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EC number: 269-246-6 | CAS number: 68201-95-6
- 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
Endpoint summary
Administrative data
Description of key information
Not skin irritating
Not eye irritating
Key value for chemical safety assessment
Skin irritation / corrosion
Link to relevant study records
- Endpoint:
- skin corrosion: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From December 08th, 2015 to January 22nd, 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- test procedure in accordance with generally accepted scientific standards and described in sufficient detail
- Remarks:
- The test was performed according to internationally accepted guidelines and in accordance with internationally valid GLP principles.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
- Version / remarks:
- Adopted: 28th July, 2015
- Deviations:
- yes
- Remarks:
- not impacting the test results
- GLP compliance:
- yes (incl. QA statement)
- Test system:
- human skin model
- Source species:
- human
- Cell type:
- non-transformed keratinocytes
- Details on animal used as source of test system:
- The reconstructed human epidermal model EpiDerm™ (EPI-200, MatTek, Ashland, USA) 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. - Vehicle:
- physiological saline
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: reconstructed human epidermis (RhE) model.
- Viable tissues: the reconstructed human epidermal model EpiDerm™ (EPI-200 ver. 2.0, MatTek, Bratislava, Slovakia)
Lots Nos. 23306 and 23308
- Frozen tissues: the reconstructed human epidermal model EpiDerm™ killed by freezing
Lot No.: 16888 FRZN EA
DIRECT MTT REDUCTION - functional check in tubes
50 mg of the test substance was added to 2.0 ml of MTT medium. Solution was incubated for 1 hour (37 ± 1 °C, 5 ± 1 % CO2, humidified).
DIRECT MTT REDUCTION - test in frozen tissues
The test substance (25 mg) was applied to three freeze-killed tissues for 60 min exposition. In addition, two freeze-killed tissues were treated with PBS for 60 min. After 60 min of incubation (37 ± 1 °C, 5 ± 1 % CO2, moistened), the test substance was rinsed off and tissues were incubated with MTT solution in the same manner as viable tissues in MTT test. Two hours extraction in isopropylalcohol with shaking and OD measuring at 570 nm followed consequently.
MTT VIABILITY ASSAY
The test substance (25 mg of substance/surface ratio 39.7 mg/cm2) is placed directly atop to the tissue moistened with 25 µl of PBS. The material is spread on the tissue surface.
A single testing, composed of three replicate tissues, was run.
On the day of receipt, EpiDerm tissues are conditioned by incubation to release transport stress related compounds and debris. After pre-incubations durable for approximately 1 and 18 hours, tissues are topically exposed to the test chemicals for 1 hour (25 minutes at room temperature and the remaining 35 minutes at 37 °C, 5 % CO2). Three tissues are used per the test substance, for the positive (PC) and negative (NC) controls. Tissues are then thoroughly rinsed with PBS, blotted to remove the test substances, and transferred to fresh medium.
After 24 ± 2 hours post-incubation period, the medium is replaced by fresh one. Tissues are incubated for another 18 ± 2 hours. Afterwards, the MTT assay is performed by transferring the tissues to 24-well plates containing MTT medium (1 mg/ml). After 3 hour MTT incubation, the blue formazan salt formed by cellular mitochondria is extracted with 2.0 ml/tissue of isopropyl alcohol and the optical density of the extracted formazan is determined using a spectrophotometer at 570 nm.
OD570 MEASURING
OD570 is measured on a spectrophotometer Libra S22. Isopropyl alcohol serves as a blank. Allowed band width is 2-3 nm. No external filter is used.
VIABILITY CALIBRATION
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.
ASSAY ACCEPTANCE CRITERIA
When any of the acceptance criteria is not met the experiment has to be repeated.
Negative Control
The absolute OD of the negative control (NC) tissues (treated with sterile PBS) in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after shipping and storing procedures and under specific conditions of use. The assay meets the acceptance criterion if the mean OD570 of the NC tissues is ≥ 0.8 and ≤ 2.8
OD570 historical negative control range is 1.470-2.342.
