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EC number: 288-927-9 | CAS number: 85940-40-5
- 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
In order to replace animal studies, tests on skin and eye irritation were performed as in vitro studies. The EpiDerm system was used to evaluate the skin corrosion and the skin irritation potential according to OECD guideline 431 and 439. A combination of the BCOP test (bovine corneal opacity, OECD guideline 437) and the EpiOcular assay was used to evaluate the eye irritation potential of the test material. The test material is not considered to be irritating to skin. The test item is considered to be irritating to eyes and is not considered to cause serious damage to eyes.
Key value for chemical safety assessment
Skin irritation / corrosion
Link to relevant study records
- Endpoint:
- skin irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2012/13
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
- Version / remarks:
- April 13, 2004
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
- Version / remarks:
- July 22, 2010
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Test system:
- human skin model
- Remarks:
- EpiDerm™ 200
- Source species:
- human
- Cell type:
- other: human-derived epidermal keratinocytes
- Cell source:
- other: commercially available kit (EpiDerm™ 200)
- Source strain:
- other: not applicable
- Details on animal used as source of test system:
- not applicable
- Justification for test system used:
- Based on the results of ECVAM (European Center for Validation of Alternative Methods) funded validation studies, it was concluded by the ECVAM Scientific Advisory Committee that the EpiDerm™ human epidermis model is suitable to be used for distinguishing between corrosive and non-corrosive chemicals (ECVAM: ESAC statement on the application of the EpidermTM human skin model for skin corrosivity testing of 14-15 Mar 2000) as well as between irritant and non-irritant chemicals (ECVAM: ESAC statement on the scientific validity of in-vitro tests for skin irritation testing of 5 Nov 2008).
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ 200
- Tissue batch number(s): not specified
- Production date: not specified
- Shipping date: not specified
- Delivery date: not specified
- Date of initiation of testing: 25 Sep 2012
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 min at room temperature or 1 h at 37 °C (corrosion test); 25 min at room temperature followed by 35 min at 37 °C (irritation test)
- Temperature of post-treatment incubation (if applicable): 42 h at 37 °C (irritation test)
REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 1 washing step, volume not specified
- Observable damage in the tissue due to washing: not specified
- Modifications to validated SOP: not specified
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL assay medium
- Incubation time: 3 h
- Spectrophotometer: SunriseTM Absorbance Reader
- Wavelength: 570 nm (OD570)
- Filter: no reference filter (not further specified)
- Filter bandwidth: not specified
- Linear OD range of spectrophotometer: not specified
FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: not specified
- Barrier function: not specified
- Morphology: not specified
- Contamination: not specified
- Reproducibility: These historical control data demonstrate the reproducibility of results and robustness of the procedures.
NUMBER OF REPLICATE TISSUES: 2 (corrosion test); 3 (irritation test)
CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues
- Procedure used to prepare the killed tissues (if applicable): Epi-200 tissue was killed by freezing at –20 °C
- N. of replicates: 1
- Method of calculation used: In case of direct reduction of MTT by the test substance, the OD570 values measured in the freeze-killed control tissues (KC) would be used to correct the mean OD570 of the testsubstance treated tissues (mean OD570 KC corrected). Since killed tissue might still have a residual enzyme activity that is able to produce some formazan net OD570 KC is calculated by subtracting the OD570 KC of the NC from the OD570 KC of the test substance. In case the net OD570 KC is greater than 0.1 it is subtracted from the respective mean OD570 to result in the mean OD570 KC corrected. The mean OD570 KC corrected represents the formazan production linked to the tissue viability and therefore indicates the cytotoxic potency of the test substance.
NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1 (corrosion test); 2 (irritation test)
PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
- The test substance is considered to be irritating to skin if the viability is less than or equal to 50%.
