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EC number: 236-283-4 | CAS number: 13280-60-9
- 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
Skin irritation / corrosion
Administrative data
- Endpoint:
- skin irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 25 September - 27 September 2019
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 019
- Report date:
- 2019
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
- Version / remarks:
- June 2019
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
- Version / remarks:
- April 2017
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: EURL ECVAM DB-ALM Protocol n° 131
- Version / remarks:
- June 2012
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
Test material
- Reference substance name:
- 5-amino-2-nitrobenzoic acid
- EC Number:
- 236-283-4
- EC Name:
- 5-amino-2-nitrobenzoic acid
- Cas Number:
- 13280-60-9
- Molecular formula:
- C7H6N2O4
- IUPAC Name:
- 5-amino-2-nitrobenzoic acid
- Reference substance name:
- Water
- EC Number:
- 231-791-2
- EC Name:
- Water
- Cas Number:
- 7732-18-5
- Molecular formula:
- H2O
- IUPAC Name:
- Oxidane
- Reference substance name:
- Unknown impurities.
- Molecular formula:
- Not available as unknown impurities.
- IUPAC Name:
- Unknown impurities.
- Test material form:
- solid
- Details on test material:
- Batch / Lot No. 10044431
Constituent 1
impurity 1
impurity 2
- Specific details on test material used for the study:
- Batch-No.: 10044431
Storage: room temperature
In vitro test system
- Test system:
- human skin model
- Remarks:
- EpiSkin Small Model (three-dimensional human epidermis model) manufactured by EPISKIN SNC Lyon, France
- Source species:
- human
- Cell type:
- non-transformed keratinocytes
- Justification for test system used:
- The EPISKIN model has been validated for irritation testing in an international trial. After a review of scientific reports and peer reviewed publications on the EPISKIN method, it showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purposes of distinguishing between skin irritating and
no- skin irritating test substances (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007). - Vehicle:
- unchanged (no vehicle)
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTM Small Model
- Supplier: EPISKIN Laboratories 4, rue Alexander Fleming, 69366 Lyon Cedex 07 - France
- Tissue batch number(s): 19-EKIN-039
- Expiry date: 30 September 2019
- Date of initiation of testing: 25 September 2019
PRE-INCUBATION
The “maintenance medium” was pre-warmed to 37 °C. The appropriate number of an assay plate wells were filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated overnight at 37±1 °C in an incubator with 5±1 % CO2, =95 % humidified atmosphere.
NUMBER OF REPLICATE TISSUES FOR TEST ITEMS AND CONTROLS
Three replicates were used for the test item and positive and negative controls, respectively.
REMOVAL OF TEST MATERIAL AND CONTROLS (day 0)
After the incubation time, the EpiSkinTMSM units were removed and rinsed thoroughly with approximately 25 mL PBS 1x solution to remove all of the test material from the epidermal surface. The rest of the PBS was removed from the epidermal surface with suitable pipette tip linked to a vacuum source (care was taken to avoid the damage of epidermis).
POST-INCUBATION (day 0-2)
After rinsing the units were placed into the plate wells with fresh pre-warmed “maintenance medium” (2 mL/well) below them and then incubated for 42 hours (± 1 h) at 37±1 °C in an incubator with 5±1 % CO2, >=95 % humidified atmosphere.
MTT TEST (day 2)
After the 42 hours (± 1h) incubation, the EpiSkinTMSM units were transferred into the MTT solution filled wells (2 mL of 0.3 mg/mL MTT per well) and then incubated for 3 hours (± 5 min) at 37±1°C in an incubator with 5±1 % CO2 protected from light, >=95 % humidified atmosphere.
FORMAZAN EXTRACTION (day 2)
At the end of incubation with MTT a formazan extraction was undertaken:
A disk of epidermis was cut from the unit (this involves the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube of 500 µL acidified isopropanol (one tube corresponding to one well of the tissue culture plate).
The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material with the acidified isopropanol then incubated for approximately four hours at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction. At the middle and at the end of the incubation period, each tube was additionally mixed using a vortex mixer to help extraction.
CELL VIABILITY MEASUREMENTS (day 2)
Following the formazan extraction, 2×200 µL sample from each tube was placed into the wells of a 96-well plate (labelled appropriately) and read the OD (Absorbance / Optical Density) of the samples in a 96-well plate spectrophotometer (Thermo Scientific; Multiscan FC) at 570 nm (±10 nm; Read out range: 0-3.5 Abs, Linearity range: 0.2136 – 3.1752) using acidified isopropanol solution as the blank (6×200 µL).
PREDITCTION MODEL / DECISION CRITERIA
The test chemical is identified as requiring classification and labelling according to UN GHS (Category
2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation
is less or equal to 50% of the negative control. - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- Amount/concentration applied:
- TEST MATERIAL
- Test item: The epidermal surface was first moistened with 5 µL deionised water* in order to improve further contact between powder and epidermis. Subsequently, approximately 10 mg of the test item was applied evenly to the epidermal surface. The test item was spread gently with a curved flat spatula in order to cover evenly all the skin surface if necessary.