Positive Control
A 5 % SDS (in H2O) solution is used as positive control (PC) and tested concurrently with the test chemicals. Concurrent means here the PC has to be tested in each assay, but not more than one PC is required per testing day. Viability of positive control should be within 95 ± 1 % confidence interval of the historical data.
The assay meets the acceptance criterion if the mean viability of PC tissues expressed as % of the negative control tissues is ≤ 20 %.
OD570 historical positive control range is 0-0.203.
Standard Deviation (SD)
Since in each test skin irritancy potential is predicted from the mean viability determined on 3 single tissues, the variability of tissue replicates should be acceptably low. The assay meets the acceptance criterion if the SD calculated from individual % tissue viabilities of the 3 identically treated replicates is < 18 %.
EVALUATION OF RESULTS
In vitro alternatives that have been validated and accepted may also be used to help in classification decisions making (Regulation (EC) 1272/2008, 3.2. Skin corrosion/ irritation, 3.2.2. Classification criteria for substances).
The cut-off values for the prediction of irritation are given below:
- In case the test chemical is found to be non-corrosive (e.g., based on TG 430, 431 or 435), and shows tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50 %, the test chemical is considered to be irritant to skin in accordance with UN GHS (3) Category 2.
- The test chemical may be considered as non-irritant to skin in accordance with UN GHS No Category if the tissue viability after exposure and post-treatment incubation is more than (>) 50 %.
A single testing run composed of three replicate tissues should be sufficient for a test chemical when the classification is unequivocal. However, in cases of borderline results, such as non-concordant replicate measurements and/or mean percent viability equal to 50 ± 5 %, a second run should be considered, as well as a third one in case of discordant results between the first two runs. - Amount/concentration applied:
- 25 mg of substance/surface ratio 39.7 mg/cm2
- Duration of treatment / exposure:
- 1 hour (25 minutes at room temperature and the remaining 35 minutes at 37 °C)
- Duration of post-treatment incubation (if applicable):
- 24 ± 2 hours
- Number of replicates:
- Three replicates
- Irritation / corrosion parameter:
- % tissue viability
- Value:
- > 50
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Other effects / acceptance of results:
- MTT TEST - VIABILITY ASSAY
In the first experiment tissues could be damaged at washing: wiping and multiple washing together with longer staying out of media probably decreased the cell viability in one of the three replicates.
Criterium #3 of assay acceptance criteria for SD among tissues was not fulfilled in the first experiment. The first experiment was then repeated.
In the second experiment longer time gaps were let between treatment of tissues to obtain more time for rinsing and tissues were not wiped, only rinsed. In the second experiment the first tissue of negative control was lost by technical mistake during processing. Therefore, no results from this tissue have been acquired.
Direct MTT reduction-functional check in tubes: the test substance changed colour to red.
Direct MTT reduction - test in frozen tissues: average OD570 value of treated tissues after 60 min treatment (0.055) was lower than that in negative control (PBS-0.058), so there is no interference with evaluation and correction of results of MTT test is not necessary.
EVALUATION OF RESULTS AND CLASSIFICATION
Under the above-described experimental design, average viability of tissues treated by the test substance was 88.1 % of negative control average value, i.e. viability was > 50 %.
The effect of the test substance was negative in EpiDermTM model.
According to the classification criteria, the test substance considered as non-irritant to skin.
ACCEPTANCE CRITERIA FULFILMENT
Criterium #3 for SD of among tissues was fulfilled neither in the first nor in the second experiment.
The first experiment was repeated and its results were not taken into account for the final evaluation.
In the second experiment range of SD in % of test item was also higher than 18 % (18.8 %). As also the lowest viability was above 50 % cut-off value, this experiment was accepted and evaluated.
The other criteria were fulfilled. - Interpretation of results:
- other: not classified, according to CLP Regulation (EC 1272/2008)
- Conclusions:
- Non skin irritating.
- Executive summary:
Test substance was assayed for the in vitro skin irritation in human epidermal model EpiDermTM. The test was performed according to the OECD Test Guideline No. 439.
After pre-incubation of tissues, 25 mg of the test substance was placed directly atop to the previously moistened tissue and it was spread on the entire tissue surface. Length of exposition was 60 minutes. Three tissues were used for the test substance and every control.