- The test substance is considered to be non-irritating to skin if the viability is greater than 50%. - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- other: MTT-reduction control (De-ionized water or test substance (corrosion test); PBS or test substance (irritation test)
- Amount/concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL (corrosion test); 30 µL (irritation test)
- Concentration (if solution): undiluted test substance
NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL (corrosion test); 30 µL (irritation test)
- Concentration (if solution): not applicable
POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL (corrosion test); 30 µL (irritation test)
- Concentration (if solution): 8 n (corrosion test); 5 % (irritation test) - Duration of treatment / exposure:
- Corrosion test: 3 min at room temperature or 1 h at 37 °C
Irritation test: 25 min at room temperature followed by 35 min at 37 °C - Duration of post-treatment incubation (if applicable):
- Irritation test: 42 hours post-incubation period at 37 °C
- Number of replicates:
- see above
- Vehicle:
- unchanged (no vehicle)
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- Corrosion test, 3 min exposure
- Value:
- 113
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- Corrosion test, 1 h exposure
- Value:
- 82
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- Irritation test, 1st test run
- Value:
- 65
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- Irritation test, 2nd test run
- Value:
- 99
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Other effects / acceptance of results:
- - OTHER EFFECTS:
- Visible damage on test system: not reported
- Direct-MTT reduction: The test substance was able to reduce MTT directly. Therefore an additional MTT reduction control (KC) was introduced (performed with the corrosion test and the 2nd test run of the irritation test). As the MTT-reduction control was used for calculation of the 1-hour exposure in the corrosion test, an influence of the test substance due to direct MTT reduction had to be excluded for the irritation test. Therefore a 2nd test run of the irritation test was performed with an additional MTT reduction control. The ability of direct MTT reduction did not impair the study result as demonstrated by the concurrently performed exposure of control tissues inactivated by freezing used during the 2nd test run.
- Colour interference with MTT: not reported
DEMONSTRATION OF TECHNICAL PROFICIENCY: Based on the historical data, a profound experience with the assay is assumed
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: yes
- Range of historical values: see tables 4 and 5 - Interpretation of results:
- GHS criteria not met
- Conclusions:
- Based on the observed results and applying the evaluation criteria it was concluded, that the test item does not show a skin irritation potential in the EpiDerm™ skin corrosion/irritation test under the test conditions chosen.
Reference
Table 1: Results of the corrosion test
|
Exposure: 3 min |
Exposure: 1 hour |
|||||||
Test substance |
|
tissue 1 |
tissue 2 |
KC |
mean |
tissue 1 |
tissue 2 |
KC |
mean* |
NC |
mean OD570 |
1.902 |
1.691 |
0.254 |
1.797 |
2.253 |
2.118 |
0.250 |
2.185 |
viability [% of NC] |
105.9 |
94.1 |
- |
100 |
103.1 |
96.9 |
- |
100 |
|
Test substance |
mean OD570 |
2.041 |
2.010 |
0.335 |
2.025 |
1.943 |
1.950 |
0.412 |
1.785 |
viability [% of NC] |
113.6 |
111.9 |
- |
113 |
88.9 |
89.2 |
- |
82 |
|
PC |
mean OD570 |
0.185 |
0.277 |
- |
0.231 |
0.110 |
0.106 |
- |
0.108 |
viability [% of NC] |
10.3 |
15.4 |
- |
13 |
5.0 |
4.9 |
- |
5 |
* The mean for the test substance after 1 hour exposure is given after KC-correction.
The result of the KC did not indicate an increased MTT reduction at the exposure period of 3 minutes (difference to KC of NC is not greater than 0.1). Thus the KC was not used for viability calculation for this exposure time.