*prepared by MILLIPORE Synergy UV HF ASTM 1: F8JA80461C in Toxi-Coop ZRT.
POSITIVE CONTROL
- Amount applied: 10µL
- Concentration: 1x PBS (Phosphate Buffered Saline)
NEGATIVE CONTROL
- Amount applied: 10 µL
- Concentration: SDS (Sodium Dodecyl Sulphate) 5% aq. solution - Duration of treatment / exposure:
- 15 minutes (± 0.5 min)
- Duration of post-treatment incubation (if applicable):
- 42 hours (± 1h)
- Number of replicates:
- Three replicates were used for the test item and controls, respectively
Results and discussion
In vitro
Results
- Irritation / corrosion parameter:
- % tissue viability
- Value:
- 90
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Remarks:
- mean viability value of three tissues
- Other effects / acceptance of results:
- OTHER EFFECTS
- Possible direct MTT reduction with test item: No colour change was observed after three hours of incubation. The test material did not interact with the MTT, therefore additional controls and data calculations were not necessary. A false estimation of viability can be precluded.
- Colouring potential of test item: As the test item has an intrinsic colour (yellow), two additional test item-treated tissues were used for the non-specific OD evaluation. Mean OD (measured at 570 nm) of these tissues was determined as 0.014. The Non Specific Colour % (NSCliving %) was calculated as 1.5 %. Therefore additional data calculation was not necessary. A false estimation of viability can be precluded.
ACCEPTANCE OF RESULTS
- Acceptance criteria met for negative control: Mean OD value 0.978 and standard deviation value (SD) for the % viability 100 (The mean OD value of the three negative control tissues should be between 0.6 and 1.5 and the standard deviation value (SD) of the % viability should be = or < 18)
- Acceptance criteria met for positive control: 4.33% mean viability range standard deviation value (SD) for the % viability 16 (The acceptable mean percentage viability range for positive controls is 0-40% and the standard deviation value (SD) of the % viability should be = or < 18.)
- For test chemicals, the standard deviation value (SD) of the % viability should be = or < 18 : 10.4 % SD
Any other information on results incl. tables
Cell viability The results of the optical density (OD) measured at 570 nm of each replicate and the calculated % viability of the cells is presented below:
Negative Control (1x PBS):
Replicate | Optical Density (OD) | Viability (%) |
1 | 0.978 | 100 |
2 | 0.993 | 102 |
3 | 0.962 | 98 |
Mean | 0.978 | 100 |
Standard Deviation (SD) | 1.59 |
Positive Control (SDS 5% aq. solution):
Replicate | Optical Density (OD) | Viability (%) |
1 | 0.182 | 19 |
2 | 0.111 | 11 |
3 | 0.187 | 19 |
Mean | 0.160 | 16 |
Standard Deviation (SD) | 4.33 |
Test item:
Replicate | Optical Density (OD) | Viability (%) |
1 | 0.966 | 99 |
2 | 0.911 | 93 |
3 | 0.769 | 79 |
Mean | 0.882 | 90 |
Standard Deviation (SD) | 10.4 |
OD Values and NSCliving % of additional control:
Additional colour control | Optical Density (OD) | Non Specific Colour % (NSCliving %) |
5 -Amino-2 -Nitrobenzoic Acid (test item treated tissueswithout MTT incubation) |
1 0.018 2 0.011 mean 0.014 |
1.5 |
Applicant's summary and conclusion
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. The test item 5-Amino-2-Nitrobenzoic Acid (CAS-No. 13280-60-9) is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category).
- Executive summary:
EpiSkinTMSM test of 5-Amino-2-Nitrobenzoic Acid (CAS-No. 13280-60-9) has been performed to predict its irritation potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 439,18 June 2019.
Disks of EPISKIN (three units) were treated with test item and incubated for 15 minutes (± 0.5 min) at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated at 37±1 °C for 42 hours (± 1 h) in an incubator with 5±1 % CO2,>=95 % humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours (± 5 min) with MTT solution at 37±1 °C in 5±1 % CO2 protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically.
The test item has an intrinsic colour (yellow), therefore two additional test item treated tissues were used for the non-specific OD evaluation. SDS (5 % aq.) and 1×PBS treated (three units / positive and negative control) epidermis were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to negative control.
The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (=) to 50 % of the negative control.
In this in vitro skin irritation test using the EPISKIN model, the test item 5-Amino-2-Nitrobenzoic Acid did not show significantly reduced cell viability in comparison to the negative control (mean value: 90 %). All obtained test item viability results were far above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin.
Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.
The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. The test item 5-Amino-2-Nitrobenzoic Acid (CAS-No. 13280-60-9) is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category).
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