After removal of the test substance, tissues were post-incubated for approximately 42 hours due to leave of damage reparation. 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.
As the test item is coloured red, test with frozen tissues was performed to detect if the test item remaining in tissues interfered with measuring of OD570. Interference was not evidenced.
First time, average viability was 50.3 %, what is near of cut-off value and high SD among tissues was observed. So the experiment was repeated and average viability of treated tissues was 88.1 %, i.e.viability was > 50 %.
The effect of the test substance was negative in EpiDermTM model (tissues were not damaged).
Conclusion
The test substance is considered to be non skin irritating.
Reference
OD570 values obtained at the MTT test, their averages, standard deviations (%) and relative viabilities
First experiment
Treatment |
OD570 | Mean | SD | Average viability (NC %) | |||
1 | 2 | 3 | |||||
NC | PBS | 1.984 | 1.910 | 1.879 | 1.924 | 0.044 | 100.0 |
% | 103.10 | 99.26 | 97.64 | 100.00 | 2.289 | ||
C1 | 432/15 | 0.259 | 1.094 | 1.553 | 0.969 | 0.536 | 50.3 |
% | 13.46 | 56.85 | 80.70 | 50.34 | 27.836 | ||
PC | 5 % SDS | 0.056 | 0.077 | 0.070 | 0.068 | 0.009 | 3.5 |
% | 2.91 | 4.00 | 3.64 | 3.52 | 0.454 |
Second experiment
Treatment |
OD570 | Mean | SD | Average viability (NC %) | |||
1 | 2 | 3 | |||||
NC | PBS | 0.179* | 1.855 | 1.832 | 1.844 | 0.012 | 100.0 |
% | 9.71* | 100.62 | 99.38 | 100.00 | 0.624 | ||
C3 | 432/15 | 1.201 | 1.622 | 2.049 | 1.624 | 0.346 | 88.1 |
% | 65.15 | 87.98 | 111.15 | 88.09 | 18.779 | ||
PC | 5 % SDS | 0.075 | 0.065 | 0.069 | 0.070 | 0.004 | 3.8 |
% | 4.07 | 3.53 | 3.74 | 3.78 | 0.223 |
NC: negative control; PC: positive control; C1, C3 TS: test substance; *tissue lost during processing excluded from evaluation; mean: arithmetic mean; SD: standard deviation calculated from individual % tissue viabilities: viability (%): viability of single tissues compared with negative control; NT: not tested; NE: not evaluated
Direct MTT reduction in frozen tissues
Treatment |
Tissues | Average | SD | NC % | ||
1 | 2 | 3 | ||||
PBS 60 min | 0.055 | .0.60 | NT | 0.058 | 0.003 | 100.0 |
% | 95.7 | 104.3 | NT | 100.0 | 4.3 | - |
432/15 | 0.054 | 0.056 | 0.054 | 0.055 | 0.001 | 95.1 |
% | 93.9 | 97.4 | 93.9 | 95.1 | 1.6 | - |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not 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:
- October from 21st to 22th, 2015
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- test procedure in accordance with generally accepted scientific standards and described in sufficient detail
- Remarks:
- The test was performed according to internationally accepted guidelines and is GLP compliant.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
- Version / remarks:
- adopted 26th July, 2013
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Species:
- other: Bovine
- Details on test animals or tissues and environmental conditions:
- - Bovine eyes source: breeding service CHOVSERVIS a.s., division TORO® Hlavečník, Hradec Králové, Czech Republic.
- Collection: eyes were collected by slaughterhouse employees. The eyes were enucleated as soon as possible after death.
- Deterget: no detergent was used.
- Animals: only healthy animals (12 to 60 months old) considered suitable for entry into the human food chain were used as a source of corneas for use in the BCOP test.
- Storage: the risk of contamination was minimized (e.g., by keeping the container containing the eyes on ice, by adding antibiotics to the HBSS used to store the eyes during transport (e.g., penicillin at 100 IU/ml and streptomycin at 100 μg/ml).