Table 2: Results of the irritation test - 1st test run
Test substance |
|
tissue 1 |
tissue 2 |
tissue 3 |
mean |
SD |
NC |
mean OD570 |
2.295 |
2.144 |
2.121 |
2.187 |
|
viability [% of NC] |
105.0 |
98.1 |
97.0 |
100 |
4.33 |
|
Test substance |
mean OD570 |
1.367 |
1.174 |
1.700 |
1.414 |
|
viability [% of NC] |
62.5 |
53.7 |
77.7 |
65 |
12.16 |
|
PC |
mean OD570 |
0.059 |
0.063 |
0.065 |
0.062 |
|
viability [% of NC] |
2.7 |
2.9 |
2.9 |
3 |
0.13 |
Table 3: Results of the irritation test - 2nd test run
Test substance |
|
tissue 1 |
tissue 2 |
tissue 3 |
KC |
mean |
SD |
NC |
mean OD570 |
2.404 |
2.415 |
2.401 |
0.057 |
2.407 |
|
viability [% of NC] |
99.9 |
100.3 |
99.8 |
- |
100 |
0.29 |
|
Test substance |
mean OD570 |
2.347 |
2.414 |
2.384 |
0.067 |
2.381 |
|
viability [% of NC] |
97.5 |
100.3 |
99.0 |
- |
99 |
1.38 |
|
PC |
mean OD570 |
0.096 |
0.090 |
0.105 |
- |
0.097 |
|
viability [% of NC] |
4.0 |
3.7 |
4.4 |
- |
4 |
0.32 |
The result of the KC did not indicate an increased MTT reduction (difference to KC of NC is not greater than 0.1). Thus the KC was not used for viability calculation.
Table 4: Historical control data - Corrosion test
Historical Range of NC |
|
||||
OD570 |
|||||
Exposure Time |
Historical Period |
Mean OD |
SD |
Mean + 2 SD |
Mean - 2 SD |
3 minutes |
Feb 2010 - Aug 2012 |
1.901 |
0.200 |
2.30 |
1.50 |
60 minutes |
Feb 2010 - Aug 2012 |
1.867 |
0.203 |
2.27 |
1.46 |
Historical Range of PC |
|
|
|
|
|
OD570 |
|
|
|
|
|
Exposure Time |
Historical Period |
Mean OD |
SD |
Mean + 2 SD |
Mean - 2 SD |
3 minutes |
Feb 2010 - Aug 2012 |
0.361 |
0.087 |
0.54 |
0.19 |
60 minutes |
Feb 2010 - Aug 2012 |
0.155 |
0.048 |
0.25 |
0.06 |
Viability (%) |
|
|
|
|
|
Exposure Time |
Historical Period |
Mean % |
SD |
Mean + 2 SD |
Mean - 2 SD |
3 minutes |
Feb 2010 - Aug 2012 |
19.16 |
4.73 |
28.62 |
9.69 |
60 minutes |
Feb 2010 - Aug 2012 |
8.47 |
2.82 |
14.12 |
2.82 |
Table 5: Historical control data - Irritation test
Historical Range of NC |
|
|||
OD570 Historical Period |
Mean OD |
SD |
Mean + 2 SD |
Mean - 2 SD |
Feb 2010 - Nov 2012 |
2.060 |
0.281 |
2.62 |
1.50 |
Historical Range of PC |
|
|||
OD570 Historical Period |
Mean OD |
SD |
Mean + 2 SD |
Mean - 2 SD |
Feb 2010 - Nov 2012 |
0.111 |
0.052 |
0.22 |
0.01 |
Viability (%) |
|
|||
Historical Period |
Mean % |
SD |
Mean + 2 SD |
Mean - 2 SD |
Feb 2010 - Nov 2012 |
5.5 |
2.47 |
10.45 |
0.56 |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not irritating)
Eye irritation
Link to relevant study records
- Endpoint:
- eye irritation: in vitro / ex vivo
- Remarks:
- In vitro eye irritation EpiOcular assay
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 2012/13
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- test procedure in accordance with generally accepted scientific standards and described in sufficient detail
- Qualifier:
- no guideline available
- Principles of method if other than guideline:
- MatTek Corporation, Ashland, MA 01721, USA: EpiOcularTM human cell construct: Procedure details, Version 3.1 a of February 10, 2010.
