical density (OD) at a wavelength of 540 nm.
The test item was applied to the model's skin surface for an exposure time of 20 minutes. D-PBS was used as a negative control and 5 % aqueous SDS was used as a positive reference control.
The mean viability of cells exposed to the test item was 98 % of the negative controls. The OD540 value was above the percentage cut-off value for cell viability of 50 %, following 20 minutes exposure. The negative control and positive controls produced valid results (positive control: 6.9 % OD540; i.e. below the 50 % cut off for cell viability).
It was concluded based on the conditions of the test, that the test item was not cytotoxic and not irritating to the skin and would not be classified under CLP Regulation (EC) 1272/2008.
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