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EC number: 944-073-5 | CAS number: -
- 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:
- 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 016
- 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)
- Version / remarks:
- adopted: 28th July, 2015
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
Test material
- Reference substance name:
- Reaction products of 1,3-dihydroxybenzene, 4-amino-5-hydroxynaphthalene-2,7-disulphonic acid, 4-methoxy-1-aminobenzene, 2-(4-aminoanilino)-5-nitrobenzenesulphonic acid, sodium salt
- EC Number:
- 602-814-7
- Cas Number:
- 12269-90-8
- IUPAC Name:
- Reaction products of 1,3-dihydroxybenzene, 4-amino-5-hydroxynaphthalene-2,7-disulphonic acid, 4-methoxy-1-aminobenzene, 2-(4-aminoanilino)-5-nitrobenzenesulphonic acid, sodium salt
- Test material form:
- solid: particulate/powder
- Details on test material:
- Batch No.: 7012/2007
Appearance: brown powder
Composition of test substance:
Main component:
2-(4-aminoanilino)-5-nitrobenzenesulphonic acid 5.5 area %
4-[[2,4-dihydroxy-3,5-bis[[4-(4-nitro-2-sulphonatophenyl)amino]phenyl]azo]phenyl]azo]-5-
hydroxynaphtalen-2,7-disulphonate, sodium salts 13.9 area %
4-[[2,4-dihydroxy-3-[(4-methoxyphenyl)azo]-5-[[4-[(4-nitro-2-sulphonatophenyl)amino]phenyl]azo]phenyl]azo]-
5-hydroxynaphthalene-2,7-disulphonate, sodium salts 42.4 area %
4-[[2,4-dihydroxy-3,5-bis[(4-methoxyphenyl)azo]phenyl]azo]-5-
hydroxynaphthalene-2,7-disulphonate, sodium salts 18.2 area %
1
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Test substance name: Acid Brown 235
- Other name: Korostan Brown DGR
- Source and lot/batch No. of test material: 7012/2007
- Expiration date of the lot/batch: May 2018
- Appearance: dark brown powder
In vitro test system
- Test system:
- human skin model
- Source species:
- human
- Cell type:
- non-transformed keratinocytes
- Vehicle:
- physiological saline
- Details on test system:
- TEST SYSTEM
The reconstructed human epidermal model EpiDerm (EPI-200, MatTek, Bratislava) consists of normal human-derived epidermal keratinocytes, which have been cultured to form a multilayered highly differentiated model of the human epidermis. The EpiDerm system is manufactured according to defined quality assurance procedures. The EpiDerm tissues (surface 0.63 cm²) are cultured on specially prepared cell culture inserts and shipped as kits, containing tissues on shipping agarose together with the necessary amount of culture media.
DIRECT MTT REDUCTION - FUNCTIONAL CHECK IN TUBES
Some test substances may interfere with the MTT endpoint if it is able to directly reduce MTT. Therefore, functional check for this possibility was performed as follows: 25 mg of the test substance was added to 1 mL MTT medium (red) and incubated in the incubator (37±1°C, 5±1 % CO2, moistened) for 1 hour. At the end of the exposure time, the presence and intensity of the staining (if any) were observed. If the solution changed colour from red to blue, other steps (test in frozen tissues) to correction have to be done.
COLOUR INTERFERENCE
To identify potential interference by coloured test chemical and decide on the need for additional controls, OD570 (optical density at 570 nm) of the test chemical in water (environment during exposure) and/or isopropyl alcohol (extracting solution) is measured.
25 mg of the test substance was mixed with 2 mL of water for injection and shaken for 2-3 hours. The same procedure was performed for isopropyl alcohol. After that, test tubes were centrifuged and decanted. OD570 of supernatant was then measured. If, after subtraction of the OD for isopropanol, the OD of the test article solution is > 0.08 (approximately 5% of the mean viability of the negative control) the test substance has to be considered as possibly interacting with the MTT measurement and an additional test on colorant controls has to be performed.
MTT TEST
Application:
25 mg of the test substance (25 mg of substance/surface ratio 39.7 mg/cm2) was dosed directly on tissue moistened with 25 µL of PBS (phosphate buffered saline). The substance was spread on the tissue surface. A single test, composed of three replicate tissues, was run.
Procedure:
On the day of receipt, EpiDerm tissues were conditioned by incubation to release transport stress related compounds and debris. Duration of pre-incubations before treatment was 60 minutes.
After pre-incubations, tissues were topically exposed to the test chemicals for 60±1 minutes (25 minutes at room temperature and the remaining 35 minutes at culture conditions). Three tissues were used per the test substance, three for the positive (PC) and three for the negative (NC) controls. Tissues are then thoroughly rinsed with PBS. Inserts were then transferred to fresh medium.