Harbell J.W. et al. (2009): COLIPA Program on Optimization of Existing ln Vitra Eye Irritation Assays for Entry into Formal Validation: Technology Transfer and Intra/Inter Labaratory Evaluation of EpiOcular Assay for Chemicals. Poster # 378, Society of Toxicology, March 2009. - GLP compliance:
- yes (incl. QA statement)
- Species:
- other: EpiOcular™ OCL-200 kit
- Strain:
- other: 24 OCL-200 tissues (reconstructed cornea): surface 0.6 cm2 cultured in Millicells® diameter 1 cm, MatTek Corp., Ashland MA, USA
- Details on test animals or tissues and environmental conditions:
- - Justification of the test method and considerations regarding applicability
: The EpiOcularTM model (OCL-200) is a three-dimensional non-keratinized tissue construct composed of normal human derived epidermal keratinozytes used to model the human corneal epithelium. The EpiOcularTM tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs, 10 mm ∅) and are commercially available as kits (EpiOcular™ 200), containing 24 tissues on shipping agarose.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live
- Tissue for MTT reduction control: OCL-200 tissue that is killed by freezing at -20°C - Vehicle:
- unchanged (no vehicle)
- Controls:
- yes, concurrent positive control
- yes, concurrent negative control
- other: MTT reduction control (KC): de-ionized water or test substance
- Amount / concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL
- Concentration (if solution): undiluted test substance - Duration of treatment / exposure:
- 30 min at 37 °C
- Duration of post- treatment incubation (in vitro):
- 2 h at 37 °C
- Number of animals or in vitro replicates:
- 2
- Details on study design:
- - Details of the test procedure used
: see above
- Tissue construct used, including batch number : see above, batch number not specified
- Doses of test chemical and control substances used : 50 µL
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable) : Exposure - 30 min at 37 °C, post incubation - 2 h at 37 °C
- Description of any modifications to the test procedure : not applicable
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable) : see above
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer) : wavelength 570 nm, no further information available
- Description of the method used to quantify MTT formazan : The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 4 microtiter wells filled with isopropanol for each microtiter plate.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model : see below
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria : see table 2
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals : Based on the historical data, a profound experience with the assay is assumed
- Positive and negative control means and acceptance ranges based on historical data : see table 2
- Acceptable variability between tissue replicates for positive and negative controls : Tissue viability is acceptable if the mean OD570 of the NC is ≥ 1.0. The mean OD570 of the NC
should not exceed 2.5. Methyl acetate used as PC usually leads to a tissue viability of approx. 25%. A viability of < 50% is acceptable.
- Acceptable variability between tissue replicates for the test chemical: A variability between the tissues is considered to be acceptable if the difference of the viability is ≤ 20%. - Irritation parameter:
- in vitro irritation score
- Remarks:
- Mean tissue viability
- Value:
- 5
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of irritation
- Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: not reported
DEMONSTRATION OF TECHNICAL PROFICIENCY: Based on the historical data, a profound experience with the assay is assumed
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values: see table 2 - Interpretation of results:
- Category 2 (irritating to eyes) based on GHS criteria
- Conclusions:
- Based on the observed results and applying the evaluation criteria it was concluded, that the test item shows an eye irritation potential in the EpiOcular™ eye irritation test under the test conditions chosen.
- Endpoint:
- eye irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 2012/13
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
- Version / remarks:
- September 07, 2009
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Species:
- other: lsolated bovine cornea
- Strain:
- other: not applicable
- Details on test animals or tissues and environmental conditions:
- SOURCE OF COLLECTED EYES
- Source: Schlachthof Bensheim, Am Schlachthof 7-9, 64625 Bensheim
- Number of animals: not specified
- Characteristics of donor animals (e.g. age, sex, weight): age of the animals: minimum 12 months, maximum 60 months
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): not specified
- Time interval prior to initiating testing: not specified
- indication of any existing defects or lesions in ocular tissue samples: no, corneas were free of defects (opacity, scratches, pigmentation etc.)