The time interval between collection of the eyes and use of corneas in the BCOP was minimized, so the collected eyes were processed on the same day. - Vehicle:
- physiological saline
- Controls:
- yes
- Amount / concentration applied:
- Application form preparation: the test substance was tested as suspension prepared from test substance at 20 % concentration in a 0.9 % sodium chloride solution. 2 g of the test substance was suspended in 10 ml of 0.9 % sodium chloride solution.
Closed-chamber method was used, because the test substance was applicable by micropipette. The test substance (750 µl of application form) to cover the epithelial side of the cornea is introduced into the anterior chamber through the dosing holes on the top surface of the chamber, and the holes were subsequently sealed with the chamber plugs during the exposure. - Number of animals or in vitro replicates:
- Exposed group (test substance) - 3 corneas
- Details on study design:
- EYE SELECTION AND EXANMINATION
The eyes, once they arrive at the laboratory, were carefully examined for defects including scratched, and neovascularisation. Only corneas from eyes free of such defects were used. The isolated corneas, after achieve normal metabolic activity (inductive incubation at 32 ± 1°C for one hour), were examined again. The corneas that show macroscopic tissue damage (e.g., scratches, pigmentation, neovascularization) or an opacity >7 opacity units were discarded.
From 20 eyes the 3 eyes were eliminated after inductive incubation, because the baseline opacity values were >7.
EYE PREPARATION
Corneas free of defects were dissected with a 2 to 3 mm rim of sclera remaining to assist in subsequent handling, with care taken to avoid damage to the corneal epithelium an endothelium. Isolated corneas were mounted in specially designed corneal holders that consisted of anterior and posterior compartments, which interfaced with the epithelial and endothelial sides of the cornea, respectively. Both chambers were filled to excess with pre-warmed Eagle's Minimum Essential Medium (EMEM). The device was then equilibrated at 32 ± 1°C for at least one hour in water bath to allow the corneas to equilibrate with the medium and to achieve normal metabolic activity, to the extent possible. Following the equilibration period, fresh pre-warmed EMEM was added to both chambers and baseline opacity readings were taken for each cornea. Any corneas that showed macroscopic tissue damage (e.g., scratches, pigmentation, neovascularization) or an opacity >7 opacity units were discarded.
Each test group (test substance, concurrent negative and positive controls) consisted of the three eyes. The three corneas with opacity values close to the median value for all corneas were selected as negative control corneas. The remaining corneas were then distributed into treatment and positive control groups.
POST EXPOSURE
After the exposure period, the negative control and the positive control substance was removed from the anterior chamber with EMEM (containing phenol red - the effectiveness of rinsing acidic or alkaline materials). The corneas were given a final rinse with EMEM (without phenol red). The EMEM (without phenol red) was used as a final rinse to ensure removal of the phenol red from the anterior chamber prior to the opacity measurement. The anterior chamber was then refilled with fresh EMEM without phenol red. The opacity and permeability of each cornea were recorded.
The test substance was removed from the anterior chamber with EMEM (containing phenol red). The corneas (applied the test substance) were also repeatedly rinsed with EMEM (without phenol red), because the test substance is coloured . Rinsing was finalized after complete removal of the test substance. The EMEM (without phenol red) was used as a final rinse to the opacity measurement. The anterior chamber was then refilled with fresh EMEM without phenol red. The opacity and permeability of each cornea were recorded.
CONTROLS
- Positive control group: 20 % Imidazole in 0.9 % NaCl; 3 corneas
- Negative control group: 0.9 % NaCl; 3 corneS
MEASUREMENTS
Opacity: the amount of light transmission through the cornea. Corneal opacity was measured quantitatively with the aid of an opacitometer resulting in opacity values measured on a continuous scale.
Permeability: the amount of sodium fluorescein dye that penetrates all corneal cell layers (i.e., the epithelium on the outer cornea surface through the endothelium on the inner cornea surface) measured indirectly using visible light spectrophotometry. 1 ml sodium fluorescein solution (5 mg/ml) was added to the anterior chamber of the corneal holder, which interfaced with the epithelial side of the cornea, while the posterior chamber, which interfaced with the endothelial side of the cornea, is filled with fresh EMEM. The holder was incubated in horizontal position for 1.5 hours at 32 ± 1 °C.