After 23 hours and 48 minutes of post-incubation period, the medium was replaced by fresh one. Tissues were incubated for another 18 hours, 21 minutes. Afterwards, the MTT assay was performed by transferring the tissues to 24-well plates containing MTT medium (1 mg/mL). After 185±5 minutes MTT incubation, the blue formazan salt formed by cellular mitochondria was extracted with 2.0 ml/tissue of isopropyl alcohol (for minim. 120 minutes, room temperature, shaking) and the optical density of the extracted formazan was determined using a spectrophotometer at 570 nm.
OD570 measuring:
OD570 was measured on a spectrophotometer Libra S22. Isopropyl alcohol served as a blank. Allowed band width is 2-3 nm. No external filter was used.
Viability calculation:
Relative cell viability is calculated for each tissue as % of the mean of the negative control tissues. Than the mean relative tissue viability of three individual tissues exposed to the test substance is calculated – this value is used for the comparison with limit. - Control samples:
- yes, concurrent vehicle
- yes, concurrent positive control
- yes, concurrent MTT non-specific colour control
- Amount/concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg Acid Brown 235
VEHICLE
- Amount(s) applied (volume or weight with unit): 25 µL phosphate buffered saline
NEGATIVE CONTROL
- Amount(s) applied (volume or weight): phosphate buffered saline
POSITIVE CONTROL
- Concentration (if solution): 5% sodium dodecyl sulphate - Duration of treatment / exposure:
- 60 min
- Duration of post-treatment incubation (if applicable):
- 42 hours
- Number of replicates:
- 3
Results and discussion
In vitro
Results
- Irritation / corrosion parameter:
- % tissue viability
- Value:
- 94
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Other effects / acceptance of results:
- DIRECT MTT REDUCTION - FUNCTIONAL CHECK IN TUBES
25 mg of the test substance was added to 1.0 mL of MTT medium. Suspension was incubated for 1 hour at culture conditions. After incubation, the medium was brown-red coloured. The test substance does not reduce MTT directly.
COLOUR INTERFERENCE
The test substance is soluble in water for injection. OD570 of solution in water for injection was >3. The test substance is not soluble in isopropyl alcohol. Average OD570 value from 2 wells was 0.008 what is < 0.08. On the basis of results obtained, it was decided do not use concurrent colorant control in the MTT test.
ACCEPTANCE CRITERIA FULFILMENT
The mean OD570 of the NC tissue was 2.373 ± 0.115 which meets the acceptance criteria of ≥ 0.8 and ≤ 2.8.
The mean viability of the PC tissues expressed as % of the negative control tissues is 3.3 % which meets the acceptance criterion of ≤ 20 %.
The SD calculated from individual % tissue viabilities of the 3 identically treated replicates for the positive control, negative control and test substance was < 18 %.
All study acceptance criteria were fulfilled.
Any other information on results incl. tables
Table 1: Optical density and viability
|
Treatment |
OD570 |
Avg |
SD |
Average viability (% NC) |
||
1 |
2 |
3 |
|||||
NC |
PBS |
2.450 |
2.458 |
2.211 |
2.373 |
0.115 |
100.0 |
% |
103.2 |
103.6 |
93.2 |
100.0 |
4.8 |
||
C4 |
390/16 |
2.412 |
2.187 |
2.096 |
2.232 |
0.133 |
94.0 |
% |
101.6 |
92.2 |
88.3 |
94.0 |
5.6 |
||
PC |
5% SDS |
0.063 |
0.087 |
0.082 |
0.077 |
0.010 |
3.3 |
% |
2.7 |
3.7 |
3.5 |
3.3 |
0.4 |
NC - negative control
PC - positive control
C4 - test substance
SD - standard deviation
SDS - sodium dodecyl sulphate
Applicant's summary and conclusion
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- Under the test conditions, average viability of tissues treated by the test substance, Acid Brown 235, was 94.0 % of negative control average value i.e. viability was > 50 %.
The effect of the test substance was negative in EpiDerm model.
According to the classification criteria given in this assay, the test substance, Acid Brown 235, is considered to have no category in accordance with UN GHS and is therefore considered a non-irritant. - Executive summary:
The test substance, Acid Brown 235, was assayed for in vitro skin irritation in the human epidermal model EpiDerm.The test was performed according to the OECD Test Guideline No. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method (2015) and protocol for: In Vitro EpiDerm Skin Irritation Test For use with MatTek Corporation’s Reconstructed Human Epidermal Model EPI-200-SIT.
In preliminary experiment neither colour interference with the endpoint nor direct MTT reduction were found.
After pre-incubation of tissues, 25 mg of the test substance was placed directly on previously moistened tissue and spread on the entire tissue surface. The length of exposure was 60 minutes. Three tissues were used for the test substance and for positive and negative controls.
After removal of the test substance, tissues were post-incubated for approximately 42 hours. Three hours incubation with MTT and two hours extraction period with shaking followed then. Optical density (OD570) of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.
Under the experimental design average viability of treated tissues was 94.0%, i.e. viability was >50 %.
The effect of the test substance was negative in EpiDerm model (tissues were not damaged).
According to the classification criteria, thetest substance, Acid Brown 235, is considered to have no category in regard to skin irritation.
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