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EC number: 248-387-7 | CAS number: 27287-73-6
- 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:
- 05 May 2016 to 10 May 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 017
- Report date:
- 2017
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
- Deviations:
- no
- GLP compliance:
- yes
Test material
- Reference substance name:
- 2-amino-6-methyl-1,2,4-triazolo[1,5-a]pyrimidin-5(1H)-one
- EC Number:
- 248-387-7
- EC Name:
- 2-amino-6-methyl-1,2,4-triazolo[1,5-a]pyrimidin-5(1H)-one
- Cas Number:
- 27287-73-6
- Molecular formula:
- C6H7N5O
- IUPAC Name:
- 2-amino-6-methyl-1,2,4-triazolo[1,5-a]pyrimidin-5(1H)-one
- Test material form:
- solid: particulate/powder
- Details on test material:
- -Storage conditions: room temperature in the dark
-Colour: beige
Constituent 1
In vitro test system
- Test system:
- human skin model
- Remarks:
- EPISKIN™ reconstructed human epidermis
- Source species:
- human
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ Reconstructed Human Epidermis Model
- Tissue batch number(s): 16-EKIN-018
- Delivery date: 03 May 2016
- Date of initiation of testing: 05 May 2016
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C
REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing Dulbecco's Phosphate Buffered Saline (DPBS) with Ca and Mg. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item.
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562 nm
- Filter: no reference filter
NUMBER OF REPLICATE TISSUES: three per treatment
PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after 15 minutes exposure period followed by the 42-Hour post-exposure incubation period is less than 50%
- The test substance is considered to be non-irritant to skin if the viability after 15 minutes exposure period followed by the 42-Hour post-exposure incubation period is equal to or more than 50% - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- Amount/concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg (26.3 mg/cm2)
NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µl sterile distilled water
POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µl Sodium Dodecyl Sulphate
- Concentration (if solution): 5% v/v - Duration of treatment / exposure:
- 15 minutes
- Duration of post-treatment incubation (if applicable):
- 42 hours
- Number of replicates:
- three per treatment
Results and discussion
In vitro
Resultsopen allclose all
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- Tissue Sample 1
- Value:
- 95.2
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- Tissue Sample 2
- Value:
- 103.7
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- Tissue Sample 3
- Value:
- 104.4
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- Mean (of 3 test tissue samples)
- Value:
- 101.1
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: No. Before removal from the transport plate each tissue was inspected for any air bubbles between the agarose gel and the insert and also that the temperature indicator colour and agar medium colour were satisfactory so as to confirm the tissues were acceptable for use in the study. The tissue model supplier also demonstrated the batch of tissues was acceptable by determining the barrier function and also performed histological examination demonstrating appropriate morphology of the tissues.
- Direct-MTT reduction: The MTT solution containing the test item did not turn blue or purple. Therefore the test item did not directly reduce MTT.
- Colour interference with MTT: The solution containing the test item was colourless. It was therefore unnecessary to use additional colour correction tissue controls.
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The assay establishes the acceptance criterion for an acceptable test if the mean OD562 for the negative control treated tissues is ≥0.6 and ≤1.5, and the SD value of the percentage viability is ≤18%. The mean OD562 for the negative control treated tissues was 0.730 and the standard deviation value of the viability was 5.0%. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues is ≤40% relative to the negative control treated tissues, and the standard deviation (SD) value of the percentage viability is ≤18%. The relative mean tissue viability for the positive control treated tissues was 7.9% relative to the negative control treated tissues and the standard deviation value of the viability was 4.4%. The positive control acceptance criteria were therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues is ≤18%. The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 5.1%. The test item acceptance criterion was therefore satisfied.
Any other information on results incl. tables
The mean OD562values and viabilities for the negative control, positive control and test item are presented in the table below:
Item | OD562 of tissues | Mean OD562 of triplicate tissues | ± SD of OD562 |
Relative individual tissue viability (%) |
Relative mean viability (%) |
± SD Relative mean viability (%) |
Negative Control Item
|
0.701 |
0.730
|
0.037
|
N/A |
100*
|
N/A
|
0.718 |
N/A |
|||||
0.771 |
N/A |
|||||
Positve Control Item |
0.072 |
0.058 |
0.032 |
9.9 |
7.9 |
4.4 |
0.022 | 3.0 | |||||
0.081 | 11.1 | |||||
Test Item | 0.695 | 0.738 | 0.037 | 95.2 | 101.1 | 5.1 |
0.757 | 103.7 | |||||
0.762 | 104.4 |
* The mean viability of the negative control tissues is set at 100%
OD = Optical Density
SD = Standard deviation
Applicant's summary and conclusion
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- Under the conditions of this EPISKIN(TM) assay according to OECD Guideline 439, the results indicate that the test item is non-irritant to the skin.
- Executive summary:
The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKIN(TM) reconstructed human epidermis model according to OECD Guideline 439. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls.
Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. After MTT loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals from the MTT-loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μL samples were transferred to a 96-well plate. The optical density was measured at 562 nm. Data was presented as percentage tissue viability (MTT reduction in the test item treated tissues relative to negative control tissues).
The relative mean viability of test item treated tissues was 101.1% compared to the negative control after the 15-minute exposure period and 42-hours post-exposure incubation period. This is above the 50% threshold for classification and therefore the test item was considered to be non-irritant to the skin under the conditions of this assay. The quality criteria required for acceptance of results in the test were met, therefore the study is considered valid.
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