The amount of sodium fluorescein that crosses into the posterior chamber was quantitatively measured with the aid of UV/VIS spectrophotometry. The values of absorbance measured at 490 nm were recorded as optical density (OD490) values. This term was used because the measuring is performed with visible light spectrophotometer using a standard 1 cm path length.
STUDY ACCEPTANCE CRITERIA
A test is considered acceptable if the positive control gives IVIS that falls within one standard deviations of the current historical mean, which is to be updated at least every three months, or each time an acceptable test is conducted in laboratories where tests are conducted infrequently. The negative or solvent/vehicle control responses should result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneas treated with the respective negative or solvent/vehicle control.
SCORING SYSTEM
The IVIS cut-off value for identifying the test substance as including serious eye damage (UN GHS Category 1) and the test substance not requiring classification for eye irritation or serious damage (UN GHS No Category) will be given hereafter:
≤ 3: no Category
> 3; ≤ 55: no prediction can be made
≥ 55: category 1 - Irritation parameter:
- in vitro irritation score
- Value:
- < 3
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Other effects / acceptance of results:
- IVIS: 2.36 not irritating
No macroscopic damage was observed on corneas before application. Corneal opacity was observed on the corneas treated by positive control. The corneas treated by negative control were without macroscopic damage. The corneas treated by the test substance were without macroscopic damage.
NEGATIVE CONTROL
The value of opacity for negative control (0.9 % NaCl) obtained during the study was 1.00 and value of permeability was 0.032.
The values obtained during this study not exceeded upper limits, so the study is considered acceptable.
POSITIVE CONTROL
The value of IVIS for positive control (20 % imidazole in 0.9 % NaCl) obtained during the study was 75.45. This value is within the acceptance limit (one standard deviations of the current historical mean), so the study is considered acceptable. - Interpretation of results:
- not classified
- Remarks:
- according to the CLP Regulation (EC 1272/2008)
- Conclusions:
- Not irritating
- Executive summary:
The substance was tested for the evaluation the potential ocular corrosivity or severe irritancy as measured by its ability to induce opacity and increased permeability in an isolated bovine cornea. The test was performed according to the OECD Test Guideline No. 437, Bovine Corneal Opacity and Permeability Test Method. The test was performed using nine isolated bovine corneas. The testing was performed on three groups of corneas: test substance treatment group, positive control group and negative control group. Three corneas per group were used. Closed-chamber method was used, because the test substance was applicable by micropipette. The opacity and permeability of each cornea were measured. The In Vitro Irritancy Score (IVIS) was calculated from the values of opacity and permeability.
The In Vitro Irritancy Score (IVIS) for test item was 2.36. This value of IVIS is lower than 3 therefore the classification according to UN GHS criteria for eye irritation or serious eye damage is: no category.