- Indication of any antibiotics used: not specified - Vehicle:
- unchanged (no vehicle)
- Controls:
- yes, concurrent positive control
- yes, concurrent negative control
- Amount / concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 μL
- Concentration (if solution): undiluted test substance - Duration of treatment / exposure:
- 10 min at 32°C, horizontal, corneas are incubated for further 2 hours at about 32 °C (post exposure period for liquids)
- Duration of post- treatment incubation (in vitro):
- 2 h
- Number of animals or in vitro replicates:
- 3 corneas each for test item, positive control and negative control, respectively
- Details on study design:
- SELECTION AND PREPARATION OF CORNEAS
: Corneas free of defects (opacity, scratches, pigmentation etc.) were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in specially designed corneal holders that consists of anterior and posterior chambers. Both chambers were filled to excess with prewarmed Eagles’s MEM (without phenol red) and then equilibrated in a vertical position at about 32 °C for at least 1 hour. After the equilibration period the medium in both chambers was replaced with fresh prewarmed
medium and initial corneal opacity readings were taken for each cornea with an opacitometer. Any corneas that show macroscopic tissue damage or an opacity value < 512 opacity units1 were discarded. Corneas with opacity values close to the median value of all corneas were selected as negative control (NC). The remaining corneas were then distributed into treatment and positive control groups.
QUALITY CHECK OF THE ISOLATED CORNEAS : see above ("SELECTION AND PREPARATION OF CORNEAS")
NUMBER OF REPLICATES : see above
NEGATIVE CONTROL USED : see above
POSITIVE CONTROL USED : see above
APPLICATION DOSE AND EXPOSURE TIME : 750 µL undiluted test substance, 10 min at 32 °C
TREATMENT METHOD: open chamber method
POST-INCUBATION PERIOD: no
REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: according to open chamber method (not further specified)
- POST-EXPOSURE INCUBATION: 2 h at 32 °C
METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Final corneal opacity readings were taken for each cornea with an opacitometer.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a microtiter plate reader (OD490)
SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used (see "Any other information on materials and methods incl. tables") - Irritation parameter:
- in vitro irritation score
- Value:
- 19.1
- Irritation parameter:
- other: Mean permeability value
- Value:
- 0.409
- Irritation parameter:
- other: Mean opacity value
- Value:
- 13.4
- Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: not reported
DEMONSTRATION OF TECHNICAL PROFICIENCY: Based on the historical data, a profound experience with the assay is assumed
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values: see table 2 - Interpretation of results:
- study cannot be used for classification
- Remarks:
- the result of the BCOP test alone is not sufficient to establish a definitive classification
- Conclusions:
- Based on the observed results and applying the evaluation criteria it was concluded, that the test item does not cause serious eye damage in the Bovine Corneal Opacity and Permeability Test (BCOP Test) under the test conditions chosen.
Referenceopen allclose all
Table 1: Findings
Test substance |
|
tissue 1 |
tissue 2 |
mean KC |
mean* |
inter-tissue variability [%] |
NC |
mean OD570 |
2.101 |
2.106 |
0.047 |
2.104 |
|
viability [% of NC] |
99.9 |
100.1 |
- |
100 |
0.3 |
|
Test substance |
mean OD570 |
0.941 |
0.759 |
0.790 |
0.107 |
|
viability [% of NC] |
44.7 |
36.1 |
35.3 |
5 |
8.6 |
|
PC |
mean OD570 |
0.593 |
0.494 |
- |
0.544 |
|
viability [% of NC] |
28.2 |
23.5 |
- |
26 |
4.7 |
*For the test substance the mean is given after KC-correction.
The MTT reduction control (KC) showed high values of the test substance (mean formazan production of the KC: 35%). Thus the acceptance criterion of ≤ 30% of the NC was not met. However, as all other acceptance criteria were met in the test and because the test substance would have been evaluated as irritant even without KC-correction, the test was considered valid despite this deviation.
Table 2: Historical control data
Historical Range of NC | |||||
OD570 | |||||
Protocol | Historical Period | Mean OD | SD | Mean + 2 SD | Mean - 2 SD |
Protocol for liquids | Apr 2010 - Aug 2012 | 1.532 | 0.213 | 1.96 | 1.11 |
Protocol for solids | Apr 2010 - Aug 2012 | 1.35 | 0.168 | 1.69 | 1.01 |
Historical Range of PC | |||||
OD570 | |||||
Protocol | Historical Period | Mean OD | SD | Mean + 2 SD | Mean - 2 SD |
Protocol for liquids | Apr 2010 - Aug 2012 | 0.390 | 0.151 | 0.69 | 0.09 |
Protocol for solids | Apr 2010 - Aug 2012 | 0.284 | 0.113 | 0.51 | 0.06 |
Viability (%) | |||||
Protocol |
Historical Period |
Mean OD |
SD |
Mean + 2 SD |
Mean - 2 SD |
Protocol for liquids |
Apr 2010 - Aug 2012 |
24.94 |
6.99 |
38.91 |
10.96 |
Protocol for solids |
Apr 2010 - Aug 2012 |
21.01 |
7.09 |
35.20 |
6.83 |
Table 1: In Vitro Irritancy score (IVIS)
Test substance |
Cornea- No. |
Opacity per cornea |
Permeability per cornea |
per cornea |
IVIS per group |
|
mean |
SD |
|||||
|
10 |
17.3 |
0.393 |
23.2 |
|
|
Test substance |
11 |
15.0 |
0.422 |
21.3 |
19.6 |
4.7 |
|
12 |
8.0 |
0.412 |
14.2 |
|
|
|
1 |
1.6 |
0.001 |
1.6 |
|
|
NC |
2 |
1.0 |
0.005 |
1.1 |
1.6 |
0.5 |
|
3 |
2.0 |
0.006 |
2.1 |
|
|
|
4 |
126.3 |
2.809 |
168.4 |
|
|
PC |
5 |
135.9 |
2.689 |
176.2 |
180.2 |
14.2 |
|
6 |
163.5 |
2.166 |
196.0 |
|
|
Table 2: Historical control data
Historical range of NC (protocol for liquids and surfactants)
Historical period
Jan 2010 - Aug 2012 |
|
||
Opacity |
Mean 1.3 |
SD 1.5 |
Mean + 2 SD Mean - 2 SD 4.2 -1.7 |
Permeability |
Mean 0.005 |
SD 0.016 |
Mean + 2 SD Mean - 2 SD 0.037 -0.027 |
Historical range of PC (1% NaOH) |
|
|
|
Historical period
Jan 2010 - Aug 2012 |
|
|
|
Opacity |
Mean 118.1 |
SD 20.4 |
Mean + 2 SD Mean - 2 SD 159.0 77.2 |
Permeability |
Mean OD 2.814 |
SD 0.897 |
Mean + 2 SD Mean - 2 SD 4.607 1.021 |
In Vitro Irritation Score (IVIS) |
Mean 160.3 |
SD 22.9 |
Mean + 2 SD Mean - 2 SD 206.1 114.6 |
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (irritating)
Respiratory irritation
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
In order to replace animal studies, tests on skin and eye irritation were performed as in vitro studies. The EpiDerm system was used to evaluate the skin corrosion and the skin irritation potential by examination of the viability of the cells of the reconstructed skin model after 3 min and 1h or after 35 min exposure according to OECD guideline 431 and 439.
A combination of the BCOP test (bovine corneal opacity, OECD guideline 437) and the EpiOcular assay was used to evaluate the eye irritation potential of the test material.
The cell viability values obtained each for the skin corrosion and the irritation test are clearly above the threshold of 15% and 50%, respectively. The test material is therefore not considered to be irritating to skin. Measurement of the permeability and opacity of the bovine corneas after treatment with the test item gave IVIS values which were below the threshold of 55. The viability after exposure of EpiOcular tissue cells was 5%. Thus, the test item is considered to be irritating to eyes and is not considered to cause serious damage to eyes.
Justification for classification or non-classification
Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is considered to be classified for irritation to eyes (cat. 2) under Regulation (EC) No. 1272/2008, as amended for the 14th time in Regulation (EU) 2020/217.
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