Conclusion
Not irritating
Reference
IVIS values
Group | IVIS | |
Calculation | Result | |
NC (0.9 % NaCl) |
1.00 + 15 x 0.032 | 1.48 |
PC (20 % Imidazole in 0.9 % NaCl) |
48.00 + 15 x 1.830 | 75.45 |
EXP (test item) |
2.33 + 15 x 0.002 | 2.36 |
NC- negative control; PC- positive control; EXP – test substance application form
Appearance of Corneas after the Test Substance Exposure
Group | Cornea No. | Appearance after exposure |
Negative control | 1 | Without macroscopic damage |
4 | Without macroscopic damage | |
5 | Without macroscopic damage | |
Positive control | 11 | Corneal opacity |
12 | Corneal opacity | |
14 | Corneal opacity | |
Test substance | 3 | Without macroscopic damage |
6 | Without macroscopic damage | |
7 | Without macroscopic damage |
Opacity
Group | Cornea No. | Baseline opacity | Opacity after treatment | Opacity difference | Mean opacity difference | Mean Opacity(corrected) |
NC (0.9 % NaCl) |
1 | 5 | 7 | 2 | 1.00 | - |
4 | 5 | 6 | 1 | |||
5 | 6 | 6 | 0 | |||
PC (20 % Imidazole in 0.9 % NaCl) |
11 | 3 | 48 | 45 | 49.00 | (49.00 – 1.00) 48 |
12 | 6 | 57 | 51 | |||
14 | 5 | 56 | 51 | |||
EXP (test item) |
3 | 4 | 7 | 3 | 3.33 | (3.33 – 1.00) 2.33 |
6 | 5 | 9 | 4 | |||
7 | 5 | 8 | 3 |
NC- negative control; PC- positive control; EXP – test substance application form
Permeability
Group | Cornea No. | Values of Permeability (Optical density at 490nm) | Mean Permeability | Mean Permeability(corrected) |
NC (0.9 % NaCl) |
1 | 0.038 | 0.032 | - |
4 | 0.026 | |||
5 | 0.032 | |||
PC (20 % Imidazole in 0.9 % NaCl) |
11 | 2.135 | 1.862 | (1.862 – 0.032) 1.830 |
12 | 1.722 | |||
14 | 1.729 | |||
EXP (test item) |
3 | 0.032 | 0.034 | (0.034 – 0.032) 0.002 |
6 | 0.035 | |||
7 | 0.034 |
NC- negative control; PC- positive control; EXP – test substance application form
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not irritating)
Respiratory irritation
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
SKIN IRRITATION
Direct Red 253 was assayed for the in vitro skin irritation in human epidermal model EpiDermTM. The test was performed according to the OECD Test Guideline No. 439. After pre-incubation of tissues, 25 mg of the test substance was placed directly atop to the previously moistened tissue and it was spread on the entire tissue surface. Length of exposition was 60 minutes. Three tissues were used for the test substance and every control. As the test item is coloured red, test with frozen tissues was performed to detect if the test item remaining in tissues interfered with measuring of OD570. Interference was not evidenced. First time, average viability was 50.3 %, what is near of cut-off value and high SD among tissues was observed. So the experiment was repeated and average viability of treated tissues was 88.1 %, i.e. viability was > 50 %. The effect of the test substance was negative in EpiDermTM model (tissues were not damaged) (Täublová, 2016).
EYE IRRITATION
Direct Red 253 was tested for the evaluation the potential ocular corrosivity or severe irritancy as measured by its ability to induce opacity and increased permeability in an isolated bovine cornea. The test was performed according to the OECD Test Guideline No. 437, Bovine Corneal Opacity and Permeability Test Method. The In Vitro Irritancy Score (IVIS) was calculated from the values of opacity and permeability and resulted to be 2.36. This value of IVIS is lower than 3, therefore the substance does not meet the criteria to be classified for eye irritation or serious eye damage, according to UN GHS criteria.
Justification for classification or non-classification
According to the CLP Regulation (EC 1272/2008), 3.2 Skin corrosion/irritation section, skin Irritation means the production of reversible damage to the skin following the application of a test substance for up to 4 hours. However, in vitro alternatives that have been validated and accepted may also be used to help make classification decisions.
Direct Red 253 has been assayed by validated and accepted in vitro skin irritation in human epidermal model EpiDermTM. The average viability of treated tissues was 88.1 %, i.e.viability was > 50 %, thus the effect of the test substance was judged as negative.
According to the CLP Regulation (EC 1272/2008), serious eye damage means the production of tissue damage in the eye, or serious physical decay of vision, following application of a test substance, which is not fully reversible within 21 days of application. Eye irritation means the production of changes in the eye, which are fully reversible within 21 days of application. In vitro alternatives that have been validated and accepted can be used to make classification decisions.
Direct Red 253 was assayed using the accepted Bovine Corneal Opacity and Permeability Test Method. The In Vitro Irritancy Score (IVIS) was calculated from the values of opacity and permeability and resulted to be 2.36. This value of IVIS is lower than 3, therefore the substance does not meet the criteria to be classified for eye irritation or serious eye damage, according to UN GHS criteria, which agree with the CLP Regulation principles.
In conclusion, the substance does not meet the criteria to be classified for the eye/skin irritation, according to the CLP Regulation (EC 1272/2008).
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